Orchiectomy upregulates rabbit prostate peripheral benzodiazepine receptors. The effect of orchiectomy on peripheral benzodiazepine receptors (PBZr) in the rabbit prostate was studied. The mean PBZr density in the mitochondrial fraction isolated from prostates of intact (non castrated) rabbits was 4066 fmol/mg protein which following castration increased to 7236 fmol/mg protein (p less than 0.005). The apparent dissociation constant (KD) of prostatic PBZr was higher in castrated rabbits (4.2 nM) than in intact animals (2.7 nM). These data suggest a role of androgen in the regulation of prostatic PBZr.


Stimulatory effect of thymic factor(s) on steroidogenesis in cultured rat granulosa cells. Thymic cells from immature female rats were isolated and used for production of thymic cell culture conditioned medium (TCM). Granulosa cells were obtained from immature diethylstilbestrol (DES)-treated rats. TCM stimulated basal progesterone and estradiol secretion from the granulosa cells in a dose and time dependent manner. Maximal stimulation of progesterone production occurred at 48 hours of incubation, during which period TCM caused approximately 5 times more progesterone secretion than heart cell conditioned medium (HCM) or mock extract (ME). The maximum progesterone secretion by granulosa cells occurred when they were exposed to 48% TCM causing 7 times more progesterone secretion than controls. Under the same maximum stimulatory conditions, however, TCM only approximately doubled estradiol secretion compared to concentrations secreted in the presence of HCM or ME. Thus, the effect of TCM on progesterone secretion was more prominent than its effect on estradiol secretion. The stimulatory action of TCM was not mimicked by HCM, thymosin-alpha 1 or thymulin. Furthermore, the stimulatory action of TCM on steroidogenesis did not appear to be mediated by the cAMP system. The stimulatory factor(s) in TCM were heat, acid and acetone labile, but could not be sedimented by activated charcoal. Thus, the present study demonstrates that the secretory product(s) of thymic epithelial cells can stimulate steroidogenesis in cultured rat granulosa cells. Our data imply that thymic factor(s) may have a direct effect on ovarian function.


The effect of active serum albumin on PC12 cells: I. Neurite retraction and activation of the phosphoinositide second messenger system. Vertebrate blood sera contain a factor that triggers oscillatory chloride currents in Xenopus oocytes through activation of the phosphoinositide/Ca2+ second system. The active serum component consists of lipids bound to an isoform of serum albumin that we have named active serum albumin (ASA). In undifferentiated PC12 cells, micromolar concentrations of ASA inhibit the early morphological changes induced by NGF, whereas in differentiated PC12 cells ASA caused a rapid withdrawal of neurites, which was reversible and dependent upon culture age. In contrast to normal serum, plasma and thrombin did not cause neurite retraction. Preincubation of ASA with monospecific antibodies to serum albumin suppressed its ability to induce neurite retraction in a dose dependent fashion. As in the oocyte, ASA activated the phosphatidylinositol second messenger system of PC12 cells, causing a several fold increase in Ins1,4,5P3 levels within minutes of application. The Ins1,4,5P3 increase was also blocked, in a titratable fashion, when ASA was preincubated with monospecific antibodies to serum albumin. This suggests that ASA-induced neurite retraction in PC12 cells may depend, at least in part, on activation of the phosphatidylinositol second messenger system. Results involving albumin-depleted sera show that ASA is the main factor responsible for serum vulnerability of neurites in PC12 cells. These findings point to some limitations in the use of serum in culture media, and raise the possibility that the serum factor may impair neuronal plasticity in disorders that are accompanied by the activation of blood coagulation together with a breakdown of the blood-brain barrier.


The effect of active serum albumin on PC12 cells: II. Intracellular Ca2+ transients and their role in neurite retraction. In the preceding paper it was shown that an isoform of serum albumin (ASA; active serum albumin) causes a rapid retraction of neurites and increases intracellular content of Ins1,4,5P3 in PC12 cells. Here we examined whether ASA's effects in nerve growth factor-differentiated PC12 cells were mediated through the Ins1,4,5P3/Ca2+ second messenger pathway by monitoring intracellular Ca2+ (Ca2+i) with Fura2. It was found that ASA caused a dose-dependent increase in Ca2+i. In Ca(2+)-free medium, the increase in Ca2+i elicited by ASA was smaller, but the rise in Ins1,4,5P3 content was not appreciably changed. The small Ca2+i increase seen in Ca(2+)-free medium was probably due to the release of Ca2+ from Ins1,4,5P3-sensitive intracellular stores. In Ca(2+)-containing medium the Ca2+ transient induced by ASA was not affected by organic Ca2+ channel blockers, but decreased when Co2+, Mn2+ or Zn2+ were present in the extracellular medium. The effect of other ligands, such as carbachol and bradykinin, whose receptors are coupled to the phosphoinositide system was also investigated. Carbachol at concentrations from 2 to 200 microM, and bradykinin at a concentration of 2 microM did not cause neurite retraction, whereas 200 microM bradykinin caused an approximately 40% decrease in neurite length. Thapsigargin, a Ca(2+)-ATPase inhibitor, caused a sustained elevation of Ca2+i and retraction of neurites, whereas depolarization of the cells by high K+ gave only a transient elevation of Ca2+i, and no neurite retraction. Therefore, a sustained elevation in Ca2+i might be a sufficient trigger to induce neurite retraction in differentiated PC12 cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Expression of glutamic acid decarboxylase messenger RNA in rat medial preoptic area neurones during the oestrous cycle and after ovariectomy. Evidence suggests that medial preoptic area (MPOA) neurones containing gamma-aminobutyric acid (GABA) are modulated directly by oestrogen. We have used an alkaline phosphatase-labelled antisense oligonucleotide probe to examine glutamic acid decarboxylase67 (GAD) mRNA expression within individual cells of the MPOA, diagonal band of Broca (DBB) and parietal cortex in rats killed at noon on each day of the oestrous cycle and after ovariectomy (n = 4-5). As a fall in extracellular GABA concentrations occurs in the MPOA on the afternoon of proestrus, the GAD67 mRNA content of cells was also examined in proestrous rats at 15:00h immediately prior to the preovulatory luteinising hormone (LH) surge. The MPOA was found to have an intermediate number of GAD67 mRNA-containing cells compared with the DBB and cortex (P less than 0.01) but expressed the lowest mean hybridisation signal (P less than 0.01). The parietal cortex had significantly fewer (P less than 0.01) GAD mRNA-containing cells than either the MPOA or DBB but these contained higher mean density of signal (P less than 0.01). The hybridisation signal for GAD mRNA was abolished by either ribonuclease pre-treatment or the use of excess non-labelled probe. No significant (P greater than 0.05) differences in GAD67 mRNA were detected in animals killed at noon throughout the oestrous cycle or after ovariectomy. On the afternoon of proestrus (15:00h) there was a significant 40% reduction in mean GAD67 mRNA content within cells of only the MPOA compared with noon (P less than 0.05). The numbers of cells in the MPOA expressing GAD67 mRNA were not significantly different.(ABSTRACT TRUNCATED AT 250 WORDS)


Visualization of cholinoceptive neurons in the rat neocortex: colocalization of muscarinic and nicotinic acetylcholine receptors. The present investigation analyzes the cellular distribution of muscarinic and nicotinic acetylcholine receptors in rat neocortex, by use of monoclonal antibodies raised against purified receptor proteins. The degree of colocalization of both types of receptors was determined by way of immunofluorescent double-labeling techniques. For both classes of receptors, pyramidal and nonpyramidal cells were found immunostained and an identical laminar distribution pattern of immunopositive neurons in the rat neocortex became apparent. A striking similarity in distribution of the two cholinergic receptor types was found in the frontal/motor and parietal cortex. Accordingly, we observed a high degree of colocalization of muscarinic and nicotinic acetylcholine receptors within immunopositive cortical neurons. Approximately 90% of the cholinoceptive neurons expressed both types of receptors. The current data demonstrate that (i) the distribution of muscarinic and nicotinic cholinoceptive neurons in the neocortex is present in identical laminar patterns and represent the same type of cells, (ii) both classes of cholinergic receptors are highly colocalized within cholinoceptive neurons, which points at individual neurons as a likely site of interaction between muscarinic and nicotinic acetylcholine receptor-mediated processes.


Localization of gene expression of calreticulin in the brain of adult mouse. The localization of gene expression of calreticulin, a calcium-binding protein in the endoplasmic reticulum, was examined throughout the entire brain of adult mice by in situ hybridization. Calreticulin mRNA is expressed widely and heterogeneously in discrete neurons throughout the brain, but the white matters expressed it weekly or faintly. In the olfactory bulb, the mRNA is expresses moderately in the mitral cells, but weakly in the periglomerular cells and internal granule cells. In the cerebrum, the gene is expressed intensely in the piriform cortex, but weakly in neocortex, the entorhinal cortex and the amygdaloid nuclei. In the hippocampal formation, calreticulin mRNA is expressed intensely in the CA1-CA3 regions but less intensely in the granule cells of the dentate gyrus. The caudate-putamen, thalamic and hypothalamic nuclei, and mammillary nuclei express the mRNA weakly or faintly. In the mesencephalon, pons and medulla, moderate expression of the mRNA is detected in the pontine nuclei and the locus ceruleus. Weak expression of the mRNA is detected in several discrete nuclei and zones such as the substantia nigra, the superior colliculus and the central gray. Expression signals of calreticulin mRNA are faint in the inferior olive. In the cerebellum, calreticulin mRNA is expressed moderately in the Purkinje cells whereas no significant expression is detected in the granule cells. The plexus choroideus of the lateral, third and fourth ventriculi express calreticulin mRNA intensely although no distinct expression of the mRNA is discerned in the ependyma.


Nerve growth factor modulates the expression of muscarinic cholinergic receptor messenger RNA in telencephalic neuronal cultures from newborn rat brain. The effect of nerve growth factor (NGF) on muscarinic receptor subtypes was investigated in a primary culture of telencephalic neurons prepared from neonatal rats. The treatment with 100 ng/ml of NGF significantly enhanced choline acetyltransferase (ChAT) activity and intracellular acetylcholine (ACh) content during cultivation. The same treatment induced an early transient increase of the number of muscarinic cholinergic receptors (mAChR), as measured by [3H]quinuclidinyl benzilate binding to cell homogenate, that was followed by a dramatic decrease of the receptor density from the 9th day of culture. Atropine completely prevented the decrease of the maximal number of muscarinic recognition sites induced by NGF. Prolonged exposure of telencephalic neurons to NGF also induced a significant reduction of the relative content of the messenger RNA (mRNA) encoding m1 and m3 receptors, while the m4 transcript was increased by the treatment. We suggest that the prolonged stimulation of cholinergic neurons by NGF induces a downregulation of m1 and m3 mAChR and their mRNAs on the postsynaptic site, while it increases the synthesis of the functionally distinct m4 receptor subtype, which might be presynaptically localized on cholinergic neurons. The transient increase of the receptor number that occurs at the first days of culture was not paralleled by changes in the relative content of mAChR mRNAs and might be associated with the trophic activity of NGF on cholinergic synapses during early development.


Delayed c-fos expression in sensory cortex following sexual learning in male rats. An elevated expression of c-fos protooncogene, encoding transcription factor Fos, is known to serve as a useful marker of neuronal activation. In the studies reported in this communication we have used Northern and dot blot techniques to analyze c-fos mRNA levels in male rat brain during the learning of the copulatory behavior. The animals were trained (single ejaculation in a training/testing session) for up to 7 sessions. C-fos mRNA levels have been found to increase in the sensory cortex area following the third and fifth session but not after the first and the last one.


Effect of the selective lesion of serotoninergic neurons on the regional distribution of 5-HT1A receptor mRNA in the rat brain. The effects of the selective lesion of serotoninergic neurons by an intra-raphe administration of 5,7-dihydroxytryptamine on the 5-HT1A receptor protein and the 5-HT1A receptor mRNA were examined in various regions of the rat brain using specific antibodies and an antisense riboprobe, respectively. Twenty one days after the treatment, the 5-HT1A receptor protein was no longer detected within the dorsal raphe nucleus but was still present in the hippocampus and entorhinal cortex. Quantitative in situ hybridization showed an 85% decrease in the levels of 5-HT1A receptor mRNA within the dorsal raphe nucleus, but no significant change in the hippocampus, interpeduncular nucleus and entorhinal cortex of 5,7-dihydroxytryptamine-treated rats. These data demonstrate that 5-HT1A receptors are synthesized by serotoninergic neurons in the dorsal raphe nucleus, and by neurons located postsynaptically with regard to serotoninergic projections in other areas. The unchanged levels of 5-HT1A receptor mRNA in the hippocampus, interpeduncular nucleus and entorhinal cortex three weeks after the extensive lesion of serotoninergic neurons are consistent with the absence of 5-HT1A receptor up regulation already reported under this condition.


Hormone replacement therapy: lipid responses to continuous combined oestrogen and progestogen versus oestrogen monotherapy. Fifty-five postmenopausal women with climacteric complaints were randomly assigned to treatment with either 2 mg oestradiol valerate (E2V) (cyclic regimen: 21 days of treatment followed by a 7-day treatment-free interval), or 2 mg E2V combined with 1 mg cyproterone acetate (E2V+CPA) daily, over a 6-month period. Treatment was by the oral route in both cases. The aim was to compare the influence of these two hormone replacement therapy regimens on lipid metabolism. Blood samples were obtained before and after 1 and 6 months of treatment. Serum was analyzed for total cholesterol (TC), high-density lipoproteins (HDL), apolipoproteins A1 and B and triglycerides. The low-density lipoprotein (LDL) concentrations were derived by calculation. All parameters were evaluated in terms of mean +/- S.D. There was no significant difference in the response of the blood lipids to the two treatments, as assessed by analysis of variance (P greater than 0.05). Serum levels of TC were found to have fallen after month 1 and 6 by 5.3 and 5.6%, respectively, during E2V treatment and by 2.4 and 0.2% during E2V+CPA treatment. Serum HDL levels had increased after months 1 and 6 by 9.7 and 5.2%, respectively, in the E2V group and by 6.9 and 2% in the E2V+CPA group, which was also confirmed by the increase in apolipoprotein A1 levels. There was, however, a borderline increase in LDL and apolipoprotein B in the E2V+CPA group. Serum triglycerides were reduced and serum levels of SHBG increased during treatment in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)


Frequency of curettage in middle-aged women treated with sequential preparations versus untreated women. Over a 2-month period a register was kept of all dilatation and curettage procedures performed in Frederiksborg County, Denmark, involving women aged 40-59 years. The total recorded was 284. In the same period, questionnaires were sent out to 1200 women in the county who fell within the same, randomly selected age group, to establish the number of women treated with sequential oestrogen/progestogen and those who had been hysterectomized. Based on the results and the total female population in the county, it was calculated that the frequency of the procedure in sequentially-treated women as compared with untreated women was 3.1 times higher in the 55-59 age group. In the 40-54 age group no difference in the incidence of curettage in the sequentially-treated women could be demonstrated.


[Detection of BK virus activation in renal and bone marrow transplantation patients using serological and virological methods]. In this study, 10 renal and bone marrow transplant recipients' sera samples taken before and after transplantation and urine samples taken after transplantation were investigated for evidence of reactivation of BK virus, a human polyoma virus. BK virus reactivation was detected in 4 transplant patients serologically by hemagglutination inhibition, ELISA-IgG and ELISA-IgM tests. However we did not succeed to isolate BK virus in Vero cell lines from any urine samples. Only one patient's urine sediment whom serologic reactivation was detected, contained uroepithelial cells with intranuclear inclusion bodies characteristic for BK virus when examined by Giemsa staining.


New clues into the etiology of osteoporosis: the effects of prostaglandins (E2 and F2 alpha) on bone. A new hypothesis is presented for the first time to explain the etiology of osteoporosis. Prostaglandins (E2 and F2 alpha) at precise concentrations, have been observed to be involved in bone formation. A close association exists between levels of prostaglandins (E2 and F2 alpha) demonstrated in the neonatal mouse leading to bone formation, with estimated prostaglandins (E2 and F2 alpha) concentrations reported in man. Several hormones (vasopressin, oxytocin, luteinizing hormone, follicle-stimulating hormone, cortisol, estradiol, and testosterone) can indirectly affect prostaglandin formation leading to reduced bone formation. The association between these hormones and prostaglandins (E2 and F2 alpha) explains the physiological mechanism whereby estradiol can be effective for the treatment of osteoporosis. This association also explains the etiology of lumbar spondylitis/spondylodynia, reasons for complaints of increased pain in wet cold weather among arthritics and a multitude of other events. Mechanisms related to this interaction between various hormones and the effect of prostaglandins (E2 and F2 alpha) on bone formation are discussed.


The dystrophin connection--ATP?

The dystrophin connection--ATP? Clinical evidence is presented supporting the hypothesis that the metabolic abnormality in the dystrophin-defective muscular dystrophies (DMD and BMD) involves the ATP pathway. Objective laboratory data show corrective trends in the abnormal values of parameters relating to creatine and calcium metabolism (ATP) by use of glucagon-stimulated c-AMP and by use of synthetically produced adenylosuccinic acid (ASA). Disease accelerating mechanisms as suggested by analysis of the clinical features, and the therapeutic potential of ASA are discussed.


Famine-affected, refugee, and displaced populations: recommendations for public health issues. During the past three decades, the most common emergencies affecting the health of large populations in developing countries have involved famine and forced migrations. The public health consequences of mass population displacement have been extensively documented. On some occasions, these migrations have resulted in extremely high rates of mortality, morbidity, and malnutrition. The most severe consequences of population displacement have occurred during the acute emergency phase, when relief efforts are in the early stage. During this phase, deaths--in some cases--were 60 times the crude mortality rate (CMR) among non-refugee populations in the country of origin (1). Although the quality of international disaster response efforts has steadily improved, the human cost of forced migration remains high. Since the early 1960s, most emergencies involving refugees and displaced persons have taken place in less developed countries where local resources have been insufficient for providing prompt and adequate assistance. The international community's response to the health needs of these populations has been at times inappropriate, relying on teams of foreign medical personnel with little or no training. Hospitals, clinics, and feeding centers have been set up without assessment of preliminary needs, and essential prevention programs have been neglected. More recent relief programs, however, emphasize a primary health care (PHC) approach, focusing on preventive programs such as immunization and oral rehydration therapy (ORT), promoting involvement by the refugee community in the provision of health services, and stressing more effective coordination and information gathering. The PHC approach offers long-term advantages, not only for the directly affected population, but also for the country hosting the refugees. A PHC strategy is sustainable and strengthens the national health development program.


Ontogeny of class II MHC mRNA in the mouse small intestinal epithelium. MHC Class II (Ia) and invariant chain cooperate in the presentation of exogenous antigen by antigen presenting cells to T-helper cells. Both glycoproteins have been identified in the small intestine of the mature mouse. In this study, we examine the ontogeny of mRNA for three molecules; (Ii31, Ii41 and I-A beta) in whole intestine and in isolated epithelial cells. When RNA from whole intestine was analysed in northern blots using cDNA probe, Ii31 mRNA was present in Day 10 mice and at each 5 day time point thereafter; Ii41 and I-A beta were not detected by this technique. To examine ontogeny of Ii chain mRNA in enterocytes, RNA was purified from an enriched population of epithelial cells isolated after systemic perfusion with 30 mM EDTA in Day 21 and Day 28 and adult mice. Ii chain mRNA was not detected until Day 28 by blot hybridization. Reverse transcription of mRNA and amplification of the resultant cDNA by PCR revealed Ii41 and I-A beta as well as Ii31. RNA from Day 21 epithelial cells required five additional amplification cycles to attain cDNA levels equivalent to those found in Day 28 cells for Ii chain, and 10 additional cycles for I-A beta. In conclusion, Ii31, Ii41 and I-A beta mRNA increase rapidly in the enterocyte after weaning.


Detection of antibodies to the E4 or E7 proteins of human papillomaviruses (HPV) in human sera by western blot analysis: type-specific reaction of anti-HPV 16 antibodies. To determine the cross-reactivity between early (E) proteins of different human papillomavirus (HPV) types, 346 serum samples were tested with E4 and E7 of HPV 16. Two hundred and sixteen of them were also tested with HPV 1 E4, 21 with HPV 11 E4 and E7, and 109 with HPV 18 E4 and E7. Viral fusion proteins were expressed in Escherichia coli and used as antigens in Western blot experiments. The sera were obtained from patients with HPV-associated genital lesions or cervical cancer, from renal transplant recipients and from patients hospitalized for reasons unrelated to HPV infections (the controls). In contrast to findings relating to HPV 16 E4 specific antibodies, the prevalence of anti-HPV 1 E4 antibodies was not greater in renal transplant recipients than in the controls. In each age group of the control population more sera reacted with HPV 1 E4 than with HPV 16 E4. Sera of patients with HPV-associated cervical diseases and cervical cancer reacted less frequently with HPV 11 E4 or E7 and HPV 18 E4 or E7, respectively, than with the corresponding HPV 16 proteins. Thirty of 117 HPV 16 E4 or E7 positive sera showed reactivity to the corresponding protein of either HPV 1, 11 or 18. As demonstrated by cross-absorption experiments performed with 26 of the double-reacting sera, 24 contained two populations of antibodies reacting with proteins of different HPV types whereas only two contained cross-reacting antibodies. We concluded that in the majority of sera antibodies to the HPV 16 E4 and E7 proteins are type-specific.


Prenatal diagnosis by enzymatic amplification and restriction endonuclease digestion for detection of haemoglobins A, S and C. The detection of the single base pair mutations at codon 6 of the beta-globin gene is important for the prenatal diagnosis of sickle-cell anaemia and SC disease. A novel procedure has been designed to create a restriction site at both the beta A and beta C alleles to facilitate the discrimination of haemoglobins A, S and C. The general principle of this procedure is to enzymatically amplify genomic DNA using a modified primer containing an altered 3'-terminal nucleotide to create these restriction sites. After this modified primer has been efficiently incorporated into amplified DNA, the PCR products are digested with the restriction enzymes Ava I and Sty I. Ava I recognizes a site in amplified DNA containing a beta A allele, and Sty I recognizes a site in DNA containing a beta C allele. Since the beta A and beta C alleles can be distinguished directly by the presence of a restriction site, the beta S allele can be identified indirectly. All three beta-globin alleles are easily distinguished by size and pattern of electrophoresed fragments on agarose gels. This procedure is specific and sensitive, thus permitting rapid, economical diagnosis of sickle-cell anaemia and SC disease.


5-HT1-like receptor mediated changes in porcine carotid haemodynamics: are 5-HT1D receptors involved? 5-Hydroxytryptamine (5-HT) reduces porcine arteriovenous shunting in the carotid vascular bed by stimulation of both 5-HT1-like and 5-HT2 receptors and increases capillary flow to some tissues, like the skin and ears, by different 5-HT1-like receptors. In view of the heterogeneous nature of the 5-HT1-like receptors and the relative selectivity for the 5-HT1D binding sites of sumatriptan, which also reduces porcine arteriovenous shunting and slightly increases capillary blood flow towards skin and ears by 5-HT1-like receptors, we have attempted to determine whether one or both of these carotid 5-HT1-like receptors belong to the 5-HT1D subtype. Pentobarbitone anaesthetized pigs, subjected to bilateral cervical vagosympathectomy, received either 5-HT (2 micrograms.kg-1.min-1) in the carotid artery or cumulative i.v. doses of sumatriptan (10, 30, 100 and 300 micrograms.kg-1). Their effect on the total carotid blood flow and its distribution into capillary and arteriovenous anastomotic parts was determined with radioactive microspheres. The effect of metergoline (1 mg.kg-1), a substance with a very high affinity for the 5-HT1D receptor as well as for the 5-HT1A, 5-HT1B, 5-HT1C and 5-HT2 receptors, was studied on the responses to 5-HT and sumatriptan. Both 5-HT and sumatriptan reduced carotid arteriovenous anastomotic blood flow. 5-HT and, to a lesser extent, sumatriptan also increased capillary blood flow towards some tissues. Metergoline by itself did not affect the distribution of porcine carotid blood flow.(ABSTRACT TRUNCATED AT 250 WORDS)


Adrenoceptor-mediated effects on calcium channel currents are antagonized by 5'-(N-ethyl)-carboxamido-adenosine in guinea-pig atrial cells. In guinea-pig atrial myocytes, the effects of the adenosine analogue 5'-(N-ethyl)-carboxamido-adenosine (NECA) in the presence of isoprenaline (ISO) on Ca2+ channel activity were analyzed. Single Ca2+ channel currents were recorded from cell-attached patches by application of several hundred 100 ms depolarizing steps. Under control conditions, burstlike activity of channel openings during some depolarizing steps were followed by variably long periods of quiescence (blank sweeps). During superfusion with ISO (100 nmol/l), ensemble-averaged (mean) current was increased by about 150%. The underlying mechanism was found to be a significant increase in the channel availability, defined as the ratio of current-containing sweeps to the total number of sweeps. In addition, the ISO-induced reduction of blank sweeps was combined with slightly but not significantly higher values of the open probability in the current-containing sweeps. Open time and shut time histograms could be fitted by single and double exponential curves, respectively, which remained rather unaffected in the presence of ISO; accordingly, mean open time and mean shut time of the channel were not significantly changed by ISO. After the addition of NECA (1 mumol/l) in the presence of ISO, the ISO-induced increase in mean current was abolished. This effect of NECA on mean current was due to a reduction of the channel availability and a slight decrease in the open probability. The purinoceptor blocking agent 8-phenyltheophylline (10 mumol/l) antagonized the inhibitory action of NECA on the ISO-induced increase in Ca2+ channel activity.(ABSTRACT TRUNCATED AT 250 WORDS)


Potentiation of cardiodepressive action among calcium antagonists from different classes: evidence for a mechanism at the single calcium channel level. The ability of calcium antagonists and antiarrhythmic agents to potentiate the negative inotropic effects of calcium antagonists was investigated in guinea-pig left atria. The potency of nitrendipine was enhanced by several amphiphilic agents by one order of magnitude or more (by pretreatment with quinidine or bepridil). The effect of preincubation with bepridil was investigated for a larger number of dihydropyridines. Only some of them were potentiated like nitrendipine. There was no potentiation between any two members of the same chemical group, i.e. between two dihydropyridines or two catamphiphilic calcium antagonists. The interaction between bepridil and nitrendipine was studied in more detail. In atria, its extent was influenced by several conditions, such as the stimulus frequency, the incubation temperature, or the extracellular K+ concentration. In measurements of whole-cell calcium currents in guinea-pig myocytes, the interaction was found to take place in a quantitatively similar manner. At the single channel level, an enhancement of the effects could also be demonstrated. It appears here that both drugs interact by binding to the same channel molecule. We conclude that the interaction may be due to 1.: an amphiphilic drug (like bepridil) binding to the channel very transiently and thus briefly favouring the inactivated channel state, which means that 2.: the other drug (like nitrendipine) has a higher chance to be bound because of its high affinity towards inactivated channels. Alternative explanations are also discussed.


In vitro effect of indomethacin on mitogen-induced lymphoproliferative response in lung cancer patients. There is some evidence that prostaglandin (PGE)-secreting cells may have a role in immunosuppression in cancer patients. In this work we investigated the effects of indomethacin--a PGE synthesis inhibitor, on PHA-induced lymphoproliferative response in vitro. Twenty patients with lung cancer before therapy were included in this study. When compared to controls, the patients had significant decrease of T cell number and proliferative response to PHA (p less than 0.001) and increased number of mononuclear phagocyting cells (p less than 0.001). The degree of depression of lymphocyte response did not correlate with the number of mononuclear phagocytes. The presence of indomethacin in the culture induced significant (p less than 0.01) improvement of the reactivity in high percentage (75%) of patients with diminished lymphoproliferative response to PHA. In the patients with normal lymphocyte response, indomethacin did not change reactivity to PHA. These results indicate that PGE-secreting cells may contribute to the immune depression in lung cancer patients, and that indomethacin may have therapeutical potential in some patients.


The protective activity of Thymex L against radiotherapeutically-induced cellular immunodepression in lung cancer patients. In order to prevent the radiotherapeutically-induced aggravation of initial immunodeficiency, a thymic preparation (Thymex L) was given to lung cancer patients simultaneously with irradiation. The parameters of both cellular and humoral nonspecific immunity were evaluated in two groups of patients: one was treated with radiotherapy only (60 Gy in 30 fractions); the other one received Thymex L (100 mg 3 times a week, total dose 1800 mg, i.m.) simultaneously with radiotherapy. The significant decrease of B and T cell number, and decreased lymphoproliferative response to PHA were found in all patients before therapy; the number and phagocyting capacity of blood monocytes, as well as the concentrations of circulating IgG, IgA and immunocomplexes, were all significantly increased. Immediately after irradiation the patients had even lower number of T and B cells, diminished reactivity to PHA and higher number of mononuclear phagocytes when compared to the values before therapy. In patients treated with Thymex L, the number of B and T cells and PHA-induced proliferative response were significantly higher than in those treated with radiotherapy only. No effect of this therapy was seen on active T cells, on high number and function of mononuclear phagocytes and on elevated concentrations of serum immunoglobulins and immune complexes. Our results indicate that Thymex L can successfully prevent the harmful effect of radiation therapy on cellular immunity in a majority of lung cancer patients.


In chronic uremia, insulin activates receptor kinase but not pyruvate dehydrogenase. We studied in vivo biochemical effects of insulin in the skeletal muscle of chronically uremic and control rats. The rate of disappearance of blood glucose (determined with a short intravenous test) was reduced by 38% in uremia (p less than 0.05). Intraperitoneal treatment with insulin plus glucose for 30 min caused a 3-fold increase in the activity of insulin receptor tyrosine kinase in the skeletal muscle of both rat groups. Conversely, pyruvate dehydrogenase activity increased by 115% in controls but only by 26% in uremics (p less than 0.01). Exercise (swimming for 30 min) increased muscle pyruvate dehydrogenase activity approximately 2-fold in both groups of animals. These experiments show that in uremic rats, insulin binds normally to its muscle receptors and adequately activates receptor tyrosine kinase but fails to activate an otherwise responsive pyruvate dehydrogenase.


Adenosine 5'-monophosphate transport across the membrane of synaptosomes and myelin. Synaptosome-enriched preparations from rat and guinea pig brain tissue vigorously accumulated [3H]-adenosine 5'-monophosphate ([3H]AMP). When the accumulation of [3H]AMP was determined using incubation periods of 30 s or less, high concentrations of adenosine, dipyridamole and soluflazine did not inhibit the accumulation of label appreciably. The accumulation of [3H]AMP was saturable, temperature-dependent, osmotic-sensitive and exhibited structural specificity. Based on the kinetics of uptake by different subcellular fractions, and the inhibitory effects of other nucleotides, the uptake of AMP appeared to be mediated by three saturable systems with Kt values of approximately 0.2, 6, and 100 microM. The transport system with the highest affinity for AMP was selectively inhibited by guanosine 5'-monophosphate, and its Vmax was several fold higher in a myelin-enriched fraction than in synaptosome-enriched fractions. The transport system with the Kt approximately 6 microM was selectively inhibited by alpha, beta-methylene adenosine diphosphate, and its Vmax was several times higher in a fraction enriched in high-density synaptosomes than in fractions enriched in low-density synaptosomes or myelin. Both of these transport systems were potently inhibited by ATP and ADP. Nucleotides that were either weak or inactive as inhibitors of AMP transport included 3'-AMP, cyclic AMP, guanosine 5'-diphosphate, and the 5'-mononucleotides of cytosine, inosine, and uridine. GTP consistently enhanced uptake at concentrations greater than or equal to 1 microM. The transport of AMP was not Na(+)-dependent and was not inhibited by membrane depolarization. This transport system may mediate the release of AMP for subsequent conversion to adenosine extracellularly.


IgG subclass responses in brain and serum in Semliki Forest virus demyelinating encephalitis. The response of IgG subclasses within the central nervous system (CNS) of the mouse to Semliki Forest virus (SFV), an alphavirus associated with meningoencephalitis and primary immune mediated demyelination, has been measured using immunocytochemistry. The subclass response in serum has been assessed using virus specific enzyme linked immunosorbent assays. In the CNS IgG1 was poorly represented throughout the sampling period of 28 days with a maximum of 3% of the total number of positive cells on day 21 after infection. Of the few IgG positive cells present on day 6, 2a and 3 positive cells were dominant. From day 9 onwards the numbers of 2b positive cells rose and by day 28 IgG2a, 2b and 3 subclasses showed roughly equal percentages of total cells counted. By contrast, in serum, anti-SFV IgG 2a and 2b were the first to appear and were dominant on day 12 and 21. Levels of anti-SFV IgG1 did not rise until after day 12 but rose steeply thereafter. IgG3 was weakly positive at days 9 and 12, rising slightly on day 21. Clearly there are differences in the patterns of subclass response between the CNS and the periphery. This may be important in the context of neurotropic viral infection.


The action of GABAB antagonists in the trigeminal nucleus of the rat. The iontophoretic administration of the GABAB antagonists (P-(3-aminopropyl)-P-diethoxymethyl-phosphinic acid (CGP 35348) and 2-hydroxy-saclofen blocked the action of iontophoretically applied L-baclofen on neurons in the trigeminal nucleus of rats, anesthetized with halothane. The substance CGP 35348 appeared to be more potent than 2-hydroxy-saclofen. The iontophoretic administration of GABA resembled L-baclofen in depressing excitatory transmission and facilitating segmental inhibition in the trigeminal nucleus. The depression of excitatory transmission was also blocked by CGP 35348 and the facilitation of segmental inhibition produced by GABA was partially blocked. These observations indicate that CGP 35348 is not only a baclofen antagonist but actually a GABAB receptor antagonist and the baclofen was acting at GABAB receptors in the trigeminal nucleus. The portion of the effect of GABA, not blocked by CGP 35348, was probably mediated by GABAA receptors, since it was previously found that segmental inhibition in the trigeminal nucleus could be modulated by GABAA agonists and antagonists as well.


The neuropharmacology of loperamide-induced emesis in the ferret: the role of the area postrema, vagus, opiate and 5-HT3 receptors. Loperamide, an opiate receptor agonist, commonly used in the treatment of diarrhoea, reliably induced emesis in the ferret, when given subcutaneously. The response latency was short (less than 10 min) and the emesis lasted for approx 70 min. The dose-response curve for the emetic response was "bell-shaped" and all animals responded at 0.5 mg/kg but none at 5 mg/kg (s.c.). The response was unaffected by dopamine D2 receptor antagonism (domperidone 1.0 mg/kg, s.c.) or 5-HT3 receptor antagonism (granisetron or ondansetron 1.0 mg/kg, s.c.). The onset of the response was delayed for about 60 min by naloxone or naloxone methiodide (1.0 mg/kg, s.c.) and abolished by naloxanazine (1.0 mg/kg, s.c.), reported to be relatively selective for mu receptors. The results implicate mu receptors (possibly mu 1) in the induction of emesis by loperamide and provide some support for activation of opiate receptors also having anti-emetic effects, as suggested in previous studies. The emetic response to loperamide was unaffected by abdominal vagotomy but was abolished by ablation of the area postrema, indicating that loperamide-induced emesis may be used as a test for ablation of the area postrema in studies of the emetic mechanism in the ferret.


[Current views on the structure and function of the hippocampus]. The aim of our study was to present the actual views on the structure and function of the hippocampus. Recent evidence shows that internal connections within this structure do not agree with Andersen's lamellar hypothesis of hippocampal organization. The fibres course seems to prefer longitudinal flow of impulses. There are discussed functions of the hippocampus, particularly these which are involved in memory processes. We also present recent view points on the relationships between changes in hippocampal structure and Alzheimer disease.


Desensitization of GABAB receptors and antagonism by CGP 35348, prevent bicuculline- and picrotoxin-induced antinociception. The effect of the GABAA antagonists, bicuculline and picrotoxin, in the hot plate and writhing tests in mice and the paw-pressure test in rats was assessed. Subconvulsant doses of bicuculline (1.3-4 mumol kg-1, s.c.) or picrotoxin (0.8-2.5 mumol kg-1, s.c.) induced a dose-related increase in latency of licking in the hot plate test in mice, whereas subconvulsant doses of strychnine and thiosemicarbazide (0.9 and 6 mg kg-1, s.c. respectively), did not modify the threshold to thermal stimuli in mice. The effects of bicuculline and picrotoxin were not modified by naloxone (3 mg kg-1, i.p., a dose which inhibited the antinociceptive effect of morphine) or by atropine (5 mg kg-1, i.p., a dose which prevented oxotremorine-induced antinociception) but were antagonized by the GABAB antagonist CGP 35348 (2.5 micrograms, i.c.v., a dose which prevented (+/-)baclofen-induced antinociception). Mice, rendered tolerant to baclofen-induced antinociception by twice daily injection of increasing doses of baclofen (5-18 mg kg-1, s.c.), were unresponsive to the antinociceptive effects of bicuculline and picrotoxin but still responded to morphine. Bicuculline and picrotoxin, in the same range of doses which affected the three models of antinociception used, inhibited pentobarbital-induced hypnosis. Large doses of bicuculline and picrotoxin (4 and 2.5 mumol kg-1, s.c. respectively), reduced locomotor activity and impaired rota-rod performance in mice. The changes in response to noxious stimuli, induced by bicuculline and picrotoxin, are interpreted as an antinociceptive effect. It is then suggested that this effect might depend on an indirect activation of GABAB receptors through release of GABA.(ABSTRACT TRUNCATED AT 250 WORDS)


Relapsing acute disseminated encephalomyelitis associated with chronic Epstein-Barr virus infection: MRI findings. A 25-year-old women had a fever, left cervical lymphadenopathy, neurological symptoms and signs, CSF pleocytosis and persistent high serum antibodies to the Epstein-Barr virus (EBV); she had a recurrence 1 year later. She was thought to have relapsing acute disseminated encephalomyelitis associated with chronic EBV infection. MRI revealed abnormalities, mainly in the right basal ganglia and left midbrain. At the time of the recurrence, further abnormalities appeared in the opposite basal ganglia and right cerebral white matter.


The surgical significance of Persian Gulf sand. Foreign bodies, in particular sand and soil particles, can cause considerable reaction in wounds and remain in tissues indefinitely. The introduction of foreign material as a contaminant in wounds and intracavitary injuries to military personnel deployed in the Persian Gulf region can be a complicating factor in treatment. Samples of desert sand from the military operational areas of the Persian Gulf were analyzed and the acute local and systemic responses of intraperitoneal contamination determined in an experimental rat model.


Morphological features of cat thalamo-parietal projection fibers investigated by PHA-L immunohistochemistry and electron microscopy. Thalamo-parietal fibers originating from the ventroanterior-ventrolateral (VA-VL) complex in the cat were labeled with Phaseolus vulgaris leucoagglutinin (PHA-L) and examined by light and electron microscopy. PHA-L (2.5% aqueous solution) was injected iontophoretically through micropipets with anodal current pulses into the VA-VL complex. PHA-L-labeled terminals were distributed in the lateral and the suprasylvian gyri in the superficial and deep cortical layers. In layer I, horizontal varicose fibers and terminals were conspicuous in the upper one-third and were widely distributed. In the deeper cortical layers (layers III-V), varicose fibers and terminals were detected in moderate numbers. Electron microscopic examination revealed that the labeled terminals formed asymmetrical synapses on the dendritic spines of spiny neurons. These morphological findings appeared to be consistent with our previous intracellular recordings in this cortex.


Effect of androgen on the expression of gap junction and beta-actin mRNAs in adult rat motoneurons. Expression of gap junction and beta-actin mRNAs was examined in androgen-sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB) in adult male rats by in situ hybridization histochemistry using complementary DNAs encoding rat liver gap junction protein (connexin 32) and chick beta-actin. Hybridizable gap junction and beta-actin mRNAs were localised on the somata and proximal dendrites of SNB motoneurons. Removal of androgen by castration significantly reduced the expression levels of both gap junction and beta-actin mRNAs in the SNB motoneurons, whereas these changes were prevented by testosterone treatment. On the contrary, castration or testosterone treatment did not induce any changes in the expression levels of gap junction and beta-actin mRNAs in the motoneurons of the retrodorsolateral nucleus (RDLN), which accumulate androgen less frequently and sparsely than those in the SNB. These results suggest that androgen regulates the expression of both gap junction and beta-actin genes in the SNB motoneurons and may provide evidence for the molecular mechanisms of hormonally induced neuronal plasticity in the SNB motoneurons.


Physiological activities of late-gestation rat fetuses in vitro. A novel method for the in vitro maintenance of rat fetuses was used to study their physiological properties. Rat fetuses together with the uterus were isolated from anesthetized pregnant rats of 14.5-21.5 days gestation, placed in a chamber, perfused through a cannula inserted into the uterine artery with a physiological solution, and decerebrated. In this condition, it was possible to maintain heartbeats of rat fetuses for 24 h. The in vitro fetuses showed various spontaneous body movements including those that resembled respiration. Various body movements were also observed when mechanical or electrical stimulation was applied to the skin. These body movements were abolished by application of general anesthetics. It was shown that reflex-like electrical activities were retained in spinal cords isolated from fetuses which had been maintained in vitro for 10 h. These results show that in vitro rat fetuses retain abilities to generate various body movements that are presumably mediated by the electrical activities of the central nervous system. The presence of various neural activities, some of which are known to be particularly susceptible to poor psychological conditions, suggests that the condition of the in vitro fetuses is good and that the method may be applied to various studies on late-gestation mammalian fetuses.


Randomized phase II trial of high-dose 4'-epi-doxorubicin + cyclophosphamide versus high-dose 4'-epi-doxorubicin + cisplatin in previously untreated patients with extensive small cell lung cancer. One hundred and eleven previously untreated patients with extensive small cell lung cancer were included in a prospective randomized study with the aim to assess the efficacy and tolerance of high-dose epirubicin (120 mg/m2) in combination with either cyclophosphamide (800 mg/m2; arm 1) or cisplatin (60 mg/m2; arm 2). Ninety-six patients were evaluable for response and toxicity and additional 12 patients for toxicity only. The overall response rate (CR+PR) in arm 1 and 2 were 61.4 (27/44) and 67.3% (35/52), respectively. The mean duration of remission was 4.4 months (arm 1) and 4.9 months (arm 2). The mean survival time was 6.6 months in arm 1 and 7.7 months in arm 2. WHO grade 4 toxicity was encountered in 25.5 and 15.8% of patients in arm 1 and 2, respectively. One case of cardiotoxicity resulting in the patient's death was observed in arm 1. Both combinations showed considerable antitumor activity. Toxicity was acceptable.


Mutations on free and integrated hepatitis B virus DNA in a hepatocellular carcinoma: footprints of homologous recombination. Hepatitis B virus nucleotide sequences derived from a hepatocellular carcinoma with free and multiply integrated viral DNAs were determined. Based on a comparison within the X-gene region, cloned free viral DNA previously had been attributed to two distinct groups of preC minus genomes. The comparison of the complete sequence identified one of the genome equivalents as a recombinant between members of these groups. Four different integrated viral DNA elements were cloned and analysed. Similarity to either one of two DNAs representing the two groups of free viral DNA on one hand and the presence of certain mutations only on integrated DNA on the other hand, allowed to recognize distinct segments within the integrants. The data suggest a contribution of different but related genotypes to contiguous stretches of integrated viral DNA via homologous recombination. On this basis an evolutionary relationship between free and integrated DNAs of the preC and the preC minus genotype could be recognized when short sequence segments were compared. The observed coexistence on a given integrated DNA of segments homologous to free viral DNA and of segments homologous to another integrated DNA is consistent with (1) a long lasting association of individual genotypes with dividing cells and (2) multiple integration events being the result of a series of steps not separated by a long time span.


Effects of active oxygen species on damage to and prostaglandin synthesis in cultured rat gastric cells. Active oxygen species cause gastric mucosal damage in vivo. However, it is not known if these species are directly cytotoxic toward gastric cells. Prostaglandins have important physiological roles in the gastric mucosa, including direct cell protection against damaging factors. So, to find if active oxygen species affect prostaglandin synthesis in gastric mucosal cells is important, but this also is not known. This study was done to investigate the effects of such species on damage to and prostaglandin synthesis in cultured mucus-producing cells from rat gastric mucosa. Active oxygen species were produced by the addition of xanthine and xanthine oxidase to the culture medium. Cytotoxicity was assayed by 51Cr release. Xanthine (1 mM) and xanthine oxidase (100 mU/ml) increased specific 51Cr release as the thiobarbituric acid reactants increased. This increase in 51Cr release was inhibited by catalase, a scavenger of hydrogen peroxide, or dimethyl sulfoxide, a scavenger of hydroxyl radicals, but not by superoxide dismutase, a scavenger of superoxide, nor deferoxamine, an inhibitor of hydroxyl radical generation. Catalase, dimethyl sulfoxide, and superoxide dismutase each had no effect on prostaglandin E2 synthesis when xanthine and xanthine oxidase were not added. In the presence of xanthine and xanthine oxidase, catalase and dimethyl sulfoxide stimulated the synthesis of prostaglandin E2 and superoxide dismutase inhibited it. Indomethacin, a prostaglandin synthetase inhibitor, did not affect the decrease in 51Cr release caused by catalase in the presence of xanthine and xanthine oxidase, but it abolished the decrease caused by dimethyl sulfoxide. These results suggest that hydrogen peroxide, but not superoxide nor hydroxyl radicals, is involved in damage to cultured rat gastric cells, and that superoxide stimulates prostaglandin E2 synthesis, but that hydrogen peroxide inhibits it. Protection of the cells by dimethyl sulfoxide may be related to stimulation of prostaglandin E2 synthesis in the cells, but not via scavenging hydroxyl radicals.


[Childhood dancing eye syndrome in occult neuroblastoma]. The case of a two year-old boy is described with dancing eyes syndrome (DES) together with ganglioneuroblastoma. Surgical removal of the tumour and ACTH therapy resulted in rapid improvement, and an almost symptome-free condition. The literature is reviewed. Need of vigorous search for an occult neuroblastoma in DES is pointed out, since the early diagnosis and tumour's surgery can significantly improve the outcome.


Dot blot hybridization in detection of human papillomavirus (HPV) infections in the oral cavity of women with genital HPV infections. The presence of human papillomavirus (HPV) types 2, 6, 7, 11, 13 and 16 DNA in cytologic scrapings of oral mucosa was studied in 309 women with genital HPV infections. The objective was to test the usefulness of oral mucosal scrapings (3 sequential swabs) in HPV DNA detection by dot blot hybridization. Based on hybridization with the 32P-labelled Alu-repeat probe, most samples contained more than 10(5) cells, which is an adequate number of cells for dot blot hybridization. Hybridization with 32P-labelled HPV DNA probes showed that 3.8% of the 309 women had an oral HPV infection. Of these, only 2 had clinical lesions indicative of HPV. All other oral HPV positive subjects had clinically healthy mucosa. HPV 6 was the most common (3.1%) type, followed by HPV 11 and 16 (1.1%). In 3 cases the genital mucosa harboured the same HPV type as found in the oral cavity. The results indicate that oral mucosal scraping results in adequate number of cells for dot blot hybridization with HPV DNA. Although the method is likely to result in an underestimation of latent and subclinical HPV infections, it is useful for studying the clinical HPV infections as well as other viral infections known to be present in exfoliated cells.


Bacterial synergy of Treponema denticola and Porphyromonas gingivalis in a multinational population. Treponema denticola and Porphyromonas gingivalis have been associated with human adult severe periodontitis. In this study, we quantified these putative pathogens in subgingival plaque samples collected from 74 Fijians, 74 Colombians and 73 U.S. Americans stationed at the Multinational Force and Observers encampment in the Sinai Desert, Egypt. A contingency table of T. denticola and P. gingivalis frequency revealed a highly significant synergistic relationship. We discovered that the occurrence of T. denticola apparently requires the presence of P. gingivalis. This represents the first observation of a synergistic relationship between these putative oral pathogens associated with adult severe periodontal disease.


Tetracycline inhibition identifies the cellular origin of interstitial collagenases in human periodontal diseases in vivo. Mammalian interstitial collagenases (E.C.3.4.24.7) are considered as key initiators of collagen degradation in periodontal diseases. However, the cellular sources of collagenases present in gingival crevicular fluid have not been completely clarified. Resident fibroblasts and epithelial cells as well as infiltrating neutrophils and monocyte/macrophages are potential sources of the enzymes. We have recently found significant differences in tetracycline inhibition between human neutrophil and fibroblast interstitial collagenases. To address the cellular source of collagenase present in gingival crevicular fluid in 2 distinct periodontal diseases, we studied the tetracycline inhibition of collagenase in gingival crevicular fluid of patients with localized juvenile periodontitis and adult periodontitis. Gingival crevicular fluid samples were collected from deep (greater than 5 mm) periodontal pockets and assayed for collagenase in the presence of 0-1000 microM doxycycline as well as a chemically modified tetracycline devoid of antimicrobial activity (4-de-dimethylaminotetracycline). The drug concentration required to inhibit 50% of collagenase activity (IC50) in localized juvenile periodontitis gingival crevicular fluid was 280 microM for doxycycline and 470 microM for 4-de-dimethylaminotetracycline. Significantly lower values, 10-20 microM, were obtained for collagenase in gingival crevicular fluid of patients with adult periodontitis. We propose that systemic tetracycline levels are efficient inhibitors of collagenase in gingival crevicular fluid in affected sites of patients with adult periodontitis but not of patients with localized juvenile periodontitis and that the fibroblast type interstitial collagenase is the predominant collagenase type in gingival crevicular fluid in affected sites of patients with localized juvenile periodontitis and the neutrophil collagenase in adult periodontitis gingival crevicular fluid.(ABSTRACT TRUNCATED AT 250 WORDS)


Protective efficacy against serotype 1 rotavirus diarrhea by live oral rhesus-human reassortant rotavirus vaccines with human rotavirus VP7 serotype 1 or 2 specificity. Rhesus-human rotavirus (RV) reassortant vaccine strains D x RRV or DS 1 x RRV with VP7 serotype 1 or 2 specificity were evaluated for safety, immunogenicity and protective efficacy in a double blind placebo-controlled three cell trial involving 359 infants ages 2 to 5 months. The titer of the D x RRV vaccine was 10(4) and that of the DS 1 x RRV vaccine was 10(5) plaque-forming units/1-ml dose. The vaccines were acceptably reactogenic, each inducing a transient febrile response in fewer than one-third of the vaccinees. Seroconversion by RV enzyme-linked immunosorbent assay IgA antibody was detected in 61 and 75% of the vaccinees receiving a single dose of the serotype 1 or 2 reassortant vaccine, respectively. Efficacy against RV diarrhea was evaluated in two successive epidemic seasons; RV serotype 1 was prevalent in both. Clinical efficacy was observed with both vaccines and was associated with seroconversion after vaccination; considering only such vaccinees both vaccines showed equal efficacy. The overall rates of protection for the two vaccines combined against clinical RV disease in children with seroconversion after vaccination were 92 and 59% in the first and second RV epidemic seasons, respectively. Protection against asymptomatic RV infection, as measured by serologic responses, was 59% in the first season and nil in the second season. It is concluded that each of the reassortant RV vaccines was effective in inducing protection against symptomatic RV disease associated with RV serotype 1.


Inhibition of voltage-dependent Ca2+ channels via alpha 2-adrenergic and opioid receptors in cultured bovine adrenal chromaffin cells. Adrenal chromaffin cells secrete catecholamines and opioids. The effects of these agents on whole-cell Ca2+ channel currents were studied, using bovine adrenal chromaffin cells kept in short term culture. Ca2+ channel currents recorded during voltage-clamp pulses from a holding potential of -80 mV to 0 mV were reversibly reduced by 10 microM epinephrine (in the presence of 1 microM propranolol) or 5 microM of the synthetic opioid, d-Ala2-d-Leu5-enkephalin (DADLE) by approximately 35% and 25%, respectively. The inhibitory action of epinephrine was mimicked by clonidine, reduced by yohimbine but not affected by prazosin. The DADLE-induced reduction of the Ca2+ channel current was antagonized by naloxone. The dihydropyridine (+)PN 200-110 (5 microM) reduced the Ca2+ channel current by approximately 40%; the Ca2+ channel current inhibited by (+)PN 200-110 was not further reduced by epinephrine. Intracellular infusion of guanosine-5'-O-(2-thiodiphosphate) and pretreatment of cells with pertussis toxin abolished the inhibitory effect of both epinephrine and DADLE. In membranes of adrenal chromaffin cells, four pertussis-toxin-sensitive G-proteins were identified, including Gi1, Gi2, Go1 and another Go subtype, possibly Go2. The data show that activation of alpha 2-adrenergic and opioid receptors causes an inhibition of dihydropyridine-sensitive Ca2+ channels in adrenal chromaffin cells. These inhibitory modulations are mediated by pertussis-toxin-sensitive G-proteins and may represent a mechanism for a negative feedback signal by agents released from the adrenal medulla.


The effect of phosphatase inhibitors and agents increasing cyclic-AMP-dependent phosphorylation on calcium channel currents in cultured rat dorsal root ganglion neurones: interaction with the effect of G protein activation. Ca2+ channel currents have been recorded in cultured rat dorsal root ganglion neurones. The amplitude of IBa(GTP gamma S), recorded in the presence of GTP[ gamma S] (200 microM) in the patch pipette solution, is enhanced by external application of forskolin (10 microM), and there is an increase in the proportion of the rapidly activating component of the current. When forskolin (1 microM) is present in the bathing solution at the start of recording, or when 8-bromocyclic AMP (100 microM) is present in the patch pipette solution, the amplitude and rate of activation of IBa(GTP gamma S) are also increased compared to control IBa(GTP gamma S). The effect is mimicked by internal application of a 5 microM solution of a phosphopeptide fragment of inhibitor 1 (I1 PP), which inhibits phosphatase 1. The enhancement of IBa(GTP gamma S) caused by I1PP is not additive with that due to forskolin. Furthermore, the enhancement due to I1PP is reversibly lost when the holding potential is shifted from -80 mV to -30 mV, as was the enhancement due to forskolin and 8-bromocyclic AMP. I1PP also produced a less marked stimulation of the control Ca2+ channel current in the absence of G protein activation. The results suggest that phosphorylation regulates the interaction between calcium channels and G proteins in these neurones, and that phosphatase 1 is tonically active to dephosphorylate the relevant protein(s).


Endothelin-1 stimulates chloride and potassium secretion in rabbit descending colon. The vasoactive peptide endothelin-1 (ET-1) which is present in high concentrations in the colon, causes concentration-dependent electrogenic Cl- secretion in rabbit descending colon. This effect is half-maximal at 0.11 mumol/l. Like other secretagogues, ET-1 also stimulates K+ secretion. The secretory effect of ET-1 is associated with increased release of prostaglandin E2 from the serosal surface of the mucosa. ET-1-induced Cl- secretion is completely inhibited by the loop diuretic bumetanide and by indomethacin and quinacrine, inhibitors of prostaglandin synthesis. Neuronal mechanisms do not seem to be involved, as tetrodotoxin did not affect the secretory response to ET-1 significantly. On the other hand, neither the catalytic activity nor the transport function of the Na+/K(+)-ATPase of rabbit colon epithelium is affected by endothelin-1 (ET-1) in concentrations up to 10 mumol/l. It is concluded that ET-1 causes Cl- and K+ secretion by stimulating phospholipase A2 and release of prostaglandins, whereas Na+ transport is not altered.


Protein kinase C regulation of cardiac calcium channels expressed in Xenopus oocytes. L-Type cardiac Ca2+ channels expressed in Xenopus oocyte were studied following rat heart ribonucleic acid, messenger (mRNA) injection. We demonstrate that exogenous Ca2+ channels are sensitive to intracellular regulation by protein kinase C (PKC). This was performed by using two types of PKC activators [phorbol esters and a structural analogue of diacyl-glycerol (DAG)] and a specific peptidic inhibitor. Ca2+ channel modulation resulted in an initial increase of the inward current, without any modification of the voltage-dependent properties, and a second delayed phase, specifically observed with phorbol esters, characterized by a progressive decrease in current amplitude. Concomitantly, a reduction of membrane capacitance, reflecting a reduction of the total membrane surface area, was observed. We suggest that this phenomenon underlies the irreversible decrease of the expressed Ba2+ current via sequestration of Ca2+ channels and/or PKC. We also demonstrate that regulation of cardiac mRNA-directed Ca2+ channels by PKC activators was strictly dependent on intracellular Ca2+ concentration, and was partially additive with cyclic-adenosine-monophosphate-(cAMP) dependent regulation.


Characteristics of synaptic transmission in reinnervating rat skeletal muscle. Synaptic transmission, and its sensitivity to the effects of 3,4-diaminopyridine (3,4-DAP) and the phosphatase 2,3-butanedione monoxime (BDM), was examined for "crushed fiber" preparations of rat extensor digitorum longus muscle undergoing reinnervation after nerve crush. While mean quantal content (m) of endplate potentials (EPPs) was low early during reinnervation (10-24 days after nerve crush), elevation of temperature or extracellular calcium concentration restored m toward normal. However, m achieved control values for reinnervating preparations exposed to 3,4-DAP. 3,4-DAP also activated quiescent motor nerve terminals: after exposure to this drug, synaptic transmission was detected as early as 8 days after nerve crush. BDM too activated quiescent regenerating motor nerve terminals and increased m to normal. It also prolonged EPP and endplate current decay, suggesting a pre-synaptic effect on the synchrony of transmitter release and/or a post-synaptic effect on the open time of acetylcholine-gated endplate channels. While the effects of temperature, extracellular calcium, 3,4-DAP, and BDM suggest that regenerating nerve terminals can mobilise a reserve of quanta, this reserve is abnormally low, since hemicholinium-3 caused rapid rundown of EPP amplitude at repetitively stimulated regenerating endplates.


Carbodiimide modification reduces the conductance and increases the tetrodotoxin sensitivity in batrachotoxin-modified sodium channels. The relationship between the channel entrance and the tetrodotoxin (TTX) binding site was investigated by chemical modification at the extracellular surface of bilayer-incorporated batrachotoxin-(BTX) modified sodium channels using an impermeant carbodiimide in the presence or absence of exogenous nucleophiles. Two (classes of) groups could be modified such that the open-channel conductance was decreased while TTX binding was unaffected, and TTX did not protect against this modification. Because the final conductance level depends on the exogenous nucleophile, each covalent modification appears to involve a carboxyl group. In addition, a third (carboxyl) group could be modified such that TTX binding affinity was increased. These results suggest that the channel entrance and the TTX binding site are spatially separate, which supports previous suggestions that the mechanism by which guanidinium toxins close sodium channels involves a conformational change subsequent to toxin binding.


Modulation of fatty-acid-binding protein content of rat heart and skeletal muscle by endurance training and testosterone treatment. The effects of training and/or testosterone treatment and its aromatization to oestradiol on fatty-acid-binding protein (FABP) content and cytochrome c oxidase activity in heart, soleus and extensor digitorum longus (EDL) muscles were studied in intact adult female rats. One group of rats remained sedentary, whereas the others were trained for 7 weeks. Thereafter the trained rats were divided into control and testosterone-treated groups, with or without an aromatase inhibitor. Testosterone was administered by a silastic implant. Training was continued for 2 weeks. In untreated sedentary rats the immunochemically assayed FABP contents were 497 +/- 28, 255 +/- 49 and 58 +/- 17 micrograms/g wet weight for the heart, soleus, and EDL respectively. In the heart the FABP content was increased after training (29%), testosterone treatment (33%) or both manipulations (53%). In soleus muscle FABP increased only after testosterone treatment (16%), whereas in EDL no changes were found. Inhibiting the aromatase enzyme complex abolished the testosterone-induced effect on FABP content in soleus (suggesting an oestradiol effect) but not in heart muscle. Among the three muscles studied the FABP content was found to be related to the cytochrome c oxidase activity in a non-linear way. In conclusion, it is shown that the FABP contents and mitochondrial activities of heart and skeletal muscle are affected by training and sex hormones and that these effects are different for heart and skeletal muscles.


Glutathione accelerates sodium channel inactivation in excised rat axonal membrane patches. The effects of glutathione were studied on the gating behaviour of sodium channels in membrane patches of rat axons. Depolarizing pulses from -120 to -40 mV elicited sodium currents of up to 500 pA, indicating the simultaneous activation of up to 250 sodium channels. Inactivation of these channels in the excised, inside-out configuration was fitted by two time constants (tau h1 = 0.81 ms; tau h2 = 5.03 ms) and open time histograms at 0 mV revealed a biexponential distribution of channel openings (tau short = 0.28 ms; tau long = 3.68 ms). Both, the slow time constant of inactivation and the long lasting single channel openings disappeared after addition of the reducing agent glutathione (2-5 mM) to the bathing solution. Sodium channels of excised patches with glutathione present on the cytoplasmatic face of the membrane had inactivation kinetics similar to channels recorded in the cell-attached configuration. These observations indicate that redox processes may contribute to the gating of axonal sodium channels.


Effects of metabolic inhibition by sodium azide on stimulus-secretion coupling in B cells of human islets of Langerhans. Sodium azide (NaN3), a reversible inhibitor of mitochondrial respiration, blocks glucose-induced electrical activity and insulin secretion in human pancreatic islet B cells. Here we show that brief (10-15 min) application followed by removal of 3 mM NaN3 results in transient overshoot of electrical activity and insulin secretion even at substimulatory levels of glucose (3-5 mM). In addition, application of NaN3, even at very low [Ca2+]o, reversibly increases cytosolic Ca2+ to levels usually associated with substantial insulin release. These results suggest that (i) metabolic inhibition may reset B cell stimulus-secretion coupling and (ii) a rise in free cytosolic Ca2+, by itself, is not sufficient to trigger insulin secretion.


External K+ increases Na+ conductance of the hyperpolarization-activated current in rabbit cardiac pacemaker cells. The hyperpolarization-activated current (I(f)) was recorded from single myocytes dissociated from rabbit sinoatrial node. Although I(f) is usually carried by both Na+ and K+, removal of the minor K+ component from physiological saline suppresses inward component. This inward Na+ current through I(f) channel increases on raising the extracellular K+ concentration. The Na+ conductance relative to K+ conductance (PNa/PK), as measured from the reversal potential, increases and saturates near 5 mM K+. This effect is different from the current increase caused by raising the concentration of carrier ion K+, which saturates at 70 mM with a half-maximal value (K1/2) of 10 mM. It is suggested that the I(f) channel has multiple, interactive binding sites for cation permeation.


[Prevalent disseminated form of bronchiolo-alveolar cancer]. Three roentgenological variants of the course of the prevalent disseminated form of bronchoalveolar cancer were distinguished proceeding from the clinical and X-ray study: focal-disseminated (5 cases), focal-nodal (11) and focal-infiltrative (16). In the authors' opinion, the prevalent disseminated form of bronchoalveolar cancer is a terminal phase in the development of the more restricted forms of a tumour (nodal and pneumonia-like) which is formed by way of intrapulmonary aerogenic (bronchogenic) metastasis.


Isoforms of the Na,K-ATPase are present in both axons and dendrites of hippocampal neurons in culture. The distributions of isoforms of the Na,K-ATPase alpha subunit were determined in mature cultured hippocampal neurons and in a polarized epithelial cell line. We find that hippocampal neurons express the alpha 1 and alpha 3 isoforms in the membranes of both axons and dendrites. In contrast the alpha 1 and alpha 3 proteins are exclusively basolateral when expressed endogenously or by stable transfection in renal epithelial cells. These data suggest that epithelial cells and hippocampal neurons localize these proteins by different mechanisms. These observations contrast with those made for the vesicular stomatitis virus and the influenza glycoproteins, which are polarized in both epithelial and neuronal cells.


[Neuraxitis in critical exanthema: description of a case]. In this work the Authors describe the case of a 16-month-old child, who presented neuraxitis infection of cerebellum, during exanthem subitum. From the present etiological considerations about exanthem subitum, referable to human herpes virus type 6, the Authors stress the usefulness of executing the dosage of anti-HHV6 antibodies in all the cases of exanthem subitum and in all febrile convulsions, because it is possible to have atypical clinic forms of VI disease, without exanthem.


The major histocompatibility complex class I antigen-binding protein p88 is the product of the calnexin gene. A 90-kDa phosphoprotein (p90) of the endoplasmic reticulum was identified by a monoclonal antibody generated against human hepatoma cells. Pulse-chase experiments with [32P]phosphate and [35S]methionine demonstrated that p90 formed both stable and transient complexes with other cellular proteins, suggesting its role as a molecular chaperone. This protein associates with heavy chains of major histocompatibility complex class I proteins, suggesting that it is the human homolog of the recently described 88-kDa protein that transiently associates with murine class I molecules in the endoplasmic reticulum. The p90 protein also associates in B lymphocytes with membrane immunoglobulin mu heavy chains and may serve as a chaperone for many membrane-bound polypeptides. A partial human p90 cDNA was cloned from a lambda gt11 expression library and identified as the human homolog of calnexin, a major canine calcium-binding protein found to be associated with the signal-sequence receptor in endoplasmic reticulum membranes.


Feline leukemia virus subgroup C phenotype evolves through distinct alterations near the N terminus of the envelope surface glycoprotein. Feline leukemia viruses (FeLVs) belonging to the C subgroup induce aplastic anemia in domestic cats and have the ability, unique among FeLV strains, to proliferate in guinea pig fibroblasts in tissue culture. Previous studies have shown that the pathogenic and host range specificity of a prototype molecular clone of FeLV-C [FeLV-Sarma-C (FSC)] colocalize to a region encoding the 3' 73 amino acids of the pol gene product and the N-terminal 241 amino acids of the envelope surface glycoprotein named SU. Here, we amplified, via PCR, cloned, and sequenced the SU coding sequence from three additional anemia-inducing subgroup C FeLV isolates. Chimeric viruses were constructed by replacement of fragments of FeLV-C envelope genes into the FeLV-A prototype virus 61E. Using a modified vesicular stomatitis virus-FeLV pseudotype assay, we demonstrated that the subgroup C receptor specificity for each virus was determined by changes within the N-terminal 87-92 amino acids of SU, in which most changes occurred within the 15- to 20-amino-acid first variable region (V1). Determinants for growth in guinea pig cells colocalized to this region. Despite the consistent localization of biological determinants, the only consistent features that distinguished the deduced FeLV-A and FeLV-C proteins was one lysine-to-arginine change and a structural prediction of an alpha-helix in FeLV-A proteins versus random coil in FeLV-C proteins within V1. However, arginine in equilibrium with lysine substitutions were not sufficient to convert the subgroup A virus to the subgroup C phenotype or vice versa. Thus, certain distinct structural changes within the N-terminal region of FeLV SU can result in convergent viral phenotypes.


Calcium signaling and episodic secretion of gonadotropin-releasing hormone in hypothalamic neurons. Gonadotropin-releasing hormone (GnRH) is released episodically into the pituitary portal vessels and from hypothalamic tissue of male and female rats in vitro. Perifused primary cultures of rat hypothalamic neurons, as well as the GT1-1 GnRH neuronal cell line, spontaneously exhibited episodic GnRH secretion of comparable frequency to that observed with perifused hypothalami. Such pulsatile GnRH release from GT1 cells indicates that GnRH neurons generate rhythmic secretory activity in the absence of input from other cell types. In primary hypothalamic cultures, the frequency of GnRH pulses increased with the duration of culture. The spontaneous pulsatility in GnRH release was abolished in Ca(2+)-deficient medium and was markedly attenuated in the presence of nifedipine, an antagonist of voltage-sensitive Ca2+ channels. The basal intracellular Ca2+ level of perifused GT1-1 cells cultured on coverslips was also dose-dependently reduced by nifedipine. Conversely, depolarization with high K+ increased intracellular Ca2+ and GnRH release in an extracellular Ca(2+)-dependent and nifedipine-sensitive manner. The dihydropyridine Ca2+ channel agonist Bay K 8644 increased basal and K(+)-induced elevations of intracellular Ca2+ concentration and GnRH secretion. These findings demonstrate that pulsatile neuropeptide secretion is an intrinsic property of GnRH neuronal networks and is dependent on voltage-sensitive Ca2+ influx for its maintenance.


DNA helicase from mammalian mitochondria. In spite of the fact that a DNA helicase is clearly required for the predominantly leading-strand synthesis occurring during mammalian mtDNA replication, no such activity has heretofore been identified. We report the characterization of a mammalian mitochondrial DNA helicase isolated from bovine brain tissue. The sucrose gradient-purified mitochondria in which the activity was detected had less than 1 part in 2500 nuclear contamination according to Western blot analysis using nuclear- and mitochondrial-specific probes. Mitochondrial protein fractionation by DEAE-Sephacel chromatography yielded a DNA helicase activity dependent upon hydrolysis of ATP or dATP but not other NTPs or dNTPs. The mitochondrial helicase unwound 15- and 20-base oligonucleotides but was unable to unwind 32-base or longer oligonucleotides, and the polarity of the unwinding is 3'-to-5' with respect to the single-stranded portion of the partial duplex DNA substrate. This direction of unwinding would place the bovine mitochondrial helicase on the template strand ahead of DNA polymerase gamma during mtDNA replication, a situation analogous to that of the Rep helicase of Escherichia coli during leading-strand DNA synthesis of certain bacteriophages.


Expression cloning of the pancreatic beta cell receptor for the gluco-incretin hormone glucagon-like peptide 1. Glucagon-like peptide 1 (GLP-1) is a hormone derived from the preproglucagon molecule and is secreted by intestinal L cells. It is the most potent stimulator of glucose-induced insulin secretion and also suppresses in vivo acid secretion by gastric glands. A cDNA for the GLP-1 receptor was isolated by transient expression of a rat pancreatic islet cDNA library into COS cells; this was followed by binding of radiolabeled GLP-1 and screening by photographic emulsion autoradiography. The receptor transfected into COS cells binds GLP-1 with high affinity and is coupled to activation of adenylate cyclase. The receptor binds specifically GLP-1 and does not bind peptides of related structure and similar function, such as glucagon, gastric inhibitory peptide, vasoactive intestinal peptide, or secretin. The receptor is 463 amino acids long and contains seven transmembrane domains. Sequence homology is found only with the receptors for secretin, calcitonin, and parathyroid hormone, which form a newly characterized family of G-coupled receptors.


Identification and functional characterization of a TIA-1-related nucleolysin. We recently reported the molecular cloning of a cytotoxic granule-associated RNA-binding protein designated TIA-1. The ability of recombinant TIA-1 to induce DNA fragmentation in permeabilized cells suggested that this protein is the granule component responsible for inducing apoptosis in cytolytic lymphocyte (CTL) targets. Here we report the characterization of a cDNA encoding a TIA-1-related protein designated TIAR. The deduced amino acid sequence of TIAR reveals it to be a 42-kDa protein possessing three RNA-binding domains and a carboxyl-terminal auxiliary domain. Although the RNA-binding domains of TIA-1 and TIAR share greater than 85% amino acid homology, their carboxyl-terminal auxiliary domains are only 51% homologous. The carboxyl terminus of TIAR contains a lysosome-targeting motif, indicating that TIAR is probably a cytotoxic granule-associated protein. Like TIA-1, purified recombinant TIAR induced DNA fragmentation in permeabilized target cells. Although immunoblotting analysis of post-nuclear supernatants revealed TIA-1 protein to be restricted to CTLs, PCR analysis revealed the expression of TIA-1 and TIAR mRNA transcripts in a wide variety of cell types. Our data suggest that the granules of CTLs contain at least two candidate nucleolysins involved in CTL killing.


Occurrence of the alpha subunits of G proteins in cerebral cortex synaptic membrane and postsynaptic density fractions: modulation of ADP-ribosylation by Ca2+/calmodulin. We have examined the isolated postsynaptic density (PSD) fraction for the presence of a G protein. First, we found specific binding of guanosine 5'-[gamma-[35S]thio]triphosphate to the PSD. Second, pertussis toxin-activated ADP-ribosylation of the isolated PSD fraction resulted in the appearance of a G protein with an apparent molecular mass of 41 kDa, and two G proteins with apparent molecular masses of 41 kDa and 39 kDa in synaptic membrane (SM) fraction and total homogenate (H). The amount of the 41-kDa G protein per unit protein was in the order of SM greater than H greater than PSD. Anti-G(i0 antibodies recognized the 41-kDa G protein in both PSD and SM, whereas anti-G(o) antibodies reacted with the 39-kDa G protein in the SM. The absence of G(o) protein in the PSD suggested that there was no contamination with SM. Moreover, unlabeled PSD incubated with an extract of SM that contained the labeled G proteins resulted in no label in the subsequently reisolated PSD, suggesting that the G protein found in the PSD was not due to adsorption of the G protein onto the PSD during its isolation from the SM. PSD pretreated with EGTA gave an 11-fold increase in the ADP-ribosylation reaction of the G(i) protein; similar effects on the G(i) and G(o) proteins of SM were obtained. Restoration of Ca2+/calmodulin to the PSD, but not of either Ca2+ or calmodulin alone, removed the effect of EGTA, indicating a strong complex formation between G(i) and Ca2+/calmodulin that decreased the ADP-ribosylation reaction. Preincubation with the Ca(2+)-channel blocker nifedipine decreased the ADP-ribosylation reaction in the PSD. We conclude that G(i) is present in the PSD, that it may interact with calmodulin and that it is involved in the regulation of voltage-dependent Ca2+ channel. We present a theory of the involvement of the G protein and calmodulin in postsynaptic neurophysiological events.


Activity of a plasmid-borne leu-500 promoter depends on the transcription and translation of an adjacent gene. leu-500 is a chromosomal promoter mutation in Salmonella typhimurium that normally causes the promoter to be inactive in the initiation of RNA synthesis. But in a strain that has mutations in topA, the gene encoding DNA topoisomerase I, the mutant promoter becomes active. We show that the leu-500 promoter can function on a plasmid when it is adjacent to the tetracycline-resistance gene tetA. Activation of the leu-500 promoter requires that the tetA gene is transcribed and translated and that the host cell is topA. We propose that the A----G mutation in the -10 region of the leu-500 promoter is compensated by local negative supercoiling arising from transcription of the tetA gene, which may reach elevated levels in a topA background, provided that diffusional dissipation is reduced due to anchoring of the TetA peptide in the membrane. This is a clear example of the modulation of the activity of a promoter by the activity of another promoter in cis, when they can be coupled through the topology of the template.


Tagging developmental genes in Dictyostelium by restriction enzyme-mediated integration of plasmid DNA. Introduction of restriction enzyme along with linearized plasmid results in integration of plasmid DNA at genomic restriction sites in a high proportion of the resulting transformants. We have found that electroporating BamHI or EcoRI together with pyr5-6 plasmids cut with the same enzyme stimulates the efficiency of transformation in Dictyostelium discoideum more than 20-fold over the rate seen when plasmid DNA alone is introduced. Restriction enzyme-mediated integration generates insertions into genomic restriction sites in an apparently random manner, some of which cause mutations. About 1 in 400 of the Dictyostelium transformants displayed arrested or aberrant development. The integrated plasmid, along with flanking genomic DNA, was excised from some of these mutants, cloned in Escherichia coli, and used to transform other Dictyostelium cells. Homologous recombination within the flanking sequences resulted in the same phenotypes displayed by the original mutants, directly demonstrating that the affected genes were responsible for the specific morphological phenotypes. This method of insertional mutagenesis should be useful for tagging, and subsequent cloning, of many developmentally important genes that can be identified by their mutant phenotypes.


Mitogen-expanded Schwann cells retain the capacity to myelinate regenerating axons after transplantation into rat sciatic nerve. We have developed a method for genetically modifying Schwann cells (SCs) in vitro and then assessed whether these SCs could interact normally with axons in vivo. Rat SCs were transduced in vitro with the lacZ gene by using a retroviral vector and then expanded with the SC mitogens forskolin and glial growth factor. These mitogen-expanded SCs had an abnormal phenotype as compared to both SCs in vivo and primary SCs in vitro, yet when they were introduced into a regenerating rat sciatic nerve, they formed morphologically normal myelin sheaths around the axons. These results demonstrate that SCs can be genetically altered, their numbers expanded in culture, and yet respond appropriately to axonal signals in the peripheral nervous system. This approach offers a plausible way to manipulate genes involved in axon-SC interactions, including genes that may be defective in some inherited peripheral neuropathies.


Intrinsic relaxation factors and length-dependent sensitivity in the rat aorta. Possible mechanisms for length-dependent sensitivity may involve intrinsic relaxation factors of the arterial wall. The role of the endothelium, beta-receptor activity, and atrial natriuretic factor were tested. ED50 and ED10 (concentrations for 50% and 10% maximum response, respectively) were significantly lower at the length of maximum force (Lmax) than at 80% Lmax for phenylephrine stimulated WKY rat aorta rings. The same result was found for norepinephrine (NE)-stimulated rings before and after endothelium removal, and for NE stimulation in the presence of propranolol. The ED10 for NE, but not its ED50, was significantly decreased by endothelium removal at both lengths. The addition of propranolol decreased the ED50 of NE at 80% Lmax, but not at Lmax. Relaxation sensitivity to atrial natriuretic factor was the same at Lmax and at 80% Lmax in phenylephrine-contracted WKY rings. For the rat aorta, it may be concluded that: (i) the mechanism for length-dependent sensitivity does not depend upon the endothelium or beta-receptor activity, however, (ii) basal levels of endothelium-derived relaxing factor and beta-receptor activity appear to modulate length-dependent sensitivity at low levels of activation, and (iii) sensitivity to atrial natriuretic factor relaxation does not depend upon length.


Ro 15-4513 binding to GABAA receptors: subunit composition determines ligand efficacy. The bidirectional modulation of ligand binding to benzodiazepine receptors (BzR) by GABA (the "GABA shift") has been widely used to predict ligand efficacy. The present study examined the effects of GABA and muscimol on [3H]Ro 15-4513 binding to "diazepam-insensitive" (DI) and "diazepam-sensitive" (DS) BzR. Neither GABA nor muscimol significantly altered [3H]Ro 15-4513 binding to DI in cerebellum, while both compounds inhibit [3H]Ro 15-4513 binding to cerebellar DS in a concentration-dependent fashion. The maximum reductions in [3H]Ro 15-4513 binding to cerebral cortical and hippocampal membranes elicited by GABA were comparable to those obtained in cerebellar DS, but significantly less than obtained with the full inverse agonist [3H]3-carbomethoxy-beta-carboline. The qualitatively different effect of GABAmimetics on [3H]Ro 15-4513 binding to DS and DI is not species specific since identical effects were obtained in rat and mouse brain. Based on previously established criteria, Ro 15-4513 can be classified as a "GABA-neutral" (antagonist) ligand at DI and "GABA negative" (inverse agonist) at other BzR. These findings suggest that GABAA receptor subunit composition determines not only ligand affinity but also ligand efficacy.


Platelet alpha 2-adrenergic receptors in depressed patients and healthy volunteers: the effects of desipramine. Binding parameters (Bmax and Kd) of alpha 2-adrenergic receptors were studied in platelets from 14 depressed patients and 18 control subjects. Using 3H-clonidine (a partial alpha 2-adrenergic agonist) as the ligand and membranes, prepared from platelets isolated under physiological conditions, we found no significant differences in Bmax and Kd between medication free patients and control subjects. Platelet binding parameters in the depressed patients did not correlate with plasma levels of norepinephrine, epinephrine or MHPG. Age had a significant positive effect on platelet alpha 2-adrenergic receptor Bmax in both groups, and may have masked the patient-control differences. Treatment with desipramine for 28 days had no effect on the binding parameters in depressed patients when compared to pretreatment values. Adding desipramine to platelets of control subjects 'in vitro' did also not affect binding parameters. Our findings suggest that receptor binding studies with a partial alpha 2-adrenergic agonist in platelet membranes are not a useful model to test the hypothesis of a central supersensitive adrenergic system in depression.


LiCl aversive conditioning has transitory effects on pheromonal responsiveness in male house mice (Mus domesticus). Appetitive and aversive experiences influence whether odors elicit precopulatory behavior from male rodents. A role for aversive experience in odor-elicited reproductive behaviors had been demonstrated for hamsters and rats, but similar work on house mice had not been performed. Four experiments examined whether lithium chloride (LiCl) aversive conditioning would alter two precopulatory behaviors (ultrasonic vocalizations and olfactory preference) that male house mice normally exhibit to female urine. Lithium chloride was used to aversively condition male house mice to either female urine odor, female urine in drinking water, the female herself, or a novel odor. Independent tests of taste aversion establishment were also conducted. In these experiments, LiCl aversive conditioning produced robust taste aversions to water adulterated with either female urine or a novel odorant/tastant (isoamylacetate), but only transitory decrements in odor-elicited, male-typical precopulatory behaviors. We conclude that aversive conditioning is unlikely to be a significant factor affecting male mouse precopulatory behavior.


Comparison of million personality profiles of chronic residential substance abusers and a general outpatient population. Several theories and studies have suggested that particular personality disorders or psychiatric symptoms should be associated with substance abuse. The current study was done to clarify these relationships by comparing scale profiles on the Millon Clinical Multiaxial Inventory obtained by 144 inpatient residentially treated substance-abuse clients and 1000 general clinical outpatient clients in psychotherapy. A surprisingly large number of significant differences were noted. Substance-abuse clients displayed relative elevations on scales for Alcohol and Drug Abuse, Hypomania, Antisocialism, Narcissism, Schizoidism, Paranoia, Psychotic Thinking, and Psychotic Delusions. General clinical outpatients showed relative elevations on Anxiety, Somatoform, Dysthymia, Borderline, and Compulsive scales. Some possible meanings of these findings were explored and suggestions for research given.


From the archives of the AFIP. Mediastinal germ cell tumors: radiologic and pathologic correlation. Germ cell tumors occur most frequently in the gonad but can rarely occur in extragonadal locations, usually in or near the midline. The most common extragonadal site of primary germ cell tumors is the anterior mediastinum. The most common histologic type of mediastinal germ cell tumor is mature teratoma, which is typically asymptomatic and incidentally discovered. Radiographically, these tumors appear as rounded, often lobulated masses; calcification may be seen. Imaging studies of mature teratoma frequently demonstrate cystic components and may demonstrate fat or calcium. Malignant germ cell tumors usually occur as large masses in symptomatic young male patients. Seminomas are typically of homogeneous soft-tissue attenuation, and nonseminomatous malignant germ cell tumors are typically of heterogeneous attenuation on computed tomographic scans. Therapy varies according to cell type and may include surgery, radiation therapy, or chemotherapy. Prognosis is excellent for patients with mature teratoma, good for patients with pure seminoma, and poor for patients with nonseminomatous malignant germ cell tumors and mixed germ cell tumors.


Liver regeneration following portacaval shunt in rats: 3',5'-cyclic AMP changes in plasma and liver tissue. Hepatic regeneration following portacaval shunt was evaluated in the light of the changes of 3',5'-cyclic AMP (cAMP) levels in plasma and liver tissue, and also plasma cAMP levels after glucagon administration, using a 70% hepatectomy and portacaval shunt model in rats. Ratios of liver to body weight recovered to 60% of pre-operative ratios 2 weeks after the operation. Plasma cAMP level decreased slightly, but insignificantly, 1 day and 3 days after the operation, then recovered to the pre-operative level. Hepatic cAMP concentration increased for 2 weeks after the operation. Plasma cAMP levels induced after glucagon administration did not increase until 3 days after the operation and were only 50% of pre-operative levels 4 weeks after the operation. From these results, liver regeneration following the portacaval shunt was remarkably delayed, but continued until at least 2 weeks after the operation.


Regulation of NK cell activity and the level of the intracellular cAMP in human peripheral blood lymphocytes by Met-enkephalin. The effect of Met-enkephalin on natural killer (NK) activity and on intracellular cyclic adenosine monophosphate (cAMP) level in human peripheral blood lymphocytes was measured 1/4, 1, 2, and 24 h after incubation. Concentrations of Met-enkephalin ranged from 10(-12) to 10(-8) M. Cyclic changes in NK activity and in the intracellular cAMP level after treatment with Met-enkephalin were observed. Kinetics of changes caused by high (10(-9), 10(-8) M) and low concentrations (10(-12), 10(-11), 10(-10) M) of Met-enkephalin differed from each other. Early, nearly threefold increase in the level of intracellular cAMP was found 15 min after treating peripheral blood lymphocytes with 10(-12) M Met-enkephalin. By contrast, a nearly 75% decrease of intracellular cAMP level was found 2 h after treatment with 10(-9) M Met-enkephalin. Generally, early intensive changes in the cAMP level in peripheral blood lymphocytes, induced by Met-enkephalin, were followed by delayed, subtle changes in NK cell activity in the opposite direction.


[Current status of low molecular weight heparin]. Low molecular weight heparins, discovered in 1976, are obtained from standard heparin by chemical or enzyme depolymerisation. Lowering of the molecular weight of polysaccharide chains has enabled dissociation of anti-IIa activity, associated with the risk of hemorrhage, from anti-Xa activity which forms the basis of anti-thrombotic activity. The anti-Xa/anti-IIa ratio varies according to low molecular weight heparin preparations from 2.5 to 10, while it is invariably 1 for standard heparin. Pharmacokinetics and a bioavailability of the order of 100 percent are undeniable advantages which distance them even farther from standard heparin. Clinical trials have provided validation as prophylaxis in general surgery and orthopedic surgery without laboratory surveillance apart from platelets, with the technique of use being very easy for nursing staff. Various trials concerned with the curative treatment of deep devnous thrombosis have shown that low molecular weight heparins are equally effective as non-fractionated heparin with, apparently, less hemorrhagic complications. However the lack of homogeneity of units, despite the international standard, requires prescribers to comply with the dosages and laboratory monitoring (anti-Xa activity) indicated by different manufacturers, which forms a barrier to their use.