Results of radiotherapy for chemodectomas. Between 1974 and 1988, 10 Mayo Clinic patients had unresectable, locally recurrent, or partially resected chemodectomas. Of these 10 tumors, 9 were confirmed pathologically, and 1 was diagnosed clinically. The chemodectoma was located in the jugular bulb in five patients, the middle ear in three, and the carotid body in two. The following symptoms were noted: tinnitus (in eight patients), loss of hearing (in six), hoarseness (in six), dysphagia (in four), pain (in three), and alteration of mental status (in one). Many patients had more than one symptom. Treatment was delivered with megavoltage photons and electrons; total doses ranged from 16.2 to 52 Gy (median, 46 Gy), and the daily doses ranged from 1.6 to 2.4 Gy. Follow-up among the nine survivors ranged from 3 1/2 to 16 years (median, 7 1/2 years). In one patient, the response could not be assessed because the patient died of renal failure 4 months after treatment. All nine assessable patients had decreased symptoms and objective control of the tumor (no evidence of progression of disease). Of the nine assessable patients, four had complete responses, one had a partial response, and four had stable disease. No patient experienced progression of disease after radiotherapy. We conclude that radiotherapy for chemodectomas yields successful results--namely, decreased symptoms and objective control of the tumor.


Management and prevention of cytomegalovirus infection after renal transplantation. We reviewed the epidemiologic characteristics, diagnosis, clinical features, and management of cytomegalovirus (CMV) infection after renal transplantation. CMV, the major viral pathogen after renal transplantation, increases patient morbidity and mortality. The spectrum of CMV infection ranges from latent infection to asymptomatic viral shedding to life-threatening multisystem disease. The two major risk factors for the development of CMV infection in renal transplant recipients are (1) preexisting CMV antibody seropositivity of either the organ donor or the recipient and (2) host immunosuppression. Blood cultures (but not urine cultures) positive for CMV predict the progression of asymptomatic infection to CMV disease, characterized by fever, malaise, myalgia, leukopenia, abnormal transaminase levels, and often involvement of the lung and gut. New genomic methods of viral detection now offer diagnostic advantages, including methods of detecting only actively replicating CMV. No evidence shows that CMV directly causes allograft rejection or glomerulonephritis, but patients with tissue-invasive CMV disease have higher rates of allograft loss and mortality than do those without the disease. Therapy for established CMV disease includes decreasing the immunosuppressive therapy and administering the antiviral agent ganciclovir sodium. Proven prophylactic strategies include limitation of exposure to the virus from CMV seropositive blood or organ donors, administration of CMV-specific immune globulin, and use of high-dose acyclovir therapy. Preemptive therapy with ganciclovir is a promising alternative to prophylaxis for patients at highest risk for progression to symptomatic CMV disease, such as those with CMV viremia and seropositive recipients receiving antilymphocyte therapy.


Oxidative stress and ageing in the fungus Podospora anserina. The ageing phenomenon exhibited by the ascomycetous fungus Podospora anserina can be either delayed or induced by either different carbon sources or effectors. As these effects seem to have analogy to catabolite-repression of respiratory genes, experiments concerning respiratory functions have been carried out. Ageing is parallelled by switching from cytochrome c-oxidase-mediated respiration to alternative, cyanide-resistant respiration for reasons still unknown. The latter is always accompanied by appearance of the phenol oxidizing enzyme laccase (EC 1.10.3.2), which seems to act as an alternative oxidase. The existence of a second, non-mitochondrially encoded respiratory pathway relieves the selective pressure on mitochondria leading to disintegrated, non-functional mtDNA and thereby whole mitochondria which accumulate in the hyphal cells. Mutants lacking cytochrome c-oxidase aa3 or laccase have stable mitochondrial populations and live eternally.


Efficacy of the rehabilitation of elderly primary health care patients after short-stay hospital treatment. The purpose of this study was to evaluate the impact of a primary home care intervention program on patient outcomes after selected patients were discharged from a short-stay hospital. Random assignment of 249 frail, elderly patients was made to a group provided with physician-led primary home care, and home assistance service on a 24-hour basis, or to a control group given standard care. At randomization, patients were considerably disabled, had a mean age of 80.5 years, and had a high likelihood of long-stay hospital care. Medical and functional data were essentially the same at baseline for both groups. At 6-months follow-up, significant improvement in instrumental activities of daily living (P = 0.04) and outdoor walking (P = 0.03), and medical condition was found in the primary care intervention group compared with the controls and less utilization of long-stay hospital facilities was displayed in the team patients (P < 0.001) than in the controls. A selection of elderly, dependent patients can be cared for in their homes after short-stay hospital discharge and benefit from this primary home care intervention program in terms of improved medical and functional outcomes and less long-stay hospitalization.


A novel insight into the mechanism of the antithrombotic action of defibrotide. Defibrotide is a polydeoxyribonucleotide sodium salt with antithrombotic properties. These properties have been attributed to its profibrinolytic activity [increase of tissue plasminogen activator (t-PA) activity, concomitant decrease of that of plasminogen activator inhibitor (PAI)], but there could conceivably be other factor(s). To look for these, we studied Defibrotide in a thrombosis model (pulmonary thromboembolism in mice) in which free radicals play a pivotal role. Defibrotide was found to be active after both intravenous and oral administration. Defibrotide behaved in vitro like a scavenger of H2O2 but not of O2.- in cell-free systems. Defibrotide added in vitro to cellular systems decreased the stimulated release of beta-glucuronidase from polymorphonuclear cells (PMNs), the luminol chemiluminescence induced by oxygen species generated by stimulated PMNs and the generation of O2.- from stimulated macrophages. We think that the antithrombotic activity of Defibrotide is based on other factor(s) in addition to profibrinolytic activity, i.e., some scavenger activity and desensitization of cells involved in thrombus formation must also be taken into account.


Evidence for tachykinin NK-2B-like receptors in guinea-pig alveolar macrophages. Mammalian tachykinins dose-dependently activate guinea-pig alveolar macrophages, by interacting with tachykinin NK-2 receptors, mainly. By evaluating the effects of different NK-2 tachykinin receptor antagonists, we now provide evidence that tachykinin NK-2 receptors in guinea-pig alveolar macrophages meet the pharmacological criteria used to define the NK-2B subtype.


Synthesis of [D-Ala2,4'-125I-Phe3,Glu4]deltorphin and characterization of its delta opioid receptor binding properties. Both [D-Ala2,Glu4]Deltorphin and [D-Ala2,4'-I-Phe3,Glu4]Deltorphin are highly selective ligands for delta, relative to mu, opioid receptors. Radiolabeled [D-Ala2, 4'-125I-Phe3,Glu4]Deltorphin ([125I]Deltorphin) was prepared with a specific activity of 2200 Ci/mmol from [D-Ala2, 4'-NH2-Phe3, Glu4]Deltorphin through a diazonium salt intermediate. The inhibition of [125I]Deltorphin binding to rat brain membranes by ligands selective for mu, delta, and kappa opioid receptors is consistent with binding by the radioligand to a single site having the properties of a delta opioid receptor. The results of these studies are in good agreement with those obtained by structurally different delta opioid receptor ligands. The similarity between the delta receptor site labeled by [125I]Deltorphin and those labeled by other delta receptor agonists, in contrast to differences seen by in vivo studies of their analgesic effects, is discussed.


Physical properties, lipid composition and enzyme activities of hepatic subcellular membranes from chick embryo after ethanol treatment. Exposure of chick embryos to ethanol resulted in significant alterations to the lipid composition of various different hepatic subcellular membranes. A marked decrease in cholesterol levels and an increase in the phospholipid content of microsomes and mitochondria was observed. Ethanol also affected the fatty acid profiles, mainly by decreasing the percentage of oleic acid in phosphatidylcholine and phosphatidylethanolamine in the mitochondria and phosphatidylethanolamine in the microsomes. In spite of these changes ethanol only induced alterations in the fluidity of the mitochondrial membranes, which showed a more rigid core, in contrast to the phospholipid-head region, which was not affected. In accordance with the changes observed in the physical state of the membrane, the enzymes involved in the microsomal electron-transport systems were not modified by ethanol, while cytochrome oxidase activity decreased by 50% compared to the activity in the mitochondria from control chick embryos. These findings establish that during the chick-embryo developmental period the mitochondria are more sensitive to ethanol than are the microsomes.


Species differences in the stereoselectivity of kappa opioid binding sites for [3H]U-69593 and [3H]ethylketocyclazocine. Stereoselectivity of the binding sites for the specific kappa-opioid agonist [3H]U-69593, a benzeneacetamido based ligand was investigated in membrane suspension prepared from frog and rat brain, as well as guinea pig cerebellum, using the pure chiral forms of different unlabelled opiates. The ligand binding sites showed stereospecificity with at least three orders of magnitude differences in the affinities (measured as Ki values) of the opioid stereoisomer pairs both in rat and guinea pig membrane fractions. However, in frog brain membranes there was no substantial difference in potencies of the (-) and (+) isomers competing for the [3H]U-69593 binding sites. Another type of the kappa-site preferring opioid ligand, [3H]ethylketocyclazocine, a benzomorphan derivative was able to discriminate between (-) and (+) forms of the same compounds even in frog brain membrane preparation. Our data concerning binding profile of [3H]U-69593 in frog brain membranes are consistent with the observation that kappa opioid binding sites in frog (Rana esculenta) brain differ from those kappa-sites found in mammalian brains.


Chronic cocaine administration decreases norepinephrine-induced phosphoinositide hydrolysis in rat aorta. The effect of chronic cocaine administration on norepinephrine stimulated hydrolysis of inositol 1,4,5-trisphosphate from the membrane phosphatidylinositol phosphate pool in isolated rat aorta was investigated. Rats received saline (controls), or 10 or 20 mg/kg cocaine once a day for 15 days. This treatment resulted in a dose-dependent reduction in norepinephrine (0.36 microM) stimulated phosphoinositide hydrolysis. The effect of acute cocaine was determined by adding 30 microM cocaine to the in vitro incubation solution. When aortas were exposed to cocaine and norepinephrine simultaneously, in vitro, inositol phosphate formation doubled. By itself, cocaine did not affect phosphoinositide hydrolysis. Contraction of aortic helical strips by norepinephrine decreased in tissues from rats chronically treated with 20 mg/kg cocaine. In vitro cocaine shifted the norepinephrine concentration/response curve to the left and increased the maximum response. Neither acute nor chronic cocaine treatment affected prazosin's apparent dissociation constant, suggesting that cocaine did not affect receptor affinity. These data suggest that chronic, but not acute cocaine administration may interfere with pharmacomechanical coupling in rat aorta.


125I-beta-endorphin binding to neuroblastoma X glioma NG108-15 cells: distribution of delta opioid receptors. The mouse neuroblastoma x rat glioma hybrid NG108-15 was previously shown to express delta opioid receptors. Because neuroblastoma cells display different phenotypes and cloned cell lines are heterogenous, we studied the characteristics and distribution of human 125I-beta-endorphin (125I-beta E) binding sites in cultures of NG108-15 cells with the use of micro-autoradiography and light microscopy. 125I-beta E labeled delta sites in NG108-15 in the presence of the non-opioid blocking peptide, beta-endorphin (6-31) (beta E (6-31)). Silver grains resulting from 125I-beta E binding to the opioid sites occurred in diffuse patches over several cells, with preferential location in dense cell patches. Pretreatment of NG108-15 with the delta agonist DADLE, previously shown to decrease beta E binding to delta sites on intact cells, also reduced silver grain density; however, some cells located in dense cell clusters were resistant to substantial agonist induced loss of labeling. These results suggest that delta opioid binding has a heterogenous cellular distribution in NG108.


Mechanism of action of capsaicin-like molecules on sensory neurons. Capsaicin produces pain by selectively activating polymodal nociceptive neurons. This involves a membrane depolarization and the opening of a unique, cation-selective, ion channel which can be blocked by ruthenium red. The capsaicin-induced activation is mediated by a specific membrane receptor which can be selectively and competitively antagonised by capsazepine. Repetitive administrations of capsaicin produces a desensitization and an inactivation of sensory neurons. Several mechanisms are involved. These include receptor inactivation, block of voltage activated calcium channels, intracellular accumulation of ions leading to osmotic changes and activation of proteolytic enzyme processes. Systemic and topical capsaicin produces a reversible antinociceptive and antiinflammatory action after an initial undesirable algesic effect. Capsaicin analogues, such as olvanil, have similar properties with minimal initial pungency. Systemic capsaicin produces antinociception by activating capsaicin receptors on afferent nerve terminals in the spinal cord. Spinal neurotransmission is subsequently blocked by a prolonged inactivation of sensory neurotransmitter release. Local or topical application of capsaicin blocks C-fibre conduction and inactivates neuropeptide release from peripheral nerve endings. These mechanisms account for localized antinociception and the reduction of neurogenic inflammation respectively.


The neurotoxicants strychnine and bicuculline protect renal proximal tubules from mitochondrial inhibitor-induced cell death. Glycine-induced cytoprotection of renal proximal tubules exposed to chemical- or hypoxic/anoxic-induced cell death is shared by a few amino acid agonists of the neuronal strychnine-sensitive glycine receptor. The goal of this study was to determine if antagonists of the strychnine-sensitive glycine receptor attenuated the cytoprotective effects of glycine. Strychnine did not antagonize the cytoprotective effects of glycine in proximal tubules exposed to antimycin A. In contrast, strychnine was cytoprotective, was equipotent as glycine (EC50 = 0.4 mM), and the combination of strychnine and glycine was additive. Likewise, bicuculline and norharmane were cytoprotective but 20-50% less potent than glycine. These results suggest that glycine and strychnine act as a common site to produce proximal tubule cytoprotection, but this site does not share the same potency and agonist/antagonist properties as the neuronal strychnine-sensitive glycine receptor.


Vasoactive intestinal peptide enhances phorbol myristate acetate-induced chemiluminescence in human lymphocytes. Phorbol-myristate-acetate (PMA) induced in lymphocytes the production or reactive oxygen intermediates in a process which was stimulated by the presence of vasoactive intestinal peptide (VIP) in a dose-dependent response at VIP concentrations in the range 10(-11)-10(-8) M. The dissociation constant for the high-affinity receptors of VIP agreed with the ID50 of the activation of adenylate cyclase, and the ID50 for the stimulation by VIP of PMA-induced chemiluminescence, which were close to 0.2 nM VIP. Forskolin produced in lymphocytes an effect quite similar to VIP. A comparison of the response to VIP and forskolin of lymphocytes and monocytes showed that, in contrast to forskolin, VIP failed to induce the above described effect in monocytes. A possible mechanism involving protein kinase C, which is activated by PMA, and an intracellular signal linked to VIP receptors is pointed out. This study further supports a role for VIP as a mediator in the neuroimmune system.


Modulation of cyclic nucleotide phosphodiesterase by dietary fats in rat heart. Feeding oils of different fatty acid composition modifies the fatty acid composition of cardiac membrane phospholipids, thereby inducing changes in cardiac contractility and altering response of adenylate cyclase to catecholamines. In the present study, the effect of such dietary manipulations on cyclic nucleotide phosphodiesterase, which is involved in the control of cyclic nucleotide intracellular levels and in the control of cardiac contractility, was investigated. Rats were fed either a saturated fatty acid-enriched diet (8 weight percent [%] coconut oil + 2% sunflower oil), an n-6 fatty acid-enriched diet (10% sunflower oil) or an n-3 fatty acid-enriched diet (8% fish oil + 2% sunflower oil). The fatty acid composition of cardiac phospholipids, as well as the nonesterified fatty acid content of heart were markedly altered by the diets. The 18:2n-6 and 20:4n-6 content of cardiac phospholipids was markedly (-49%) depressed by fish oil as compared with sunflower oil feeding, but the nonesterified fatty acid level of heart membrane was lowest in coconut oil-fed rats. In addition, fish oil feeding more drastically depressed the n-6/n-3 fatty acid ratio in the nonesterified fatty acid pool than in cardiac phospholipids. Cyclic AMP phosphodiesterase activity was the lowest in both the particulate and soluble fractions of heart from rats fed sunflower oil, whereas cyclic GMP phosphodiesterase activity was not altered by the diets. Cyclic AMP phosphodiesterase activity was decreased by 18 and 12% in heart membranes of the sunflower oil group as compared to that of the coconut oil and fish oil groups, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)


Primary tumors of the ovary and colon associated with pseudomyxoma peritonei. The patient presented had primary tumors of the ovary and colon in association with pseudomyxoma peritonei. Since pseudomyxoma peritonei has been associated with mucin-producing tumor of the genital and gastrointestinal tract, a thorough evaluation of the gastrointestinal tract should be performed in patients thought to have pseudomyxoma peritonei secondary to an ovarian neoplasm.


The transcription factors c-JUN, JUN D and CREB, but not FOS and KROX-24, are differentially regulated in axotomized neurons following transection of rat sciatic nerve. In adult rats, expression of c-JUN, JUN B, JUN D, c-FOS, FOS B, KROX-24 and CREB proteins was investigated by immunocytochemistry in L4 and L5 dorsal root ganglia and lumbar spinal cord for up to 300 days following transection of the left sciatic nerve. In dorsal root ganglia, expressions of c-JUN and JUN D were increased 10 h and 15 h after sciatic nerve transection, respectively. c-JUN was still at an elevated level after 300 days predominantly in small diameter neurons, whereas JUN D had declined to control levels after 100 days. In contrast to the JUN proteins, expression of CREB showed a delayed onset after 10 days and reached a maximum between 70 and 150 days. In motoneurons, expression of c-JUN and JUN D was increased 15 h and 25 h after sciatic nerve transection, respectively. Expression of c-JUN remained increased after 150 days, whereas JUN D had declined to control levels after 70 days. In contrast, expression of CREB declined within 30 h in axotomized motoneurons and remained on a reduced level for up to 150 days. JUN B, c-FOS, FOS B and KROX-24 were not induced either following axotomy or following a repeated nerve crush. Sciatic nerve transection including the surgical procedure transynaptically provoked a transient expression of all JUN, FOS and KROX-24 proteins in neurons of spinal dorsal horn which disappeared after 5 days except the expression of JUN D which lasted for up to 20 days. In contrast, CREB immunoreactivity was not at all altered in neurons of spinal dorsal horn. In untreated animals, CREB and to a lesser extent JUN D showed an ubiquitous expression in neurons and glia cells of spinal cord, whereas expression of c-JUN and a weak expression of FOS B were restricted to motoneurons. In neurons of the dorsal root ganglia, a basal expression was found for c-JUN, JUN D and CREB and, at a low level, for FOS B and KROX-24. c-JUN and JUN D were colocalized with CREB in many cells such as interneurons, motoneurons, dorsal root ganglion cells and glial cells indicating the possibility for both the control of c-jun and jun D expression by CREB and the competition of JUN and CREB proteins for CRE consensus sequences.


Molecular dynamics of serotonin and ritanserin interacting with the 5-HT2 receptor. A three-dimensional model of the serotonin (5-hydroxytrytamine; 5-HT) 5-HT2 receptor was constructed from the amino acid sequence by molecular graphics techniques, molecular mechanics energy calculations and molecular dynamics simulations. The receptor model has 7 alpha helical segments which form a membrane-spanning duct with a putative ligand binding site. Most of the synaptic domains and the ligand binding site were surrounded by negative electrostatic potentials, suggesting that positively charged ligands are attracted to the receptor by electrostatic forces. The cytoplasmic domains, except the C-terminal tail, had mainly positive electrostatic potentials. The molecular dynamics of the receptor-ligand complex was examined in 100 ps simulations with 5-HT or ritanserin at a postulated binding site. During the simulations the helices moved from an initial circular arrangement into a more oval arrangement, and became slightly tilted relative to each other. The protonated ligands neutralized the negative electrostatic potentials around Asp 120 and Asp 155 in the central core of the receptor. 5-HT had only weak interactions with Asp 155 but strong interactions with Asp 120 during the simulations, with the amino group of 5-HT tightly bound to the carboxylic side chain of Asp 120. Ritanserin showed similarly strong interactions with Asp 120 and Asp 155 during the simulations.


Stimulation of c-fos and c-jun gene expression and down-regulation of proenkephalin gene expression in C6 glioma cells by endothelin-1. The linkage between the transmembrane signal transduction system utilized by endothelin and alterations in gene expression has been investigated in C6 glioma cells. Treatment of C6 cells with endothelin-1 caused a rapid and transient 5-fold increase in c-fos and c-jun mRNA levels, followed by a decrease at 4 h. Dose-response studies indicated that 1 nM endothelin-1 caused half-maximal induction of c-fos mRNA 0.5 h after treatment and that maximal induction was elicited with a concentration of 10 nM. Actinomycin D totally abolished the rapid increase in c-fos mRNA caused by endothelin, indicating that the effect is at the transcriptional level. Endothelin-1 caused a decrease in proenkephalin mRNA to 50% of control levels at 4 h after treatment and had no effect on histone H4 mRNA over a 24 h period that was examined. These data indicate that receptor binding of endothelin-1 leads to rapid changes in the expression of immediate-early response genes which may cause more prolonged changes in the expression of AP-1 and/or CREB target genes in the nervous system.


Omega-conotoxin- and nifedipine-insensitive voltage-operated calcium channels mediate K(+)-induced release of pro-thyrotropin-releasing hormone-connecting peptides Ps4 and Ps5 from perifused rat hypothalamic slices. The rat thyrotropin-releasing hormone (TRH) precursor (prepro-TRH) contains five copies of the TRH progenitor sequence linked together by intervening sequences. Recently, we have shown that the connecting peptides prepro-TRH-(160-169) (Ps4) and prepro-TRH-(178-199) (Ps5) are released from rat hypothalamic neurones in response to elevated potassium concentrations, in a calcium-dependent manner. In the present study, the role of voltage-operated calcium channels in potassium-induced release of Ps4 and Ps5 was investigated, using a perifusion system for rat hypothalamic slices. The release of Ps4 and Ps5 stimulated by potassium (70 mM) was blocked by the inorganic ions Co2+ (2.6 mM) and Ni2+ (5 mM). In contrast, the stimulatory effect of KCl was insensitive to Cd2+ (100 microM). The dihydropyridine antagonist nifedipine (10 microM) had no effect on K(+)-evoked release of Ps4 and Ps5. Furthermore, the response to KCl was not affected by nifedipine (10 microM) in combination with diltiazem (1 microM), a benzothiazepine which increases the affinity of dihydropyridine antagonists for their receptor. The dihydropyridine agonist BAY K 8644, at concentrations as high as 1 mM, did not stimulate the basal secretion of Ps4 and Ps5. In addition, BAY K 8644 had no potentiating effect on K(+)-induced release of Ps4 and Ps5. The marine cone snail toxin omega-conotoxin, a blocker of both L- and N-type calcium channels had no effect on the release of Ps4 and Ps5 stimulated by potassium. Similarly, the omega-conopeptide SNX-111, a selective blocker of N-type calcium channels, did not inhibit the stimulatory effect of potassium. The release of Ps4 and Ps5 evoked by high K+ was insensitive to the non-selective calcium channel blocker verapamil (20 microM). Amiloride (1 microM), a putative blocker of T-type calcium channels, did not affect KCl-induced secretion of the two connecting peptides. Taken together, these results indicate that two connecting peptides derived from the pro-TRH, Ps4 and Ps5, are released by K(+)-induced depolarization through activation of voltage-sensitive calcium channels. The calcium channels appear to have a pharmacological profile different from that of L- and N-type channels. Although, their insensitivity to low Cd2+ concentrations and sensitivity to Ni2+ ions would support the involvement of T-type calcium channels, the lack of effect of amiloride suggests that they belong to a yet undefined class of calcium channels.


Distribution of cells containing progesterone receptor mRNA in the female rat di- and telencephalon: an in situ hybridization study. In an attempt to examine regional synthesis of the progesterone receptor (PR) in the brain, the distribution of mRNA encoding PR was investigated in the female adult rat di- and telencephalon by in situ hybridization using T7 RNA polymerase transcripts of a 320 base pair rat PR cDNA clone. The rat PR cDNA had been partially cloned and sequenced by using the reverse transcription-polymerase chain reaction (RT-PCR) method. The primer set corresponds to a part of the progesterone binding domain of human PR cDNA. Large numbers of strong labeling were observed in the arcuate nucleus, medial preoptic nucleus, and ventrolateral part of the ventromedial nucleus which are relative to sexual behavior. Moderate labeling was found in layers II and IV of the isocortex, in the pyramidal layer of the CA1 and CA3 fields of the hippocampal formation, in the cortical nucleus of the amygdala, in the nucleus of the diagonal band, and in the anterior periventricular nucleus. Weak labeling was found in many other regions. These results were largely in agreement with the distribution of PR previously reported by ligand binding assay and autoradiographic studies. This present in situ hybridization study may provide a useful tool for the analysis of the regional regulation of PR synthesis in the rat brain.


Accumulation of calcitonin-gene related peptide-like immunoreactivity after hypoxic-ischaemic brain injury in the infant rat. Unilateral carotid ligation in immature rats, followed by either 15 min (moderate group) or 90 min (severe group) of hypoxia were used to assess the effects of hypoxia-ischaemia (HI) on the accumulation of the neuropeptide calcitonin-gene related peptide (CGRP). Severe, but not moderate, HI produced a massive time-dependent increase in CGRP-like immunoreactivity throughout the damaged regions of the brain (neocortex, caudate-putamen, hippocampus) beginning at 24 h and maximal at 3-5 days after HI. By 11 days after HI levels appeared to have returned to baseline. The increased immunoreactivity was largely localized to presumed axon terminals contacting neurons, blood vessels and non-nerve cells. Scattered neurons in the cingulate cortex, piriform cortex and striatum also showed increased immunoreactivity in their soma. These results raise the possibility that CGRP may be involved in neuronal repair after HI in the infant brain.


Transient decrease in rat striatal D2 dopamine receptor mRNA level after acute haloperidol treatment. Acute haloperidol administration decreases the number of rat striatal D2 dopamine receptor (D2-receptor). The potential involvement of decreased D2-receptor gene transcription in the above process was examined using Northern blot analysis. There was a dose- and time-dependent transient decrement in the level of striatal D2-receptor mRNA after haloperidol. The decrease in transcription may be mediated via blockade of D2-receptor, since S(-)-sulpiride but not the inactive enantiomer R(+)-sulpiride produced the inhibition.


Isolation and structural characterization of peptides related to alpha- and gamma-melanocyte-stimulating hormone (MSH) from the frog brain. Peptides that are derived from the processing of proopiomelanocortin were isolated in pure form from the brain of the frog Rana ridibunda. The primary structure of the most abundant of those peptides was established as: Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val. This amino acid sequence is identical to that of mammalian and frog pituitary alpha-melanocyte-stimulating hormone (MSH) and the peptide co-eluted with synthetic desacetyl alpha-MSH, indicating that it is COOH-terminally alpha-amidated. A second component, which exhibited a shorter retention time, co-eluted with the glycine-extended form of desacetyl alpha-MSH [ACTH(1-14)]. The primary structure of the third peptide isolated in pure form from the brain extract was established as: Lys-Tyr-Val-Met-Ser-His-Phe-Arg-Trp-Asn-Lys-Phe-NH2. This sequence corresponds to Lys-gamma 1-MSH as predicted from the nucleotide sequence of frog proopiomelanocortin. The presence of substantial amounts of desacetyl alpha-MSH and Lys-gamma 1-MSH in the frog brain supports the concept that, in amphibia, melanotropins may act as neurotransmitters and/or neuromodulators as well as hormonal peptides.


An intraocular injection of kainate induces expression of c-fos-like protein and activation of protein kinase C (alpha) in specific rabbit retinal neurones. Intraocular injection of kainate into the rabbit eye causes both a translocation and transport of the bipolar cell's alpha PKC 6 h later. Although this effect is similar to what occurs for the phorbol ester, phorbol 12,13-dibutyrate (PDbut), it shows specificity in that N-methyl-D-aspartate (NMDA), 5,7-dihydroxytryptamine and 2-amino-4-phosphonobutyrate (APB) are ineffective. However, preliminary experiments suggest that, when injected into the eye, quisqualate also influences the alpha PKC of the bipolar cells. Injection of kainate into the rabbit eye shows that c-fos-like protein is expressed in certain amacrine and ganglion but not in bipolar cells 6 h later. This expression of c-fos immunoreactivity is transient because 15 h after the injection of kainate no positive staining was seen. It was not possible to analyse the kainate-induced c-fos expression for periods of less than 6 h because the anaesthetic used, Hypnorm, induced c-fos-like protein expression which lasted for 2-4 h.


Aromatic trivalent arsenicals: covalent yet reversible reagents for the agonist binding site of nicotinic receptors. The agonist binding site of nicotinic acetylcholine receptors (AChRs) includes a disulfide bond that is easily reduced with dithiothreitol to a pair of thiols, and can be then either reoxidized with dithiobis(nitrobenzoic acid) (DTNB) or irreversibly alkylated with bromoacetylcholine (BAC). Aromatic trivalent arsenicals form stable complexes with pairs of appropriately-spaced thiols, but not single thiols. Furthermore, once complexed in proteins, trivalent arsenicals can be removed with dimercaptans, such as 2,3-dimercaptopropanesulfonic acid (DMPS). In an effort to develop reagents that will covalently, yet reversibly label AChRs, we investigated the effects of two model arsenicals, p-aminophenyldichloroarsine (APA) and 4-bromoacetyl-aminophenylarsenoxide (BAPA) on two types of nicotinic receptors: AChRs from Torpedo electroplax and neuronal receptors from chick retina. APA and BAPA significantly decrease the number of 125I-alpha-bungarotoxin binding sites in reduced Torpedo AChRs. Furthermore, arsenylation of neuronal and Torpedo receptors with APA or BAPA (1) prevents reoxidation with DTNB, (2) is reversible with DMPS, and (3) protects against irreversible alkylation by BAC. In Torpedo receptors, the EC50 of protection against BAC alkylation with APA or BAPA is approximately 30 nM. APA arsenylation of Torpedo receptors persists up to 20 h, but can be reversed at any time with DMPS. These results suggest that heterobifunctional arsenicals could anchor labeling groups in the agonist binding site in order to map the agonist binding site, quantitate receptors, or purify and reconstitute functional receptors.


The GABAA receptor gamma 1 subunit is expressed by distinct neuronal populations. The distribution of GABAA receptor gamma 1 subunit was examined in the rat central nervous system using in situ hybridization histochemistry. The gamma 1 subunit was expressed in relatively limited areas compared to other subunits investigated previously. The brain regions strongly expressing this subunit were the septum, globus pallidus, bed nucleus of the stria terminalis, hypothalamic periventricular nucleus, supraoptic nucleus, medial and central nuclei of the amygdaloid complex, medial part of the medial geniculate body, substantia nigra pars reticulata, interpeduncular nucleus, lateral parabrachial nucleus, Purkinje cell layer of the cerebellum, and inferior olivary nucleus. This relatively limited expression implies a possible role of gamma 1 subunit in relation to some specific neuronal circuit.


Axotomized medial septal-diagonal band neurons express Jun-like immunoreactivity. The expression of the transcription factors Fos and Jun was studied in rat brain after transection of the fornix-fimbria (FF) using polyclonal antibodies to these proteins and immunocytochemical detection methods. FF-transection lead to a massive induction of Jun-like immunoreactivity (JLI) in neurons in the medial septal nucleus and in the vertical limb of the diagonal band of Broca, within 48 hours and lasting up to 14 days after lesion. Fos was not induced in these neurons after FF-transection. These results indicate that axotomized medial septal and diagonal band of Broca neurons selectively and rapidly express JLI. The role of Jun expression in axonal regeneration or neuronal death is discussed.


Induction of NGF by isoproterenol, 4-methylcatechol and serum occurs by three distinct mechanisms. Evidence is provided that isoproterenol, 4-methylcatechol and serum induce NGF by three separate mechanisms. Isoproterenol and 4-methylcatechol induced NGF and NGF mRNA in mouse fibroblast L929 cells in either the presence or absence of serum. Propranolol prevented NGF induction by isoproterenol, but not by 4-methylcatechol or serum. All possible combinations of these inducers resulted in additive increases in the levels of NGF and NGF mRNA.


Hypothalamic neuropeptide Y gene expression in rats on scheduled feeding regimen. Recent evidence suggests that neuropeptide Y (NPY) is an important signal in the neural circuitry that controls feeding behavior. Previously we observed that in rats entrained to 4 h daily scheduled feeding regimen (SFR), NPY content and release in the paraventricular nucleus (PVN) was elevated but decreased rapidly in association with food consumption. In the present study, we investigated the pattern of hypothalamic NPY gene expression in SFR rats before and after food consumption by measuring the content of preproNPY mRNA in the medial basal hypothalamus (MBH). Adult male rats were maintained on either ad libitum diet (control) or on SFR. Rats were killed before food presentation at 11.00 h and at the end of 4 h food consumption at 15.00 h. The levels of preproNPY mRNA in the MBH were determined by solution hybridization/RNase protection assay using a cRNA probe complementary to rat NPY precursor mRNA. We observed that, as compared to that in control rats on ad libitum diet, preproNPY mRNA levels in the MBH were increased two-fold in the SFR rat at 11.00 h and remained elevated even after 4 h of food consumption. These results show a simultaneous enhancement in PVN NPY release and hypothalamic gene expression in advance of scheduled feeding time, but food intake rapidly decreases PVN NPY release and content, with little impact on hypothalamic gene expression.


Apomorphine induction of AP-1 DNA binding in the rat striatum after dopamine depletion. The expression of AP-1 transcription factors was assessed in the dopamine-depleted rat striatum over a 1 week period of repeated apomorphine injections. A single injection of apomorphine increased the expression of a 35 kDa Fos-related antigen and Jun proteins and their expression continued to increase until day 3 of repeated apomorphine treatment in dopamine-depleted striata. Apomorphine induces AP-1 transcription factors which may be involved in modulating gene expression in the striatum.


L-glutamate transporter derived from mRNAs of primary glial cultures: expression in Xenopus laevis oocytes. A high-affinity sodium-dependent L-glutamate transporter was expressed in Xenopus oocytes after microinjection of poly(A)+ RNA from primary astrocyte cultures from rat brain cortex. mRNA-induced L-glutamate transport was saturable by substrate and shows kinetic features similar to those found in intact glial cell preparations. L-Glutamate accumulation was prevented by rising the external K+ concentration or by coincubation with L-, D-aspartate or D-glutamate. After fractionation by sucrose density gradient, the mRNA encoding for the expressed L-glutamate transporter from glial cells was found in fractions containing messages of 2.05-2.9 kilobases (kb) in length.


Co-expression of the alpha 1 and beta 2 subunit genes of the GABAA receptor in the magnocellular preoptic nucleus. Co-localization of the alpha 1 and beta 2 subunit mRNAs of the GABAA receptor was examined on serial sections of the rat magnocellular preoptic nucleus using in situ hybridization histochemistry. More than half of the labeled neurons in this nucleus contained both transcripts, while 31.9% and 17.4% of them contained only alpha 1 or beta 2 subunit mRNA, respectively. These results indicate that there may be three GABAA receptor subtypes in this nucleus, with co-localization of the alpha 1 and beta 2 subunits occurring in the most common subtype.


An NGF-inducible octamer binding protein activity in a C1300 neuroblastoma cell line. Nerve growth factor (NGF) induces a protein in the C1300 mouse neuroblastoma cell line which recognises the octamer DNA consensus sequence 'ATGCAAAT'. This protein is absent, or only minimally present, in C1300 cells prior to induction with NGF. This induced octamer binding protein is detectable by gel retardation analysis within 3 h of NGF treatment and increases progressively with 24 h and 72 h of exposure to NGF.


Regulation of aromatase cytochrome P-450 (estrogen synthetase) transcripts in the quail brain by testosterone. The aromatase cytochrome P-450 (P-450AROM) cDNA, which was identified by homologies in the DNA and in the deduced amino acid sequences with human P-450AROM cDNA, was isolated from a brain cDNA library of Japanese quail, demonstrating the presence of RNA transcripts of P-450AROM in the quail brain. To determine trace amounts of P-450AROM mRNA in the brain and to examine the effects of testosterone on its expression, a quantitative PCR method of RNA transcripts was developed. Brain total RNA was subjected to reverse transcription reaction and then quantitated by PCR from cDNA with a fluorescent dye-labeled primer. The quantity of P-450AROM mRNA was calculated by using an internal standard of modified P-450AROM (m-P-450AROM) RNA. The brain P-450AROM was primarily transcribed in the hypothalamus area (1.15 +/- 0.14 amol/micrograms of RNA) and traces of transcripts only were detected in the cerebellum (0.038 +/- 0.005 amol/micrograms of RNA). The P-450AROM mRNA in the hypothalamus of castrated quail was low (0.270 +/- 0.078 amol/micrograms of RNA) and increased 4- to 5-fold following treatment with testosterone. These results demonstrate, for the first time, that the increase in P-450AROM activity that is observed in the brain following treatment with testosterone results from a pretranslational regulation of the P-450AROM by androgens.


Role of dopamine in the regulation of proopiomelanocortin (POMC) mRNA levels in the arcuate nucleus and pituitary gland of the female rat as studied by in situ hybridization. The effects of the dopaminergic antagonist haloperidol (HAL) as well as the D2 dopamine receptor agonist bromocriptine (BRO) on proopiomelanocortin (POMC) mRNA levels in the female rat arcuate nucleus and pituitary were investigated by quantitative in situ hybridization. Since we had already shown that sex steroids could induce a decrease in POMC mRNA levels in the arcuate nucleus of castrated rats, the involvement of the dopaminergic system in the inhibitory effect of estradiol (E2) was also investigated. In situ hybridization was performed on paraformaldehyde-fixed cryostat sections through the arcuate nucleus and whole pituitary gland using a 35S-labelled cDNA probe encoding for POMC. In the arcuate nucleus of intact animals, a 14-day treatment with BRO increased by 54% the number of silver grains/unit of surface of labelled neurons while HAL decreased by 30% the value of this parameter. Hypophysectomy which induced a 20% decrease in the hybridization signal could not prevent the effects of BRO or HAL. Ovariectomy performed 14 days earlier increased by 20% the number of silver grains while a 14-day treatment of ovariectomized animals with E2 decreased the hybridization signal by 32%. On the other hand, the concomitant administration of HAL and E2 did not induce significant variations in POMC mRNA levels compared to those obtained following HAL administration, but slightly decreased the hybridization signal by 20% compared to that induced by E2 alone. In the intermediate lobe of the pituitary, BRO markedly depressed (30% of control values) and HAL increased by 50% the levels of POMC mRNA. The present data clearly demonstrate that POMC mRNA levels are differently regulated by dopamine in the intermediate lobe of the pituitary and the arcuate nucleus and that the effects of BRO and HAL on arcuate POMC mRNA are not mediated by the pituitary gland. They do not allow to draw any definite conclusion about the possible involvement of the dopaminergic system in the inhibitory role of E2 on POMC gene regulation.


Localization of mRNA for c-kit receptor and its ligand in the brain of adult rats: an analysis using in situ hybridization histochemistry. Localization of mRNA for the c-kit receptor and its ligand (Sl factor) in the brain of adult rats was studied using in situ hybridization histochemistry. The mRNA for the c-kit receptor was detected in the forebrain, the lower brain stem and the cerebellum. In the forebrain, the c-kit mRNA signals were detected in the olfactory bulb, the caudate-putamen, throughout the superficial cortex, the accumbens nucleus, the nucleus of vertical limb diagonal band, the bed nucleus of anterior commissure, Ammon's horn, the entopeduncular nucleus, the subthalamic nucleus, the dorsal raphe nucleus, the parasubiculum, the presubiculum, the ventricular nucleus of lateral lemniscus, and the entorhinal cortex. In the lower brain stem, the signals were detected in the inferior colliculus, the spinal vestibular nucleus, the spinal tract nucleus of trigeminal nerve, and the pyramidal tract. In the cerebellum, the signals were detected in the molecular layer of the cortex and cerebellar nuclei. By contrast, the signals of mRNA for Sl factor were detected in the forebrain and the cerebellum. In the forebrain, the signals were detected in the olfactory bulb, the endopiriform nucleus, the septohippocampal nucleus, the habenular nuclei, and most of the thalamic nuclei. In the cerebellum, the signals were detected in Purkinje cells. Several pairs of structures were found in which mRNA of either the c-kit receptor or the Sl factor was expressed and between which the synaptic connection had been reported, suggesting that the interaction between the c-kit receptor and the Sl factor may play some roles in the development of such synaptic connections.


Molecular characterization of a human brain adenosine A2 receptor. A cDNA encoding a G protein-coupled receptor of unknown ligand specificity was isolated from a human hippocampal cDNA library by virtue of the high degree of structural homology between members of this receptor family. The cloned receptor DNA was transfected into human embryonic kidney 293 cells. Stably transfected cell lines bound a variety of adenosine agonists and antagonists with affinities characteristic of a brain adenosine A2a receptor. The A2a specific agonist CGS21680 stimulated cAMP production but did not alter intracellular calcium concentrations in transfected 293 cells.


The human nerve growth factor gene: structure of the promoter region and expression in L929 fibroblasts. We previously studied the transcriptional mechanisms involved in expression of the murine nerve growth factor (NGF) gene. To investigate the regulation of transcription of the human NGF gene, the promoter region was cloned. The nucleotide sequences of the human and mouse genes are greater than 90% similar near their promoters. The cloned human promoter was transcriptionally active in mouse L929 fibroblasts. 5' Deletion analyses indicated that the -85 to -45 region stimulates basal transcription 6-fold. This segment is greater than 80% identical in human and mouse genes except for an AP-1 consensus sequence found only in the human gene. A second AP-1 consensus sequence at +34, previously shown to function as a regulatory element in the mouse gene, is identical in both genes. Gel shift analyses of L929 cell extracts revealed binding of protein to oligonucleotide probes spanning each of the two AP-1 consensus sequences of the human gene. The gel shift patterns differed, suggesting interaction of different proteins with the two probes. Our results demonstrate that the human NGF gene promoter is transcriptionally active in mouse fibroblasts, and implicate an upstream region in basal transcription.


Protein differences in tau mutant hamsters: candidate clock proteins. In the tau mutant hamster, the period of the circadian rhythm is shortened from about 24 h to about 22 h in heterozygotes and to about 20 h in homozygotes. Understanding the biochemical basis of the period changes in the tau mutant may elucidate the regulation of the vertebrate pacemaker. Using two-dimensional gel electrophoresis, we have found two sets of proteins that differ between the different genotypes. P33tau (about 33 kDa; pI 6.5) was found in all gels from wild type and heterozygous animals, but was absent in gels from all except one of the homozygous mutant animals. P32tau (about 32 kDa; pI 4.8) was a chain of spots, which showed a striking difference in pattern between gels from wild type animals and from mutant animals. P33tau was greatly enriched in soluble cellular fractions, whereas P32tau was found only in insoluble fractions. These differences between P33tau and P32tau were apparent in gels from both SCN and cortical tissue, suggesting that both proteins are distributed throughout the brain. These proteins should be useful as new tools to explore the biochemistry of circadian pacemakers.


Multiple nuclear factors interact with the promoter of the human neurofilament M gene. In order to identify potential regulatory elements of the human mid-sized (M) neurofilament (NF) gene we preformed DNase I footprinting, gel mobility shift assays and methylation interference studies with probes from the NF(M) immediate 5' flanking region. These studies identified multiple sites for DNA-binding proteins including four Sp1 sites, and single sites each for members of the NF-1 and AP-1 families of DNA binding proteins. In addition a binding site within a pyrimidine tract likely binds a novel DNA-binding protein which also interacts with the human NF(H) gene promoter. Factors that bind to these sites are found in both neural and non-neural cells suggesting that the NF(M) promoter may not contain tissue specific regulatory signals. In transient assays, addition of these binding sites to an NF(M) minimal promoter containing only a TATA box lead to a greater than 40-fold activation of transcription over background. Progressive 5' deletions reduced expression in a step wise manner suggesting that all the factors likely act synergistically as positive regulators of transcription.


Localization of multiple dopamine receptor subtype mRNAs in human and monkey motor cortex and striatum. Dopamine plays a critical role in motor and cognitive function through actions mediated by specific receptors, multiple subtypes of which have recently been identified. The distribution of mRNAs encoding D1, D2 and D5 receptors in the motor cortex of humans and in the motor cortex and striatum of macaque monkeys was examined using in situ hybridization. In motor cortices from both primate species, hybridization to each receptor probe resulted in numerous labeled cells throughout layers II-VI. In contrast to neocortex, in monkey striatum only the D1 and D2 receptor probes showed significant hybridization. Thus, not only does primate neocortex possess a broader representation of the dopamine receptor subtype mRNAs examined in comparison with striatum, but the unexpected presence and widespread distribution of D2 and D5 receptor mRNAs in cortex suggests that, along with D1 receptors, D2 and D5 receptors play a crucial role in the dopaminergic modulation of cognition and motor behavior, and in dopamine dysfunction associated with neuropsychiatric disorders.


Reserpine treatment stimulates enkephalin and D2 dopamine receptor gene expression in the rat striatum. We investigated the effect of catecholamine depletion on gene expression for preproenkephalin A (PPA) and D2 dopamine receptor (D2R) in the rat nigrostriatal complex, using quantitative Northern blot analysis. The D2R probe indifferently recognizes the two mRNA isoforms generated by alternative splicing from the same gene. Short-term and chronic reserpine treatment increase the level of PPA and D2R mRNA in the striatum in a complex manner. For short-term treatment, we injected 10 mg/kg of reserpine the first day, 5 mg/kg 24 h later and sacrificed the rats at various times after the last injection. This treatment resulted in an increase of the level of PPA mRNA by 50% and D2R mRNA up to 150%. For chronic treatment, we injected 0.5 mg/kg of reserpine for 21 days, sacrificed the rats one day after the last injection and observed an increase in PPA and D2R mRNA levels by 100%. Statistical analysis revealed that the PPA mRNA level after chronic treatment was significantly higher from the one obtained after short-term treatment while no such difference was observed for the D2R mRNA. In contrast, reserpine treatment does not modify the level of D2R mRNA in the substantia nigra suggesting that catecholamine depletion has postsynaptic but not presynaptic consequences in the rat nigrostriatal complex. These results demonstrate that reserpine acts at the gene or the mRNA level to induce dopamine supersensitivity in striatal dopaminoceptive neurons.


Beta-amyloid precursor protein localization in the Golgi apparatus in neurons and oligodendrocytes. An immunocytochemical structural and ultrastructural study in normal and axotomized neurons. We have used a polyclonal antibody raised against a synthetic peptide from the carboxyl terminal of the beta-amyloid precursor protein (APP) to examine the cellular and subcellular localization of this protein in the rat brain. Light and electron microscopic immunocytochemical techniques were used. Immunoreactivity was found throughout the brain in all the neurons examined as well as in oligodendrocytes. At the light microscopic level, a perinuclear filamentous distribution was seen in neurons, suggesting a concentration of the protein to the Golgi apparatus. Axotomy of motor neurons of the facial nucleus produced a decrease in choline acetyltransferase (ChAT) activity and an increase in the perineuronal microglial nucleoside diphosphatase (NDPase)-positive cells in addition to a hypertrophy of the GFAP immunoreactive astrocytes. On the other hand, increased APP-like immunoreactivity all over the neuronal cell bodies accompanied by a dispersion ('rete dispersion') of the Golgi apparatus labeling was demonstrated. In contrast, reactive microglia and hypertrophic astrocytes in axotomized facial nucleus were not immunolabeled. Oligodendrocytes showed a punctate APP immunoreactivity corresponding to the Golgi apparatus in both operated and control facial nucleus. This was further demonstrated by electron microscopic immunolabeling. These results show that the main localization of the C-terminal containing forms of the APP in the rat brain is the Golgi apparatus in both neurons and oligodendroglia and further supporting the secretory nature of these proteins. The increased synthesis of this protein after axotomy is suggestive of a role of the APPs in growth and/or regeneration.


Characterization and localization of glutathione binding sites on cultured astrocytes. Glutathione (GSH) binding sites found in brain white matter in a previous study using biotinylated GSH (Third IBRO World Congress Neurosci. Abstr., 1991, P59.17) suggested that there might GSH receptors on glial cells. In the present study, radioligand receptor assays were performed on cultured astrocytes using [35S]GSH. Scatchard analyses of saturation binding of [35S]GSH revealed two binding sites: Kd1 = 2.0 +/- 0.1 nM, Bmax1 = 89.5 +/- 1.5 fmole/2.2 x 10(5) cells and Kd2 = 12.8 +/- 0.4 nM, Bmax2 = 187.7 +/- 2.4 fmol/2.2 x 10(5) cells. The saturable and displacible high affinity [35S]GSH binding we have observed suggests that this binding is not due to GSH sequestration by uptake sites or to the association of GSH with GSH S-transferases or GSH peroxidases which have Kds in the microM range. Colloidal gold and immunofluorescence double labelling were used to visualize the binding sites at the cellular level. Positive colloidal gold decoration further suggests that these labelled binding sites are membrane receptors on astrocytes.


ProELH-related peptides: influence on bag cell cAMP levels. ProELH is the prohormone to the bag cell egg-laying peptide of Aplysia. In addition to containing the structure of the hormone (ELH) itself, proELH also contains several other secreted peptides: AP (acidic peptide) and alpha-, beta-, and gamma-bag cell peptides (BCPs). The BCPs, ranging in length from 5 to 9 amino acids, are structurally similar in that they all contain the sequence Arg-Leu-Arg-Phe. An additional peptide from the atrial gland, Atrial A, also contains this sequence. The BCPs previously have been reported to have direct feedback (autocrine) effects on the bag cells, including electrophysiological excitation and inhibition. Moreover, some of these effects are temperature-dependent. The autocrine functions of these peptides were explored here by investigating their effects on bag cell cAMP levels. In addition, we monitored the effects of Atrial A, as well as ELH and AP, which are proELH products that do not have sequence homology with the BCPs. While ELH and AP have no effect on bag cell cAMP levels, the other peptides fall into two functional classes. alpha- and gamma-BCP produce an elevation of cAMP levels at 20 degrees and a depression at 15 degrees C. The elevation in cAMP is sensitive to low Ca2+/high Mg2+. beta-BCP and Atrial A elevate cAMP levels independently of temperature, and are insensitive to low Ca2+/high Mg2+. Our results suggest that there may be multiple bag cell receptors for these peptides with the Arg-Leu-Arg-Phe sequence representing a receptor-recognition motif.


Enhanced GLUT1 glucose transporter and cytoskeleton gene expression in cultured bovine brain capillary endothelial cells after treatment with phorbol esters and serum. The in vitro angiogenesis of endothelium obtained from peripheral tissues is stimulated by phorbol esters. The present studies examine the effects of phorbol esters or serum factors on GLUT1 glucose transporter, cytoplasmic actin, and beta-tubulin messenger RNA levels and gene transcription rates in bovine brain capillary endothelial cells grown in tissue culture. Messenger RNA levels were measured by Northern blot analysis and transcription rates were quantified by nuclear run-on assays. Although cytoplasmic actin mRNA levels in cultured brain endothelium were comparable to levels found in isolated capillaries isolated in vivo, there was a profound down-regulation of the GLUT1 glucose transporter mRNA in the cultured endothelium. The GLUT1 mRNA level was increased by exposure to 12-O-tetra-decanoyl-phorbol 13-acetate (TPA). Both serum and TPA enhanced cytoplasmic actin and beta-tubulin mRNA levels in cultured cells; the serum effect on cytoskeletal mRNA persisted through at least 24 h of exposure whereas the TPA stimulation was maximal by 2 h of exposure and lost following 8 h. Both serum and TPA increased cytoplasmic actin mRNA levels approximately 2- to 3-fold greater than the increase in beta-tubulin mRNA levels. GLUT1 and actin transcription rates were measured with the nuclear run-on assay, but no stimulation was observed following 3 h exposure to 200 nM TPA. In conclusion, these studies show that GLUT1 glucose transporter, cytoplasmic actin, and beta-tubulin mRNA levels in bovine brain capillary endothelial cells are regulated by both serum factors and phorbol ester, which activates the protein kinase C pathway, and that the mechanism of the phorbol ester effect is post-transcriptional.


Gene expression of the insulin-like growth factors and their receptors in human neuroblastoma cell lines. Insulin-like growth factors (IGF) I and II are polypeptides with both growth-promoting and insulin-like metabolic effects. The developmentally specific expression of IGF I and II in the nervous system implies a role for these growth factors in neuronal growth and differentiation. In the present study, we analyzed IGF and IGF receptor mRNA transcripts from two related human neuroblastoma cell lines, SH-SY5Y and SK-N-SH. These cell lines provide a good in vitro model of neuronal development. Northern analysis of total RNA from each cell line revealed three IGF II mRNA transcripts (6.0, 4.8, and 1.8 kb), and one mRNA transcript each for the type I (11.0 kb) and type II (9.4 kb) IGF receptors. The size distribution of these multiple transcripts is similar to that found during normal human fetal development. These results establish both cell lines as good in vitro models for investigating the mechanisms which underly IGF gene expression during nervous system development.


Modulation of gamma-actin and alpha 1-tubulin expression by corticosterone during neuronal plasticity in the hippocampus. Evidence is given for altered gene expression of gamma-actin in the hippocampus in response to entorhinal cortex lesion (ECL). Time course analysis reveals a progressive repression of gamma-actin expression between 4 and 14 days post-lesion, coinciding with the early and middle phases of the hippocampal reinnervation process. RNA prevalence returns to near control values at 30 days post-lesion. Corticosterone administration, which is known to impair the reinnervation process in ECL rats, prevents the lesion-induced reduction in gamma-actin expression and blocks the induction of alpha 1-tubulin in the deafferented hippocampus. The timing of response of gamma-actin to ECL and its modulation by glucocorticoid administration support suggestions that gamma-actin has an important role to play in neuronal cytoarchitecture remodelling during hippocampal reinnervation.


Circadian and light-induced expression of immediate early gene mRNAs in the rat suprachiasmatic nucleus. Recent work has suggested that immediate early genes are involved in the phase-shifting response of the circadian pacemaker to light. In mammals, the endogenous pacemaker is located in the suprachiasmatic nucleus (SCN). We report here a systematic examination of the expression of the immediate early genes fos, NGFI-A, and NGFI-B in the rat SCN, in basal and light-stimulated conditions across the circadian cycle. The photic induction of all three genes examined was found to be gated by the circadian system with maximal induction observed during the mid to late subjective night. The photic-induced expression of NGFI-B in the SCN was considerably less than that of NGFI-A, despite previous observations suggesting greater induction of NGFI-B by membrane depolarization. The levels of immediate early gene expression induced by light do not directly correspond to the magnitude of previously reported light-induced phase-shifts of the activity rhythm. The results also suggest that fos may undergo a circadian rhythm of expression in the rat SCN.


Induction of nerve growth factor receptor (p75NGFr) mRNA within hypoglossal motoneurons following axonal injury. The hypoglossal nerve is a useful model system for analysis of gene expression in injured motoneurons. In particular, we sought to determine whether the increased appearance of the low affinity nerve growth factor receptor (p75NGFr) observed immunocytochemically following nerve injury can be directly correlated to increased levels of the p75NGFr mRNA. The present study also examined the relative effects of nerve crush versus nerve transection on the expression of p75NGFr mRNA. In sham-operated or intact animals, p75NGFr mRNA is detected rarely and then only at levels slightly higher than background. Following unilateral transection or crush of the rat hypoglossal nerve, the levels of p75NGFr mRNA increase in a time dependent fashion that parallels the appearance of the protein as reported previously. Moreover, this increase in p75NGFr mRNA following transection is dependent on a signal from the injured site, since blockage of axonal transport with vincristine also blocks the increased p75NGFr mRNA levels. When comparing the effect of nerve crush to nerve transection, we observed that the intensity of the response was greater in the crush paradigm versus that observed following transection. The duration of the response following nerve crush was shorter than that observed following transection of the nerve. The increase in p75NGFr mRNA after crush was most robust 4 days postlesion and appeared more robust primarily due to a 90-150% increased number of motoneurons expressing p75NGFr mRNA when compared to nerve transection. These data suggest that nerve crush is more effective than nerve transection in eliciting increased p75NGFr mRNA levels.


Hypothalamic mechanism for cobalt protoporphyrin-induced hypophagia and weight loss: inhibition of the feeding response to NPY. The mechanism whereby neurally or peripherally administered cobalt-protoporphyrin (CoPP) leads to transient hypophagia and prolonged weight reduction in normal and genetically obese animals is unknown. Neuropeptide Y (NPY) is a known endogenous stimulator of feeding behavior and is elevated in the hypothalamus of food-deprived rats. Accordingly, we examined the interaction between CoPP and NPY in the central nervous system. Concentrations of NPY mRNA in the hypothalami of rats treated intracerebroventricularly with vehicle or CoPP responded to decreased food intake with comparable increases. However, intracerebroventricular infusions of NPY elicited increased intake of food in vehicle-treated rats but were without effect in CoPP-treated animals. The results suggest that CoPP acts, at least in part, by blocking the feeding response to NPY.


Age-related changes in the proportion of amyloid precursor protein mRNAs in Alzheimer's disease and other neurological disorders. In the human brain, alternative splicing of amyloid precursor protein (APP) gene transcript generates at least three types of mRNA coding for APP770, APP751 and APP695. The former two types harbor, but the latter one lacks a domain of Kunitz-type serine protease inhibitor (KPI). We studied, by using the RNase protection technique, the expression of APP mRNAs in brains of Alzheimer's disease (AD) and other neurological disorders with special reference to aging. We found that the ratio of (APP770 mRNA+APP751 mRNA)/APP695 mRNA in the frontal cortex increased approximately 1.5-fold in AD compared with other neurodegenerative or cerebrovascular disorders. The ratio in other neurological disorders did not change significantly from control even in their affected brain regions. On the other hand, we found a positive correlation between the ratio and age; the ratio (y) increased gradually with the advance of age (x) as expressed by y = 0.005x + 0.014 (r = 0.372) for the AD group, and y = 0.004x -0.037 (r = 0.486) for the non-AD group. These correlations indicate that the AD brain reached the same ratio of KPI-harboring to lacking APP mRNAs a few decades earlier than the non-AD brain in senescence. This finding of AD-specific and age-related change led us to the idea that a relative increase in KPI-harboring APPs over a KPI-lacking APP may perturb normal degradation of APPs, thereby leading to deposition of beta A4 protein as amyloid.


Basic fibroblast growth factor mRNA increases in specific brain regions following convulsive seizures. Basic fibroblast growth factor (bFGF) is a trophic factor synthesized in the central nervous system (CNS), where it is believed to play a role in neuronal maintenance and repair. Little is known about the regulation of this growth factor in the CNS. To determine whether the expression of the bFGF gene in the brain of adult animals changes in response to alterations of neuronal activity, we examined bFGF mRNA levels in several brain regions of rats experiencing focally-evoked convulsive seizures. Seizures were induced by microinjecting bicuculline unilaterally into an epileptogenic site within the deep prepiriform cortex, area tempestas (AT). By 5 h after initiation of brief limbic motor seizures from AT, there was a four fold increase in the levels of bFGF mRNA in the entorhinal cortex, hippocampus and olfactory bulb, but not in the caudate-putamen. The maximal expression of bFGF mRNA was reached by 10 h after seizure onset. In the same animals, the mRNA encoding nerve growth factor (NGF) was increased in entorhinal cortex and hippocampus, but not in the olfactory bulb. Our results demonstrate that neuronal activity can influence bFGF expression in an anatomically selective fashion and that acute changes in bFGF can occur in the uninjured mature brain. The increase in bFGF expression in response to excessive activation of specific neuronal circuitry may represent an adaptive response to protect against potential injury in those circuits.


Increased phosphorylation of elongation factor 2 in Alzheimer's disease. Elongation factor 2 (EF-2) is a phosphoprotein that mediates the translocation step of elongation during protein synthesis. We investigated its phosphorylation to characterize translational regulation of gene expression in Alzheimer's disease. EF-2 was identified on two-dimensional (2D) gels of brain homogenates by analyzing immunoblots with EF-2-specific antibody (M(r) 96,000; pI 6.8). Four distinct charge variant isoforms were observed. We identified the two most acidic isoforms as being the phosphorylated forms by incorporation of radiolabeled phosphate. The phosphorylation of EF-2 in control and Alzheimer's disease (AD) brain was directly measured as the distribution of the four polypeptides on silver stained 2D gels. The ratio of the phosphorylated forms to unphosphorylated forms was elevated 45% in AD homogenates compared to controls (1.07 +/- 0.18; n = 9 vs 0.73 +/- 0.20; n = 6; P less than 0.004) which indicated an increased phosphorylation of AD EF-2. The phosphorylation exhibited specificity to the disease in that it was observed in affected areas (cortex and hippocampus) but not in an unaffected area (thalamus) of the same brains. Because phosphorylation of EF-2 inhibits protein synthesis, the observed AD-associated phosphorylation of EF-2 is consistent with the reduced in vitro activity of polysomes isolated from AD tissues that we have previously reported.


Muscimol-induced reduction of GABAA receptor alpha 1-subunit mRNA in primary cultured cerebral cortical neurons. The expression of mRNA for GABAA receptor alpha 1-subunit in mouse cerebral cortical neurons in primary culture was examined using RNA blot analysis and ribonuclease protection assay following the treatment of neurons with muscimol, a selective agonist of GABAA receptor. The level of mRNA for GABAA receptor alpha 1-subunit showed a decrease in comparison with that in non-treated cells, whereas no changes in the level of beta-actin mRNA were noted under the same experimental conditions. This muscimol-induced reduction in GABAA receptor alpha 1-subunit mRNA was counteracted by the simultaneous exposure of neurons to both bicuculline, an antagonist of GABAA receptor, and muscimol. The expression of mRNA for GABAA receptor alpha 1-subunit also showed a decline by the treatment of cells with flunitrazepam alone, an agonist of benzodiazepine receptor, and this change was also abolished by the simultaneous exposure of cells to flunitrazepam and Ro15-1788, an antagonist for central benzodiazepine receptor. These results suggest that the continuous stimulation of cerebral GABAA receptor complex may induce the reduced expression of mRNA for the receptor complex.


Phospholipase C activation by neurotensin and neuromedin N in Chinese hamster ovary cells expressing the rat neurotensin receptor. The rat neurotensin receptor cDNA sequence was transfected in Chinese hamster ovary cells and cellular clones which stably express the corresponding protein were isolated and characterized. The Scatchard analysis of the specific binding of [3H]neurotensin indicated a Kd value of 0.45 +/- 0.08 nM and a Bmax value of 3.27 +/- 0.29 pmol/mg of protein. Displacement experiments using peptidic analogs of neurotensin and levocabastine confirmed that the transfected receptor exhibits the binding properties of the neurotensin receptor characterized in the rat brain. Neurotensin stimulated the phosphoinositides hydrolysis in a time- and concentration-dependent manner and this effect was mimicked by neurotensin(8-13) and by neuromedin N. The stimulation of phosphoinositides hydrolysis was not inhibited by pertussis toxin. These results indicate that the transfected cells actively express the rat neurotensin receptor which is functionally coupled to phospholipase C through a pertussis toxin-insensitive GTP-binding protein, and that neuromedin N is able to induce the phosphoinositides turnover by interaction with the neurotensin receptor.


Further evidence against a direct automatic neuromotor link between the ACL and hamstrings. The purpose of this experiment was to determine whether reflexive, or more broadly, automatic hamstrings excitation could be elicited during isometric, maximum effort, step increases in knee extension torque. Eight healthy subjects without lower extremity dysfunction or injury performed maximum effort isometric knee extension at 15 and 85 degrees of knee flexion in minimum elapsed time. Surface electromyography was used to record medial (semimembranosis and semitendinosis) and lateral (biceps femoris--long head) hamstrings excitation that was subsequently normalized to the excitation during maximum isometric knee flexion. To assess whether automatic hamstrings excitation was elicited, the amplitude of the EMG signals was analyzed and compared for 50 ms prior to peak knee extension torque, and three consecutive 50 ms windows following peak knee extension torque. The amplitude of the EMG subsequent to the peak knee extension torque failed to support the contention of an automatic hamstrings excitation mediated by neural circuitry from the ACL. The present results, in conjunction with previous work, suggest that a protective automatic hamstrings contraction is not normally elicited in response to conditions subjecting the anterior cruciate ligament to strain.


Acute exercise attenuates phenylephrine-induced contraction of rabbit isolated aortic rings. Factors associated with a single bout of dynamic exercise (increased circulating catecholamines, increased body temperature, and decreased pH) are known to attenuate the vascular response to alpha-adrenergic receptor activation. Therefore, we postulate that an acute bout of dynamic exercise may decrease the vascular response to catecholamines. To test this hypothesis, we evaluated contractile responsiveness to phenylephrine (PE) in aortae of two groups of New Zealand white rabbits, a control group (no exercise) and an exercise group (treadmill running, 24m.min-1; 16 +/- 2.0 min). Aortic rings were prepared from age-matched control (N = 6) and exercise rabbits (N = 5) and mounted for isometric tension recording (in Krebs-Henseleit-bicarbonate solution, 37 degrees C, 1.5 g passive tension). After equilibration (2 h) a cumulative concentration-response curve to PE (10(-7) M-10(-2) M) was obtained. The results demonstrate that a single bout of dynamic exercise attenuates (P < 0.05) the maximal contractile tension (2,457 +/- 120 vs 3,620 +/- 321 mg tension.mg-1 ring wt), gain (602 +/- 31 vs 878 +/- 87 mg.M-1 PE), and rate of contraction (6.2 +/- 0.3 vs 4.7 +/- 0.3 mg tension.s-1). In addition, contraction threshold was significantly increased in exercise (2.6 +/- 0.4 x 10(-6) M) vs control aortae (1.03 +/- 0.4 x 10(-6) M). A single bout of dynamic exercise did not alter the contractile response to 70 mM KCl (3,555 +/- 270 vs 3,083 +/- 233 mg tension.mg-1 ring weight). These data suggest that an acute bout of dynamic exercise significantly attenuates alpha-adrenergic receptor-mediated contraction of vascular smooth muscle.


Opioid receptor modulation of postexercise hypotension. Previous studies of both hypertensive and normotensive individuals have indicated a prolonged reduction in blood pressure for several hours after aerobic exercise. In related studies of spontaneously hypertensive rats, this postexercise hypotension has been prevented with naloxone. The purpose of the present investigation was to examined whether the postexercise hypotension may be reversed by antagonism of opioid sensitive receptors with naloxone in normotensive humans. Eight males 22-34 yr of age, participated in two 60-min cycling trials at 60% of VO2 peak, followed by 29 min of recovery. Beginning at 7-min recovery, naloxone or saline (control) was administered intravenously through an indwelling catheter. Blood pressure and heart rate were monitored every 15 min during exercise and every 2 min during recovery. Heart rate was significantly elevated (P < 0.05) over basal levels for the first 11 min of recovery, but from 13 to 29 min was not different from that measured at rest. In both trials, after 11 min of recovery, systolic and mean arterial blood pressures were significantly (P < 0.05) lower than pre-exercise levels (9 +/- 1 mm Hg and 4 +/- 1 mm Hg, respectively). Injection of naloxone (0.1 mg.kg-1) reversed the hypotensive response. However, the reversal was transient, lasting from minutes 15 to 27. Since naloxone reverses postexercise hypotension, opioid sensitive receptors appear to be involved in the reduction in systolic blood pressure following a single bout of submaximal exercise in normotensive humans.


Electrical stimulation-induced changes in skeletal muscle enzymes of men and women. The purpose of the present study was to determine the effects of low-frequency electrical stimulation (LFES) on the skeletal muscle metabolic profile of men and women. The knee extensor muscles of sedentary men (N = 16) and women (N = 10) were submitted to 3 h.d-1 of 8-Hz neuromuscular electrical stimulation with the use of a portable stimulator (Respond II, Medtronic), 6 d.wk-1 for 6 wk. Enzyme activity levels of creatine kinase (CK), hexokinase (HK), glyceraldehydephosphate dehydrogenase (GAPDH), 3-hydroxyacyl CoA dehydrogenase (HADH), citrate synthase (CS), phosphofructokinase (PFK), and cytochrome c oxidase (COX) were determined in vastus lateralis muscle samples taken before and after the LFES protocol. The analyses of variance revealed no change in CK and in GAPDH. However, a small decrease in PFK activity, the rate-limiting enzyme of glycolysis, was observed in female (8%) and in male subjects (10%), but it reached significance in males only (P < 0.05). The activity level of HK, a regulatory enzyme of the skeletal muscle glucose phosphorylation (HK), increased significantly in female subjects only (36%; P < 0.01) in response to the stimulation protocol. Activity level of marker enzymes of the Krebs cycle (CS) and of the electron-transfert chain (COX) significantly increased in males (18% and 16%; P < 0.05) as well as in females (31% and 19%; P < 0.05). Increment in the marker enzyme activity of the fatty acid oxidation (HADH) was significant in female subjects (30%; P < 0.01) and, although significant, rather modest in male subjects (12%; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)


Solid-state 31P NMR spectroscopy of bone and bone substitutes. Using magic angle sample spinning 31P NMR spectroscopy, we studied NMR parameters of hydroxyapatite, calcium-deficient hydroxyapatite, and beta-tricalcium phosphate. These data were compared to rabbit bone parameters. Rabbit bone spectrum is similar to deficient hydroxyapatite spectrum but high-power proton decoupling did not modify its linewidth. Moreover, both bone T1 and deficient hydroxyapatite T1 were much longer than pure hydroxyapatite T1 and beta-tricalcium phosphate T1. We used these results to develop quantitative analysis of newly deposited bone inside beta-tricalcium phosphate implants in rabbit. This method made it possible to demonstrate the effect of porosity on osteoinduction of biomaterials.


1/T1 rho and low-field 1/T1 of tissue water protons arise from magnetization transfer to macromolecular solid-state broadened lines. We argue that, for solutions of immobilized protein and for tissue, the dependence of 1/T1 of solvent protons on B(0) at low fields and 1/T1 rho on B1 for all B(0) are both manifestations of the same underlying phenomena: magnetization transfer between mobile water protons and solid-state broadened protein proton levels. Broadening causes rapid mixing of spin orientation within the transverse plane, at all B(0), unless B1 is greater than the protein internal field; this affects 1/T1 rho of solvent protons by magnetization transfer. Similarly, decreasing B(0) below the internal field mixes all orientations of magnetization, which affects the solvent proton low-field 1/T1 and high-field 1/T2.


[Surveillance of the sensitivity of antibiotics to the principal germs responsible for purulent meningitis at the University Hospital Center in Treichville from 1986 to 1991]. Between January 1986 and December 1991, the sensitivity of germs isolated from purulent meningitidis diagnosed at the Universitary and Hospital Center Treichville was tested with reference to the antibiotics utilized in the treatment of purulent meningitidis (Ampicillin, cefotaxime, amoxicillin+clavulinic acid, chloramphenicol, gentamicin and Trimethoprim-sulfamethoxazole)). Cefotaxime, cephalosporin of the third generation, keeps its effectiveness on the main germs as a whole (Pneumococcus, Meningococcus, haemophilus influenzae). On the contrary, the other antibiotics undergo some fluctuations, or their activities are declining year after year. As far as etiology is concerned, pneumococcus always takes the first place, then come Haemophilus influenzae and Meningococcus.


SJL/J resistance to mouse hepatitis virus-JHM-induced neurologic disease can be partially overcome by viral variants of S and host immunosuppression. The basis of the resistance of SJL/J mice to various strains of mouse hepatitis virus (MHV) has been the subject of some debate, especially as it relates to the number and nature of the determinants involved. Our previous work demonstrated that resistance by primary SJL/J glial cultures may involve events subsequent to viral gene expression, possibly due to a defect in cell-to-cell spread of the infection. Since S, the virion's major spike glycoprotein, is known to facilitate the spread of infection due to its syncytiogenic properties, we decided to investigate the role of this viral structural protein in resistance by primary SJL/J glial cells. Variants possessing deletions within the S coding region were able to grow in SJL/J glial cells 10-100 times easier and fuse five-times more efficiently than wt virus. Induction of neurologic disease in SJL/J mice following intracranial inoculation with either wt JHMV or the S deletion variant, AT11f cord, was age-dependent, occurring only in animals inoculated under 4 weeks of age. Resistance in older animals to wt and variant viruses could be abrogated by immunosuppression with cyclosporin A. However, both disease incidence and viral brain titers were higher in animals receiving the JHM variant AT11f cord virus, suggesting that SJL/J resistance to neurologic disease may manifest itself through interactions between inefficient cell-to-cell spread of the infection and protective aspects of the immune response.


Attenuated deletion mutant of vaccinia virus IHD-W recovered virulence by reinsertion of a terminal restriction fragment. Since vaccinia virus is being considered as a potential immunization vector, it is important to identify genes responsible for pathogenicity. One approach to identify virulence genes is the use of attenuated deletion mutants with a null background to reintroduce regions of the deleted genome material. We have previously described an attenuated deletion mutant of vaccinia virus strain IHD-W (Z-19) that lost 21 kb from the left terminus comprising fragments HindIII C and N, displays rearrangements at the right terminus and is unable to grow in vivo. To establish whether the loss of genes present at the left terminus is the basis for the attenuated phenotype, rescue experiments were performed to reintroduce fragment HindIII C, using growth in vivo as a selection system to isolate recombinant viruses. Several recombinants were isolated and molecularly and biologically characterized. The results indicate that recovery of virulence in mice was correlated with the presence and expression of two genes: vaccinia growth factor and C4b binding protein, which are located at the left terminus. Serpin I and II genes, located at the right terminus, were not affected in Z-19 and seem not to play a role in virulence in the IHD-W strain; interestingly, a copy of the serpin I gene was found at the left terminus. Moreover, a gene involved in virulence in the WR strain, the 13.8 kDa virokine gene (N1L) was found to be located in fragment HindIII G in the IHD-W strain, and therefore did not contribute to the attenuated phenotype.


[Prevalence of asthma and respiratory diseases in schools in Bouake (Ivory Coast): preliminary results]. Using an auto-administered questionnaire, a cluster sample survey was carried out in January 1990 among 2433 secondary schoolchildren in Bouake (Cote d'Ivoire). The prevalence rate of asthma was fairly high (10.8%), as well as that of wheezing (19.8%), dyspnea (42.8%), broncho-pulmonary diseases (25.8%) and smoking habit (14.5%). The results are discussed with regard to the published data.


Effect of porphyrins and host iron transport proteins on outer membrane protein expression in Porphyromonas (Bacteroides) gingivalis: identification of a novel 26 kDa hemin-repressible surface protein. Porphyromonas gingivalis is capable of in vitro growth when iron sources are either complexed to hemin or host iron transport proteins, or exist in an inorganic form. This study examined the effect of these iron sources on outer membrane protein (OMP) expression in P. gingivalis W50. Hemin (iron) starved P. gingivalis was transferred into growth medium containing hemin, hemoglobin, hemin-saturated human serum albumin, hemin-free human serum albumin, transferrin, lactoferrin, or inorganic iron. Surface proteins were identified by 125I-labeling and resolved by SDS-PAGE and autoradiography. When grown under hemin starved conditions, P. gingivalis W50 and related strains expressed a major 26 kDa OMP, as revealed by 125I-autoradiography. Autoradiographic analysis demonstrated the absence of this 26 kDa OMP from the P. gingivalis surface in hemin-containing environments. Growth of P. gingivalis W50 in the presence of host iron transport proteins (hemin-free) or inorganic iron resulted in surface expression of a 26 kDa OMP. The presence of protoporphyrin IX or substitution of hemin-associated iron with zinc, resulted in continued surface expression of the 26 kDa OMP, indicating that repressibility of this OMP required the coordination of iron to the protoporphyrin IX molecule (i.e. hemin). A survey of 125I-labeled OMPs from several hemin starved P. gingivalis and related strains, demonstrated that a hemin-repressible 26 kDa OMP occurred only in P. gingivalis. We report here a newly described 26 kDa hemin-regulated surface protein occurring in several strains of P. gingivalis which is expressed on the cell surface in hemin starved conditions and is lost from the cell surface in response to an environment containing iron coordinated specifically to protoporphyrin IX (i.e. hemin).


Horizontal transfer of hobo transposable elements within the Drosophila melanogaster species complex: evidence from DNA sequencing. The hobo family of transposable elements, one of three transposable-element families that cause hybrid dysgenesis in Drosophila melanogaster, appears to be present in all members of the D. melanogaster species complex: D. melanogaster, D. simulans, D. mauritiana, and D. sechellia. Some hobo-hybridizing sequences are also found in the other members of the melanogaster subgroup and in many members of the related montium subgroup. Surveys of older isofemale lines of D. melanogaster suggest that complete hobo elements were absent prior to 50 years ago and that hobo has recently been introduced into the species by horizontal transfer. To test the horizontal transfer hypothesis, the 2.6-kb XhoI fragments of hobo elements from D. melanogaster, D. simulans, and D. mauritiana were cloned and sequenced. The DNA sequences reveal an extremely low level of divergence and support the conclusion that the active hobo element has been horizontally transferred into or among these species in the recent past.


Gene cassettes from the insert region of integrons are excised as covalently closed circles. Integrons are DNA elements which generally include one or more discrete gene cassettes inserted at a specific site. We have recently proposed a model for the acquisition and dissemination of genes found in the insert region of integrons, which requires the existence of circularized gene cassettes. Evidence for the existence of covalently closed circular molecules consisting of one or more gene cassettes has now been obtained. Low levels of small molecules which hybridize to probes specific for individual gene cassettes were detected in plasmid DNA isolated from cells containing a plasmid which includes an integron fragment with three gene cassettes aacC1, orfE and aadA2. These molecules were only detected when the gene encoding the integron DNA integrase was also present and are thus products of site-specific cassette excision. The excised cassettes have been shown to be in the form of covalently closed supercoiled circles, by digestion with restriction enzymes exonuclease III and DNase I. The circular excision products detected included either one cassette, aadA2 or orfE, two cassettes, aacC1 and orfE or all three cassettes. The predicted sequence of the recombinant junction in the excised aadA2 cassette confirmed that excision was precise. The predicted unique sequences of the 59-base elements associated with individual genes in the circular cassette form were compiled, and the sequences of the seven-base core sites which flank 59-base elements are now, with few exceptions, exact inverted repeats.


Elevation of blood vitamin D2 levels does not impede the release of vitamin D3 from the skin. The mechanism for the transfer of fat-soluble vitamin D3 from the avascular basal cellular layers of the epidermis to dermal capillaries and peripheral circulation is unknown, although vitamin D-binding protein (DBP) is thought to mediate this process. To evaluate the effect of increased occupancy of vitamin D carrier(s) on vitamin D3 removal from the skin, serial serum vitamin D2 and D3 concentrations were determined in three groups of six healthy volunteers given combinations of an oral dose of vitamin D2 (50,000 IU) and a fixed dose of UVB radiation (27 mJ/cm2). Serum vitamin D3 levels increased significantly following UVB (time effect, P < .01 by ANOVA), but the response remained unchanged after pretreatment with vitamin D2, increasing from 3 +/- 1 to 14 +/- 5 ng/mL (mean +/- SEM), versus UVB alone, 5 +/- 1 to 16 +/- 5 ng/mL. Elevation of serum vitamin D2 levels was also similar in the groups given vitamin D2 alone (< 1 to 64 +/- 8 ng/mL) and vitamin D2 + UVB (< 1 to 45 +/- 8 ng/mL). There was no time or treatment effect for changes in serum levels of 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, or (DBP) levels (P > .1). We conclude that vitamin D3 egress from the skin is not affected by elevated circulating vitamin D concentrations; thus, the cutaneous release of vitamin D is probably mediated by a protein such as DBP with a high carrying capacity for the vitamin.


Platelet factor Xa receptor.

Platelet factor Xa receptor. The assembly and function of the prothrombinase complex on the bovine and human platelet membrane is mediated through binding interactions in which factor Va bound to the platelet surface forms at least part of the "receptor" for factor Xa in a 1:1 stoichiometric complex. A model depicting these binding interactions is shown in Fig. 12. Data from our laboratory indicate that the prothrombinase catalyst assembles in an analogous manner on the surface of monocytes, lymphocytes, neutrophils, and well-defined phospholipid vesicles employed in model systems. The 74,000-Da subunit of factor Va, component E, which mediates the binding of factor Va to either bovine platelets, human monocytes, or phospholipid vesicles, is shown binding to the cell membrane through its putative "receptor." The 94,000-Da subunit of factor Va, component D, is associated with the membrane surface through its metal ion-dependent interaction with component E. Factor Va forms at least part of the receptor that mediates the binding of factor Xa to an appropriate membrane surface, because component E has been shown to contribute significantly to the interaction of factor Xa with either the platelet, monocyte, or vesicle membrane surface. Our data do not preclude the possibility that component D contributes to the binding of factor Xa and the function of the prothrombinase complex. Component D appears to be important for several reasons. Cleavage of component D by activated protein C results in the complete loss of factor Va cofactor activity. An interaction between factor Xa and component D is implied from the observation that factor Xa protects factor Va from activated protein C inactivation. Furthermore, the binding of factor Xa to platelet-bound factor Va results in the time-dependent cleavage of components D and D'. Because component D is not required absolutely for prothrombinase complex assembly, we would speculate that it may be important in mediating prothrombin binding (depicted as a three-domain molecule) and increasing the catalytic efficiency of the enzymatic complex.(ABSTRACT TRUNCATED AT 400 WORDS)


Absence of HTLV-I/II infection in blood donors with positive and inconclusive HTLV-I/II serology. The pathogenetic potential and the true extent of human T leukemia/lymphotropic virus type I (HTLV-I) and type II (HTLV-II) infection are unknown. To find out more about HTLV-I/II seroepidemiology and the risks of iatrogenic transmission, we performed a serological study, screening 4086 healthy blood donors. A surprisingly high percentage of serum reactivity to HTLV-I/II antigens was observed by commercial ELISA (2.08%) and immunoblotting (IB) (0.85%) analysis, although none of the samples satisfied the (IB) criteria for positivity based on detection of gag protein p24 and at least one env gene product, either gp46 or gp61/68. To clarify these inconclusive results, we performed polymerase chain reaction (PCR) analysis for HTLV-I and HTLV-II provirus detection in peripheral blood lymphocytes, obtained from individuals with an apparent pattern of seropositivity. The data obtained by PCR failed to reveal evidence of HTLV-I/II provirus integration in peripheral blood cells, ruling out the possibility of a viral infection in these cases, and pinpointing the limitations of both serological methods used. Our observations suggest that serological assays alone are not a reliable tool for blood donor screening of HTLV-I/II infection and raise the important question of interpreting inconclusive results.


Role of phospholipids in BK virus infection and haemagglutination. The role of phospholipids in BK virus infection and haemagglutination was studied by competition binding experiments and by treatment of susceptible cells with phospholipases. Phospholipids extracted from Vero cells and some commercial phospholipids showed an inhibiting activity on both BK virus infectivity and haemagglutination. The treatment of Vero cells with phospholipases affected the binding of BK virus, but the addition of phospholipids to enzyme-treated cells restored their susceptibility to both viral infectivity and haemagglutination.


The response of pregnant gilts previously given an inactivated preparation of porcine parvovirus (PPV) to challenge infection with a fully virulent PPV. Eight 40-day pregnant gilts, previously treated with an adjuvanted-inactivated viral preparation (AIVP) obtained with a field strain of porcine parvovirus (PPV) together with 4 pregnant untreated controls, were subjected to challenge infection with a virulent strain of PPV at the 40th day of gestation. After challenge, all controls became febrile for 2 to 8 days, whereas only one gilt among those which had been treated with the AIVP experienced fever which lasted 4 days. Virus was consistently recovered from fecal swabs obtained from the controls and only sporadically from feces of AIVP-treated gilts. When the gilts were killed 53 days after challenge infection, no macroscopic lesions were found in any of the gilts in either groups, but fetal death was observed in the two groups of animals. However, the rate of dead fetuses was much higher among the control (70.5%) than among those from the AIVP-treated gilts (10.1%). Virus was recovered from 23 of the 24 dead fetuses in the control group and only from 3 among the 8 dead fetuses which were reported for the AIVP-treated gilts.


A mechanism involved in the plaque enhancement effect of sodium thiosulfate for foot-and-mouth disease viruses. A mechanism involved in the plaque enhancement effect of foot-and-mouth disease viruses (FMDV) by the addition of sodium thiosulfate (Hypo) in the agar overlay medium (AOM) previously reported was studied. It was experimentally proved that the diffusion of virus particles through agar overlay medium was enhanced when this salt was incorporated. Accordingly, the enlarged plaque formation was assumed to be caused by the enhanced diffusion of viral progenies produced in infectious centers during plaque assays.


Evaluation of three methods for the concentration of poliovirus from oysters. Three methods for the concentration of poliovirus from oyster homogenates were compared. The adsorption-elution-precipitation method gave the lowest average virus recovery (24.1%), while the beef extract elution-acid precipitation method and the non-fat dry milk elution-acid precipitation methods gave recoveries of 47.2% and 39.6%, respectively. Although the overall recovery rates with these methods were lower than those reported in previous studies, recoveries of 40-47% obtained with the elution-precipitation methods used in the present study are considered to be above average in terms of recovery efficiency.


[Apparent simultaneous occurrence of hepatitis A and hepatitis B infections]. In this study, 92 patients sera which were found to be HBsAg positive by ELISA method were investigated for hepatitis A. In this study Anti-HAV IgM antibody was investigated to determine acute cases. Anti-HAV IgM was found to be positive in 3 (3.2%) of 92 patients sera whose HBsAg were positive.


[Distribution of HSV-1 and HSV-2 antibodies in pregnant women]. In this study, the distributions of Anti-HSV 1 and HSV 2 IgG and IgM antibodies have been investigated in sera obtained from 296 pregnant women who were admitted to the gynecology clinics of Medical Faculty of Atatürk University, Erzurum, Turkey. While the 24 (8.11%) of 287 HSV 1 IgG antibody positive patients were IgM positive but Anti-HSV 1 IgM alone was not detected in any patients. On the other hand, in 16 (5.41%) of 125 (42.23%) patients who were Anti-HSV 2 IgG antibody positive, HSV 2 IgM antibodies were detected too. In the 4 patients (1.35%), HSV 2 IgM antibodies were detected alone.