Biomedical subjects
C Bernard
Publications and source records attributed to C Bernard.
Multiple promoters of human choline acetyltransferase and aromatic L-amino acid decarboxylase genes.
The promoter regions of human choline acetyltransferase (ChAT) and aromatic L-amino acid decarboxylase (AADC) genes have been analyzed by transient transfection assays. AADC gene is transcribed from two alternative noncoding first exons, 1N and 1NN, expressed in pheochomocytoma and hepatoma cells, respectively. 5' flanking sequences of exon 1 N (from 9000 to 147 bp) display promoter activity in SK-N-BE neuroblastoma cells, but not in MC-I-XC cholinergic neuroepithelioma cells, and in AADC-rich non-neuronal cells. On the contrary, 5' flanking sequences of exon 1 NN (from 1117 to 119 bp) display high promoter activity in human hepatoma cells HepG2, but not in SK-N-BE cells, suggesting high degrees of specificity of promoters N and NN for AADC-expressing neuronal and non-neuronal cells, respectively. Preliminary evidence suggests that leukemia inhibitory factor suppresses the activity of the neuronal promoter in cultured sympathetic neurons. Two alternative first exons, R and M, have been localized in human ChAT gene, and the corresponding promoters characterized in cholinergic PC12 and NG-108-15 cells, and in non-cholinergic neuro2A cells. Several positively or negatively acting cis elements have been localized in the two promoters, as well as a cAMP-inducible, enhancer-like element in the second intron. Among the various cell lines studied, there was no correlation between promoter activities and the expression of the endogenous ChAT gene, suggesting that the fine-tuning of ChAT gene expression is controlled by silencer elements which remain to be localized.
Evaluation of Biohit HPV Screening and Typing kits in detection of human papillomavirus DNA from lesions of anogenital tract.
The Biohit HPV Screening and Typing kits for in situ hybridization of human papillomavirus (HPV) DNA are now commercially available. The HPV Screening kit contains a cocktail of HPV probes, and the Typing kit contains separated hybridization probes for HPV 6, 11, 16, 18, 31, and 33. They were evaluated by comparison with an in situ hybridization (ISH) method, using the Pathogene HPV probes 6/11, 16/18, 31/33/51. One hundred anogenital biopsies from 78 women and 22 men were tested. Among them, 43% showed normal or inflammatory mucosa, 44%, koilocytosis or mild dysplasia, and 13%, moderate to severe dysplasia. Altogether, 60 specimens were positive with the ISH reference method: 17 with the HPV 6/11 probe, 12 with the HPV 16/18 probe, 16 with the HPV 31/33/51 probe, and 15 had mixed infections. The agreement between the Screening test and the homemade ISH is 91%. The Screening test has a sensitivity of 93% and a specificity of 87%. As for the Biohit Typing test, four false-negative samples, and partial or total discordance in nine and four samples, respectively, were observed when compared to our reference method. Thus the agreement between both typing ISH tests is 92%. The sensitivity of the Biohit Typing test is 93%, and the specificity, 91%. The sensitivity decreases to 72% when the 31 and 33 probes are evaluated separately. The Biohit Screening assay is simple, reliable, reproducible, and suitable for rapid routine screening. The Biohit Typing test allows the detection of a specific type of HPV DNA and also permits, in mixed HPV infection, definition of the type of associated HPV DNA.(ABSTRACT TRUNCATED AT 250 WORDS)
A ompR gene in the plastid genome of Rhodella violacea.
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Implication of cholecystokinin in pancreatic adaptation after biliopancreatic bypass in the rat.
Biliopancreatic bypass (BPB), a bariatric surgical procedure, leads to a malnutrition-induced general visceral atrophy except for the pancreas. This work investigates the implication of cholecystokinin (CCK) in the exocrine pancreatic adaptive process using a plasma CCK assay and the CCK receptor antagonist CR 1409. No significant reduction in weight and DNA content of the pancreas was noted 36 days after BPB, while a strong decrease in protein, enzymes and RNA contents indicating cellular hypotrophy became apparent. CR 1409 treatment strongly depressed pancreatic weight and its DNA content in BPB animals, suggesting an additional hypoplasia; however, the reduction in pancreatic enzyme content was not aggravated. BPB increased plasma CCK concentrations by 160%, unrelated to CR 1409 treatment. These results indicate that: (1) CCK is involved in the pancreatic adaptive response after BPB in rats, and (2) in the context of a protein malnutrition state, CCK dissociates its pancreatic growth and enzymatic effects, favouring the former.
Stimulation of glucagon-like peptide-1 secretion by muscarinic agonist in a murine intestinal endocrine cell line.
Studies on the cholinergic regulation of intestinal L-cells have been focused on the release of enteroglucagon, but the signal transduction pathways were not defined. These were here investigated by using as index the release of immunoreactive glucagon-like peptide-1 (GLP-1) from the endocrine cell line STC-1, that has been shown to contain proglucagon mRNA transcripts. Abundant GLP-1 immunoreactivity was revealed in STC-1 cells at immunocytochemistry and by RIA. The cell content was 4927 +/- 689 pg/10(6) cells, as measured with antiserum 199D that recognizes specifically the C-terminal amidated forms of GLP-1. The secretion of GLP-1 over a 2-h incubation period amounted to 1.4 +/- 0.3% of the total GLP-1 cell content and was significantly increased by 10 microM forskolin and 100 nM 12-O-tetradecanoylphorbol 13-acetate to 206% and 574% of control values, respectively. The cholinergic agonist carbachol stimulated GLP-1 secretion in a concentration-dependent manner, maximal release was observed at 1 mM carbachol (228% of the control value). Binding of the muscarinic antagonist [N-methyl-]scopolamine ([3H]NMS) on cell homogenates was time dependent, specific, and saturable. Scatchard analysis revealed one class of receptors (Kd, 14 pM; binding capacity, 20 fmol/mg protein). Carbachol (0.1 microM to 1 mM) dose dependently displaced [3H] NMS binding and increased the intracellular calcium concentration without modification of adenylate cyclase activity. The order of potency of different antagonists, showing a preferential affinity for M1, M2, and M3 muscarinic receptor subtypes, to inhibit [3H]NMS binding, the carbachol-induced increase in intracellular calcium, and carbachol-stimulated GLP-1 secretion, was as follows: atropine (nonselective) > 4-diphenylacetoxy-N-methylpiperidine methiodide (M3) > pirenzepine (M1) > AF-DX 116 (M2). The results of the present study, therefore, demonstrate that secretion of GLP-1 induced by cholinergic agonist depends on muscarinic M3-subtype receptors in the endocrine intestinal cell line STC-1. This system may prove useful to study the cellular mechanisms of GLP-1 secretion.
Regulation of glucagon-like peptide-1-(7-36) amide secretion by intestinal neurotransmitters and hormones in the isolated vascularly perfused rat colon.
Glucagon-like peptide-1 (GLP-1) is promptly released from endocrine cells of the distal part of the gut after oral ingestion of a meal. To test the possibility that hormones produced by the proximal small intestine or transmitters of the enteric nervous system may be involved in the early phase of meal-induced GLP-1 secretion, various intestinal regulatory peptides and neurotransmitters of the gut were administered intraarterially in the isolated vascularly perfused rat colon preparation. The release of GLP-1 in the portal effluent was measured by a specific RIA. Intraarterial infusion of glucose-dependent insulinotropic peptide (GIP) over the concentration range 0.25-1 nM evoked a dose-dependent release of GLP-1, with a maximal response of 350% of the basal value. Tetrodotoxin did not modify the GIP-induced release of GLP-1. Secretin or cholecystokinin did not stimulate the secretion of GLP-1. Bombesin (10(-9)-10(-7) M) provoked a dose-dependent release of GLP-1, consisting of an early peak, followed by a sustained response. Calcitonin gene-related peptide (5 x 10(-8) M) induced a dramatic rise of GLP-1 immunoreactivity in the portal effluent (peak at 800% of the basal value 10 min after the start of infusion). Similarly, the beta-adrenergic agonist isoproterenol at concentrations of 10(-7) and 10(-6) M provoked a pronounced release of GLP-1 (peak at 500% of the basal value with 10(-6) M isoproterenol). Finally, the muscarinic cholinergic agonist bethanechol at a concentration of 10(-4) M evoked a gradual increase in GLP-1 immunoreactivity, which reached a maximal value (900% over basal) at the end of the 30-min infusion period. The lowest concentration of bethanechol used in the present study (10(-5) M) did not increase portal GLP-1 immunoreactivity over the basal value. Tetrodotoxin did not modify the bethanechol-, isoproterenol-, calcitonin gene-related peptide-, or bombesin-induced GLP-1 release. In conclusion, the present study conducted with the isolated vascularly perfused rat colon shows that there are interactions between the two most potent incretins, GIP and GLP-1, probably through an enteroendocrine pathway. Additionally, several transmitters of the gut are potent stimulants of GLP-1 release and, therefore, represent potential tools in the treatment of the noninsulin-dependent diabetes mellitus.
Higher maternal than paternal inheritance of diabetes in GK rats.
Results from crosses between Goto-Kakizaki (GK) rats, which exhibit spontaneous non-insulin-dependent diabetes mellitus (NIDDM), and outbred nondiabetic Wistar rats have demonstrated an effect of maternal inheritance on diabetes in offspring of the first generation (F1). At 6 weeks of age, F1 offspring of sex-directed crosses exhibited plasma glucose values intermediate between GK and Wistar parents. Hyperglycemia in F1 rats born of female GK rats (F1GK) was more marked than in those born of female Wistar (F1W) rats. At 3 months of age, F1 rats showed a marked impairment of both glucose tolerance and insulin secretion, which was intermediate between GK and Wistar rats. Glucose intolerance was more pronounced in F1GK rats than in F1W. By contrast, insulin secretion in F1W rats was more deteriorated than in F1GK rats. No deletion in mitochondrial DNA was observed in the GK rats, which decreased the possibility of a mitochondrial inheritance effect as an explanation of our findings. These data support a polygenic model in diabetes inheritance of NIDDM and suggest that, in addition to genetic factors, a perturbed maternal metabolism can contribute to its inheritance.
Oncogene activation: an informative marker for the human papillomavirus-lesions severity.
The aim of this study was the detection of c-Ha-ras and c-myc activation and p21ras and p62myc expression in anogenital HPV-associated lesions. Thirty-two women with cervical and vaginal lesions and forty men with anal or penile warts were enrolled for the study. Biopsies were subjected to histological assessment, in situ hybridization with biotinylated probes, and immunohistochemistry. In both women and men, most of the biopsies showing no major histological abnormalities were negative for HPV detection. The low-grade squamous intraepithel-)ial lesions (SIL) harbored HPV in more than 50% of cases. The high-grade SIL contained mostly oncogenic HPV. C-Ha-ras and c-myc activation was detected in 31/41 HPV-positive lesions, especially in lesions harboring HPV 31/35/51 or multiple HPV. Simultaneous activated oncogenes were found in 7 out of 9 high-grade SIL and in 21 out of 45 low-grade SIL. In most of the lesions, we demonstrated concurrent detection of p21ras and p62myc proteins and oncogene activation. Our study shows that c-Ha-ras and c-myc activation is correlated with oncoprotein expression and associated with the presence of oncogenic HPV. Such activation might play a role in the development of high-grade anogenital dysplasia.
Finite-size and quark mass effects on the QCD spectrum with two flavors.
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Effects of recurrent collateral inhibition on Purkinje cell activity in the immature rat cerebellar cortex--an in vivo electrophysiological study.
We present here a study on the effects of inhibitory recurrent collaterals of Purkinje cell (PC) axons on the activity of the immature rat cerebellar cortex. Simultaneous extracellular recordings of pairs of PCs were performed in rat pups aged 5-8 days postnatal. Bicuculline was applied to the surface of the cortex in order to functionally antagonize PC recurrent collaterals. At this early developmental stage these are the only inhibitory links in the network. Dye marks from the microelectrode tips and 3D serial-section reconstruction of the structure allowed the exact determination of the distance separating recorded cells and of their respective orientation in the cortex. Standard statistical tests and an informational entropy index were used to calculate levels of cooperativity. By comparing PC activity under control conditions and after bicuculline superfusion it is shown that recurrent collateral inhibition has a structurating effect on the PC activity and that it increases the informational content of the network. Inhibition decreases the activity of the cells by 35% and drastically changes the interspike interval histograms. This leads to a more constrained state of the system. Three types of coupling via recurrent collaterals are present: symmetrical, asymmetrical or non existent. The exact type of coupling follows a simple vicinity rule and strongly influences the cooperativity level between the recorded cells. This cooperativity was also found to be spatially compartmentalized. Several pairs were driven by common inputs via climbing fibers or parallel fibers. Using the predictive value of a theoretical model of this immature structure we propose a complementary explanation of the role of the recurrent collaterals at this stage of development: that of a spatial and temporal filter, specific to each different microzone.
Effects of modifications of extracellular and intracellular calcium concentrations on the bioelectrical activity of the isolated frog semicircular canal.
In the inner ear, calcium appears to play a major role in different processes including mechanoelectrical transduction, adaptation during prolonged stimulation and electrical resonance. The aim of the present study was to investigate the effect of an increase of the cytosolic calcium content and a reduction of the perilymphatic concentration of calcium, on the bioelectrical activity of the isolated frog semicircular canal. Under resting conditions, the spontaneous activity of the afferent fibers and the difference of potential between the endolymphatic and perilymphatic compartments, called endolymphatic potential, were recorded. When the sensory epithelium was mechanically stimulated three additional parameters were investigated: the variations of the endolymphatic potential (ampullar direct current), the variations of the ampullar nerve potential (nerve direct current) and the frequency of the evoked afferent spikes. Increase of the intracellular calcium concentration by administration of the calcium ionophore A23187 (3 x 10(-6) M, 20 min) into the perilymphatic compartment, caused a biphasic effect on the spontaneous activity of the ampullar nerve which increased rapidly, reaching a maximum within 15 min, and then gradually declined to stabilize at 74% of the control 1 h after withdrawal of A23187. A23187 did not induce any modifications of the endolymphatic potential, the ampullar direct current or the frequency of the evoked afferent spikes. In contrast, A23187 induced a significant reduction of the nerve direct current which decreased by 31% of the control 1 h after withdrawal of the ionophore. Gradual reduction of the perilymphatic concentration of calcium (from 2 to 1 mM) induced a dose-dependent increase of the spontaneous activity of the ampullar nerve and the frequency of the evoked afferent spikes. Reduction of the perilymphatic calcium concentration from 1.6 to 1.2 mM caused a transient increase of the endolymphatic potential, while 1 mM Ca2+ induced a decrease to 88% of the control. The nerve direct current slightly increased for calcium concentrations ranging from 1.8 to 1.4 mM and decreased in the presence of 1.2 mM CaCl2. These data suggest that an increase of calcium into the cytosol induces an alteration of the mechanisms responsible for the spontaneous release of the afferent neurotransmitter and the electrogenic spreading of the postsynaptic potentials. In contrast, an excess of calcium does not impair the mechanisms involved in the generation of the action potentials. Our results also suggest that reduction of the perilymphatic calcium concentration may lead to modifications of the physical and electrical properties of the cell membranes of the labyrinthine epithelium and/or the ampullar afferent fibers.
Effects of collateral inhibition in a model of the immature rat cerebellar cortex: multineuron correlations.
A model of the immature rat cerebellar cortex is used to simulate the effect of the inhibitory recurrent collateral axons of the Purkinje cells on the spike trains in the network. Inhibition induces an important overall change in the statistical characteristics of individual spike trains. It is also instrumental in producing a strong cooperativity between the different neurons. Moreover, a functional spatial anisotropy appears. A specific entropy index is used to analyze levels of information transfer between clustered and faraway neurons in the network. The formatting effect of recurrent collateral inhibition on spike trains and on network functional dynamics is studied by means of a model of the newborn rat cerebellar cortex. This immature structure has simpler morphological characteristics and fewer physiological parameters than the adult one. It is thus a good candidate for the comparison between experimental and theoretical data. The model network is made of 256 formal neurons (FN), arranged in a square lattice. Each neuron is coupled to its eight nearest neighbors by inhibitory links. All the parameters of the different elements of the model--in particular integration of inhibitory and excitatory inputs--are given anatomical and physiological values derived from biological data. Activities of single FNs and correlations between spatially distant ones are analyzed with classical statistical techniques as well as with a specific informational entropy method we introduce. Simulation results indicate that inhibition is instrumental in: (1) the transformation of the spike train characteristics. This includes a lengthening of the mean interspike interval as well as an overall change in the statistical distribution of intervals, with an emergence of long-lasting ones; (2) the functional structuration of the network. Inhibitory connections between nearest neighbors induce a strong cooperativity between FNs. Furthermore a clear spatial anisotropy occurs in the functioning of the network, with inhibitory effects extending beyond local connectivity in preferential directions. We propose an interpretation of this functional structuration in terms of the various routes followed by the inhibition, including relay effects. The parameters of the model (levels of activities, inhibition rules and connectivities) were varied in order to test the robustness of the above results. Finally, the results are compared with those obtained in an experimental situation.
N-ethylmaleimide-inhibited electrogenic K+ secretion in the ampulla of the frog semicircular canal.
1. The mechanisms of K+ secretion into endolymph were studied on a preparation of isolated semicircular canal with different pharmacological inhibitors. Three periods of 5 or 30 min were performed, the first as control, the second in the presence of the drugs added to the apical or the basolateral bathing solution, and the third as recovery. Apical fluid was sampled at the beginning and the end of each period, transepithelial potential was recorded, Na+, K+, and Cl- concentrations, and K+ efflux, with 86Rb+ as a tracer, were measured and K+ fluxes were calculated. 2. When both sides of the epithelium were bathed with perilymph-like solution, the epithelium absorbed Na+, secreted K+, and generated a lumen positive potential. 3. The ATPases inhibitors, ouabain (10(-5) and 10(-3) M) and N-ethylmaleimide (10(-4) and 10(-3) M) inhibited the electrogenic K+ secretion when added to the basolateral fluid. N-ethylmaleimide (10(-3) M) applied to the apical fluid during a 5 min period decreased the K+ influx by 43% and the transepithelial potential by 66%. Other ATPase inhibitors, harmaline (10(-3) M), omeprazole (10(-4) M), vanadate (10(-4) M and 10(-3) M), N,N'-dicyclohexylcarbodiimide (DCC, 10(-5) M), 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl, 5 x 10(-6) M and 5 x 10(-5) M), and bafilomycin (10(-7) M) did not affect the K+ transport nor the transepithelial potential when they were added to the apical fluid. 4. The Na(+)-K(+)-Cl- co-transporter inhibitor, bumetanide, decreased both the transepithelial potential and the K+ transport when added to the basolateral solution but not to the apical one. At 10(-6) M, bumetanide maximally decreased the K+ influx by about 60%. 5. K+ channel blockers, quinine (10(-4) M), TEA (5 x 10(-3) M), added to the apical solution and barium (2 x 10(-3) M) added to either the apical or the basolateral solutions, did not affect the K+ transport and the transepithelial potential. 6. The carbonic anhydrase inhibitor acetazolamide (10(-3) M) added to both apical and basolateral solutions did not affect the K+ transport and the transepithelial potential. 7. It is concluded that, in the ampulla of the semicircular canal, a basolateral Na(+)-K(+)-Cl- co-transporter energized by the Na+, K(+)-ATPase was involved for 60% in the K+ secretion into endolymph. The electrogenic K+ transport would partly depend on a N-ethylmaleimide-sensitive protein possibly located at the apical plasma membrane or intracellularly.
Is the endolymphatic K secretion electrogenic?
The endolymphatic potential is assumed to result from active K transport into the endolymphatic compartment and passive K diffusion in the opposite direction. However, in several in vivo experiments, changes in the endolymphatic potential differed from those in the endolymphatic K concentration. Moreover, in in vitro experiments, a negative endolymphatic potential was observed in the presence of ouabain without K gradient between the two compartments. These observations suggest that the coupling between the K transport and the genesis of the endolymphatic potential is not tight. Several factors may separately influence the endolymphatic potential and the K transport such as the acid-base equilibrium, the integrity of Reissner's membrane, the hormonal status, and the Na transport.
[Anogenital papillomavirus lesions in humans with or without HIV infection. Comparison of colposcopic, histopathological and virological results].
Anal and genital lesions caused by human papilloma virus (HPV) may be associated with severe dysplasia and cancer, chiefly in cases of "high risk" HPV types 16, 18, 31, 35, 51. The frequency of HPV infections and the severity of genital cancers seem to be increased in patients with human immunodeficiency virus (HIV) infection. PATIENTS AND METHOD. The distribution of different HPV types was compared with the anatomical and clinical features of the lesions in two populations, one HIV+ (n = 40) and the other HIV- (n = 48), who had anal and genital lesions. The HPV DNA was determined by molecular hybridization in situ, using biotinylated probes which recognized HPV types 6/11, 16/8 and 31/35/51 on 99 lesions. RESULTS. HIV+ subjects differed from HIV- subjects in that a higher proportion of them had anal lesions (50 p. 100 vs 10 p. 100) and condyloma latum (80 p. 100 vs 50 p. 100). Koilocytosis without dysplasia was more often found in HIV- subjects (12.5 p. 100 vs 55 p. 100). Conversely, dysplasia was more frequent among the lesions of HIV+ subjects: grade I 39.5 p. 100 vs 17.5 p. 100; grade II 25 p. 100 vs 4 p. 100; grade III 12.5 p. 100 vs 0 p. 100. Koilocytosis was preferentially associated with condyloma acuminatum. In HIV+ subjects the DNA of HPV, detected in 73 p. 100 of the lesions, was "high risk" HPV DNA in 86 p. 100 of the cases, whereas in HIV- subjects 51 p. 100 of the samples were positive in hybridization, and 61.5 p. 100 had "low risk" HPV DNA. In subjects of all groups "high risk" HPV was found in dysplastic lesions. CONCLUSION. HIV seropositive subjects show an imbalanced distribution of HPV with predominance of "high risk" HPV. This suggests that immunodepression encourages infection by this oncogenic virus, thereby contributing to the frequency of cancer in HIV+ subjects.
[Effect of a low-fat diet on the fasting volume and postprandial emptying of the gallbladder].
Little is known of the effects of meal composition on gallbladder emptying and cholecystokinin (CCK) release in man. Gallbladder volumes (measured by means of real time ultrasonography) and plasma CCK levels (determined by radioimmunoassay) were studied in 5 normal subjects, before and after a normal solid-liquid meal, and before and after a low-fat, low-protein, solid-liquid meal after 3 days regimen with low-fat, low-protein meal. Low-fat, low-protein regimen significantly increased gallbladder fasting volume and significantly decreased fasting plasma CCK levels. This suggests that CCK secretion regulates fasting gallbladder volume and that basal CCK secretion depends on diet composition. After a normal meal, gallbladder emptying was biphasic with a 44% volume decrease within the first 15 min followed by slower emptying during the next 60 min with a final volume reaching less than 15% of the fasting volume. After a hypolipidic, hypoproteic meal, the initial 15 min emptying (42%) was the same as after control meals but no further decrease of gallbladder volume was observed. This study shows that the initial phase of post prandial gallbladder contraction is not dependent on meal composition which affects late gallbladder emptying only. We conclude that a low-fat, low-protein diet, increasing gallbladder fasting volume and decreasing gallbladder emptying, may favor gallbladder stasis and therefore increase the risk of gallstone formation.
[Mechanisms of electrogenic secretion of potassium in endolymph].
Endolymphatic potential is assumed to result from active K transport into the endolymphatic compartment and passive K diffusion in the opposite direction. However, in several in vivo experiments, changes in endolymphatic potential differed from that in endolymphatic K concentration. Moreover, in in vitro experiments, a negative endolymphatic potential was observed in presence of ouabain without K gradient between the two compartments. These observations suggest that the coupling between the K transport and the genesis of the endolymphatic potential is not tight. Several factors may influence separately the endolymphatic potential and the K transport such as the acid-base equilibrium, the integrity of the Reissner's membrane, the hormonal status and the Na transport.