Biomedical subjects
C Dupuy
Publications and source records attributed to C Dupuy.
[Too much is too much].
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[A disoriented and jovial woman].
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The Ca2+/NADPH-dependent H2O2 generator in thyroid plasma membrane: inhibition by diphenyleneiodonium.
The thyroid plasma membrane contains a Ca(2+)-regulated NADPH-dependent H2O2-generating system which provides H2O2 for the peroxidase-catalysed biosynthesis of thyroid hormones. The electron transfer from NADPH to O2 catalysed by this system was studied by using diphenyleneiodonium (DPI), an inhibitor of flavo- and haemo-proteins. The prosthetic group of the H2O2 generator was removed by incubation with 5 mM CHAPS at 40 degrees C, and an active holoenzyme was reconstituted with FAD, but not with FMN. The H2O2-generating system also had an intrinsic Ca(2+)-dependent NADPH:ferricyanide reductase activity which is probably linked to its flavodehydrogenase component (or domain). Both activities, H2O2 production and ferricyanide reductase activity, were inhibited by DPI, with similar K1/2 (2.5 nmol/mg of protein). DPI only inhibited a system reduced with NADPH in the presence of Ca2+. NADPH could not be replaced by NADP+, NADH or sodium dithionite, suggesting the need for specific mild reduction of a redox centre in a particular conformation. Ferricyanide protected both activities against inhibition by DPI; the NADPH:ferricyanide reductase activity was completely protected at a ferricyanide concentration 20 times lower than that needed to protect the H2O2 formation, implying at least two target sites for DPI. One might be the flavodehydrogenase component; the other was beyond, on the entity which transfers the electrons to O2. This second site has not been identified.
[Secondary hemomediastinum after positioning a hemodialysis catheter].
We describe a case of late perforation of the superior vena cava by a hemodialysis catheter inserted via the left internal jugular vein. This resulted in extravasation of blood. Malposition of the catheter was confirmed by CT scan. Removal of the catheter resulted in rapid resolution of symptoms. Confirmation of correct placement of central venous catheter must be obtained by chest X-ray contrast study.
In vivo motility of rat colon chronically pretreated with sennosides.
Ceco-colonic myoelectrical activity was investigated in rats pretreated for 23 weeks by sennosides (10 or 40 mg/kg/day), Na-picosulfate (2.5 or 10 mg/kg/day) or laxative vehicle (control). On the last week of treatment the animals were equipped with Nichrome electrodes on the cecum, the proximal and distal colon. In comparison with controls, sennoside or Na-picosulfate treatment did not induce any significant (p > 0.05) change in the duration of long spike bursts (LSB) which are associated with phasic contractions. On the last 2 days of treatment the frequency of LSB for 2 h before and 2 h after laxative administration, as well as for 30 min after a 3-gram meal was not significantly (p > 0.05) different in control and treated animals. Similarly, on the first 2 days, as well as on days 13 and 14, after the end of treatment, no significant (p > 0.05) difference in the LSB frequency appeared between control and treated animals, in the fasted state or after a 3-gram meal. It is concluded that long-term treatment with sennosides or Na-picosulfate does not induce chronic changes in colonic motility in rats.
The mycotoxin, deoxynivalenol, delays gastric emptying through serotonin-3 receptors in rodents.
The effects of the trichotecene mycotoxin, deoxynivalenol, on gastric emptying and intestinal propulsion in mice and rats and gastrointestinal myoelectrical activity in rats were investigated. Gastric emptying and intestinal transit were evaluated after gavage with a milk meal containing a marker (51CrO4Na2) and radio-activity was counted in the stomach and 10 segments of the small intestine. The myoelectrical activity of the antrum, duodenum and jejunum was assessed by implanting electrodes for long-term electromyographic recordings. Deoxynivalenol given orally (50-1000 micrograms/kg) but not i.c.v. (5 micrograms/kg) 10 min before the test meal inhibited gastric emptying in a dose-related manner. Intestinal propulsion was reduced for the highest dose (1000 micrograms/kg) only. The inhibition of gastric emptying induced by deoxynivalenol was antagonized by ondansetron and granisetron given s.c. (50 micrograms/kg) but not by ondansetron i.c.v. (10 micrograms/kg). Metoclopramide, domperidone (1 mg/kg s.c.), methysergide, ritanserin and cisapride (2 mg/kg s.c.) did not modify the deoxynivalenol-induced inhibition of gastric emptying. In rats, gavage with a 2.5-ml milk meal increased the frequency of antral spike bursts from 1.9 +/- 0.9/min in the fasted state to 4.7 +/- 0.4/min and disrupted intestinal migrating motor complexes for 84.9 +/- 10.8 min. Oral administration of deoxynivalenol (50-100 micrograms/kg) 10 min before the meal did not modify the frequency of antral spike bursts but induced migrating motor complexes on the small intestine after the meal. This effect was reversed by ondansetron (10 micrograms/kg s.c.). It was concluded that, in rodents, deoxynivalenol inhibits gastric emptying by inducing intestinal migrating motor complexes through a peripheral action at the serotonin-3 receptors.
Interface measurements of heterojunction band lineups with the Vanderbilt free-electron laser.
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A method for measuring H2O2 based on the potentiation of peroxidative NADPH oxidation by superoxide dismutase and scopoletin.
NADPH oxidation catalyzed by horseradish peroxidase is considerably increased by scopoletin and superoxide dismutase. These effects were used to develop a method for measuring H2O2 in a horseradish peroxidase, superoxide dismutase, and scopoletin system by measuring the NADPH oxidation rate. The optimal concentration of each reactant was determined. H2O2 could be detected and measured when it was present free in the medium or when it was produced by an H2O2-generating system, such as glucose-glucose oxidase or NADPH oxidase from thyroid plasma membranes. H2O2 was measured either by taking aliquots of the incubation medium or by placing NADPH directly in the medium and following the kinetics of NADPH oxidation. This latter approach required smaller amounts of biological material. In contrast to other methods, the H2O2 which is measured is regenerated. This method is 10 times more sensitive than the standard scopoletin method for H2O2 measurement and will detect a H2O2 production rate as low as 0.2 nmol per hour. The method is particularly suitable for biological systems in which small quantities of biological material are available.
Activation of the NADPH-dependent H2O2-generating system in pig thyroid particulate fraction by limited proteolysis and Zn2+ treatment.
The NADPH-dependent H2O2-generating system in a pig thyroid particulate fraction requires micromolar concentrations of Ca2+ for activity. The H2O2 generator could be Ca(2+)-desensitized (i.e. made fully active in the absence of Ca2+) by limited proteolysis with alpha-chymotrypsin or by treatment with ZnCl2. The Zn2+ effect was temperature- and dose-dependent with an apparent half-maximum concentration of 0.15 mM at 40 degrees C. Ca2+ desensitization was not reversed by adding the Zn2+ chelators, 1,10-phenanthroline and EGTA, but about one-third of the Ca(2+)-sensitivity was recovered after addition of 10 mM-dithiothreitol. The proteolysed enzyme and the Zn(2+)-treated enzyme had different Km values for NADPH. The Zn2+ effect did not seem to involve proteolysis or membrane fusion. These results indicate that Ca2+ regulation occurs via an autoinhibitory domain or inhibitory protein component of the H2O2-generator system. Its inhibitory effect may be removed by proteolysis or conformational changes, making the catalytic site accessible to the substrate NADPH and/or enabling electrons to be transferred from NADPH to O2.
[The association of pulmonary hemosiderosis and celiac disease. Apropos of a new case in a child].
A new case of pulmonary hemosiderosis with coeliac disease is reported. This is an extremely rare combination of which only nine instances have been published over the last 20 years. Three of the reported cases occurred in children. Apart from a marked predominance of males, the combination has no specific features. Firm evidence of a causal relationship between the two diseases is lacking but treatment with a gluten-free diet alone apparently had beneficial effects on the lung disease in two patients. Three pathogenic hypotheses are discussed herein: deposition of circulating immune complexes involving food allergens on the basement membrane of alveolar capillaries; reaction between antireticulin antibodies and an alveolar basement membrane antigen; or effect of adenovirus 12, a potential causative factor for celiac disease. Patients with idiopathic pulmonary hemosiderosis should routinely have tests for gluten intolerance, for instance a lactulose-mannitol intestinal permeability test. Lastly, other concomitant food allergies are reported.
Mechanism of NADPH oxidation catalyzed by horse-radish peroxidase and 2,4-diacetyl-[2H]heme-substituted horse-radish peroxidase.
The mechanism of NADPH oxidation catalyzed by horse-radish peroxidase (HRP) and 2,4-diacetyl-[2H]heme-substituted horse-radish peroxidase (DHRP) was studied. The roles of the different H2O2/peroxidase compounds were examined by spectral studies. The oxidized NADPH species were identified using the superoxide dismutase effect and by measuring the stoichiometry between NADPH oxidized and H2O2 used. In the presence of a mediating molecule, like scopoletin, both enzymes acted via a similar mechanism, producing only NADP degrees, which in turn reacted with O2 producing O2-. Consequently H2O2 was completely regenerated in the presence of superoxide dismutase and partially regenerated in its absence. In the absence of a mediating molecule, the H2O2 complex of both enzymes (compound I) catalysed NADPH oxidation by single-electron transfer, producing NADP degrees; compound II of these enzymes catalyzed NADPH oxidation more slowly by a direct two-electron transfer, producing NADPH+. There were difference between HRP and DHRP. HRP compound II was produced by the oxidation of 1 mol NADPH/mole compound I, while DHRP compound II was formed by the spontaneous conversion of compound I to compound II. The NADPH oxidation catalyzed by DHRP compound I did not lead to the formation of compound II. When H2O2 was produced slowly by the glucose/glucose-oxidase system, compound II was never formed and a pure O2- adduct of DHRP (compound III) accumulated.
Mechanism of hydrogen peroxide formation catalyzed by NADPH oxidase in thyroid plasma membrane.
The thyroid plasma membrane contains a Ca2(+)-regulated NADPH-dependent H2O2 generating system which provides H2O2 for the thyroid peroxidase-catalyzed biosynthesis of thyroid hormones. The plasma membrane fraction contains a Ca2(+)-independent cytochrome c reductase activity which is not inhibited by superoxide dismutase. But it is not known whether H2O2 is produced directly from molecular oxygen (O2) or formed via dismutation of super-oxide anion (O2-). Indirect evidence from electron scavenger studies indicate that the H2O2 generating system does not liberate O2-, but studies using the modified peroxidase, diacetyldeuteroheme horseradish peroxidase, to detect O2- indicate that H2O2 is provided via the dismutation of O2-. The present results provide indirect evidence that the cytochrome c reductase activity is not a component of the NADPH-dependent H2O2 generator, since it was removed by washing the plasma membranes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid without affecting H2O2 generation. Spectral studies with diacetyldeuteroheme-substituted horseradish peroxidase showed that the thyroid NADPH-dependent H2O2 generator does not catalyze superoxide anion formation. The O2- adduct compound (compound III) was formed but was completely inhibited by catalase, indicating that the initial product was H2O2. The rate of NADPH oxidation also increased in the presence of diacetylheme peroxidase. This increase was blocked by catalase and was greatly enhanced by superoxide dismutase. The O2- adduct compound (compound III) was produced in the presence of NADPH when glucose-glucose oxidase (which does not produce O2-) was used as the H2O2 generator. NADPH oxidation occurred simultaneously and was enhanced by superoxide dismutase. We conclude that O2- formation occurs in the presence of an H2O2 generator, diacetylheme peroxidase and NADPH, but that it is not the primary product of the H2O2 generator. We suggest that O2- formation results from oxidation of NADPH, catalyzed by the diacetylheme peroxidase compound I, producing NADP degree, which in turn reacts with O2 to give O2-.
Nonenzymatic NADPH-dependent reduction of 2,6-dichlorophenol-indophenol.
The reduction of 2,6-dichloroindophenol (DCIP) by direct interaction with NADPH was studied. The results indicate that reduction proceeds via a direct electron transfer from NADPH to DCIP, with no oxygen consumption, and a rate constant of k = 4.69 M-1.s-1. The reduced DCIP can rapidly transfer its electrons to potassium ferricyanide (K3Fe(CN)6) or ferricytochrome c, but not to nitro blue tetrazolium. Superoxide dismutase inhibits DCIP reduction in an oxygen-dependent manner by favoring the reoxidation of the reduced DCIP. We therefore conclude DCIP is not suitable for detecting O2- when the nucleotides NADH or NADPH are present.