Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.6.99.3 (diaphorase)
 5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Treatment of red cell ghosts with ozone inhibited both AChE (marking the outside of the membrane) and G3PDH (marking the inside of the membrane). There was no change in tryptophan fluorescence of the ghosts after the ozone treatment. Band 3 protein was isolated from the ozone-treated ghosts. The protein was digested with trypsin to obtain water soluble peptides from the cytoplasmic N-terminal tail and the interhelical loops. Fluorescent peptides included GWVIHPLGLR from the outer loop between helices 7 and 8, and peptide WMEAAR from the N-terminal cytoplasmic tail. Neither one of these peptides was oxidized by ozone. This was true whether or not the ghosts were sealed. We conclude that the position of these tryptophans either in the membrane structure, or because of binding to other proteins in the cytoplasmic tail, protects them from oxidation by ozone. Treatment of horse heart cytochrome c with ozone did not change the absorbance spectrum in the heme region or the tryptophan absorbing region. HPLC of the ozone-treated cytochrome c showed that cytochrome c was being modified, indicated by a change in the elution time. Treatment of cytochrome c with ozone did not change the activity in the NADH-cytochrome c reductase assay. Digestion of the ozone-treated cytochrome c with trypsin gave peptides which demonstrated normal fluorescence. (Cytochrome c has abnormally low fluorescence, which is not changed by ozone exposure.) The peptides were separated by HPLC. The fluorescence of the tryptophan-containing peptide (GITWK) was not decreased by treatment of the cytochrome c by ozone. Amino acid analysis of the ozone-treated cytochrome c indicated that methionine was oxidized. We conclude that tryptophan in cytochrome c is protected from oxidation by ozone because of the interaction with the porphyrin ring. Bovine serum albumin and human serum albumin were treated with ozone. There was a monotonic decrease in tryptophan fluorescence in both cases. Digestion of BSA with trypsin produced two fluorescent peptides. The peptide FWGK was identified by coelution with the authentic peptide. The putative peptide AWSVAR was not the same as the chemically synthesized peptide. The peptide sequences FWGK and "AWSVAR" were both oxidized in ozone-treated bovine serum albumin, with no detectable discrimination. Tryptic digestion of the ozone-treated human serum albumin produced a single fluorescent peptide, which was oxidized by ozone. The putative peptide AWAVAR in the tryptic digest of HSA was distinct from chemically synthesized peptide. The oxidation of tryptophans in proteins by ozone is markedly influenced by position in tertiary structure, position in membrane structure, and by chemical interactions within the protein.
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PMID:Reaction of ozone with protein tryptophans: band III, serum albumin, and cytochrome C. 902 65

Nitric oxide has been postulated as a retrograde intercellular messenger for long-term potentiation, a form of synaptic plasticity that is associated with learning and memory processes. In the present study we investigated whether the loss or survival of nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase-containing neurons, which are known to synthesize nitric oxide, would be an useful indicator for evaluating the structural and functional state of the rat hippocampus after status epilepticus that is induced by intraperitoneal injection of kainic acid. Besides NADPH diaphorase histochemistry, two other histological parameters were studied: the grade of cell damage evaluated from silver-impregnated sections, and the number of somatostatin-containing neurons in different hippocampal subfields. We found that the number of NADPH diaphorase-containing neurons in the hilus and granule cell layer correlated well with spatial learning and memory performance as assessed by the Morris water-maze test. The extent of cell damage in the CA1 subfield analysed in silver-impregnated sections and the number of hilar somatostatin-containing neurons also significantly correlated with latencies in the water-maze test. Furthermore, linear regression analysis revealed that the number of somatostatin-containing neurons in the hilus explains about 50% of the variation in water-maze learning. These findings emphasize that although general structural preservation is of crucial importance for the function of the hippocampus also interneurons, such as somatostatin- and NADPH diaphorase-containing neurons, may play an important role during the acquisition phase and processing of information in hippocampal circuitry. Therefore, in addition to evaluating general cell damage, analysis of the cell loss that occurs in the interneuron subpopulations will be beneficial in verifying structural and functional deficits of the hippocampus after status epilepticus.
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PMID:Comparison of NADPH diaphorase histochemistry, somatostatin immunohistochemistry, and silver impregnation in detecting structural and functional impairment in experimental status epilepticus. 925 25

Rabbits given 1 ppm of vanadate in drinking water for twelve months showed (a) increased plasma levels of catecholamines (b) reduction of the arterial concentration of nitric oxide (c) lower activity of urine kallikrein and higher activities of urine kininases I and II and enkephalinase (d) reduced cardiac inotropism and augmented total peripheral resistance, with unchanged blood pressure levels (e) accumulation of the metal in the aorta and cardiac ventricles. Monoaminooxidase and glucose-6-phosphate dehydrogenase activities were increased by vanadate in both kidney and liver and that of NADH-diaphorase in the kidney, in which NADPH-diaphorase activity was reduced. Some of the above results were also obtained in rats given 10 and 40 ppm of vanadate in drinking water for six-seven months; these animals showed arterial hypertension and reduced activity of Na, K-ATPase in the kidney. Vanadium appears to act on the cardiovascular function through selective neurohumoral, autacoidal and transductional mechanisms only in part depending on the species.
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PMID:[Neurohumoral, autacoid and transductional mechanisms in the cardiovascular effects of vanadate: histochemical correlations]. 937 36

The distribution of immunoreactivity to neuronal nitric oxide synthase (nNOS) and vasopressin (AVP) was studied in the circumventricular organs of the female rat. The occurrence of NOS immunoreactivity showed correspondence to nicotinamide dinucleotide phosphate diaphorase reactivity, a previously used but less specific marker for neuronal NOS. nNOS immunolabeling was detected in the two most rostrally located circumventricular organs - the organum vasculosum of the lamina terminalis and the subfornical organ. In the latter, AVP immunoreactivity was observed in some cell bodies, which also were nNOS-immunoreactive. In the median eminence and the neurohypophysis there were large amounts of nNOS- and AVP-immunoreactive nerve fibers, which often displayed similarities in distribution and morphology. Within the pineal gland, only very few nNOS-immunoreactive varicose terminals were observed, which ran along blood vessels. nNOS immunoreactivity was also seen in the epithelium of the choroid plexus, whereas no nNOS immunoreactivity could be found in the subcommissural organ or in the area postrema. The present demonstration of nNOS and AVP immunoreactivity in the subfornical organ, median eminence, and neurohypophysis, and the occurrence of nNOS immunoreactivity also in the choroid plexus and organum vasculosum of the lamina terminalis, provides a morphological background for a functional role for nitric oxide in water homeostatic mechanisms, both as executed through the hypothalamohypophyseal system and via the production of cerebrospinal fluid.
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PMID:Nitric oxide synthase and vasopressin in rat circumventricular organs. An immunohistochemical study. 938 4

The proton-pumping NADH:ubiquinone oxidoreductase of Escherichia coli is composed of 14 different subunits and contains one FMN and up to nine iron-sulfur clusters as prosthetic groups. By use of salt treatment, the complex can be split into an NADH dehydrogenase fragment, a connecting fragment and a membrane fragment. The water-soluble NADH dehydrogenase fragment has a molecular mass of approximately 170,000 Da and consists of the subunits NuoE, F, and G. The fragment harbors the FMN and probably six iron-sulfur clusters, four of them being observable by EPR spectroscopy. Here, we report that the fully assembled fragment can be overproduced in E. coli when the genes nuoE, F, and G were simultaneously overexpressed with the genes nuoB, C, and D. Furthermore, riboflavin, sodium sulfide, and ferric ammonium citrate have to be added to the culture medium. The fragment was purified from the cytoplasm by means of ammonium sulfate fractionation and chromatographic steps. The preparation contains one noncovalently bound FMN per molecule. Two binuclear (N1b and N1c) and two tetranuclear (N3 and N4) iron-sulfur clusters were detected by EPR in the NADH reduced preparation with spectral characteristics identical with those of the corresponding clusters in complex I. The preparation fulfills all prerequisites for crystallization of the fragment.
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PMID:Characterization of the overproduced NADH dehydrogenase fragment of the NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli. 948 11

Changes of nitric oxide (NO)-producing neurons in the brain following learning is not yet clear. In present study, nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry and neuronal NO synthase (nNOS) immunohistochemistry were used to detect NOS neurons in rat brain. Results demonstrated that expression of NOS neurons in dentate gyrus and frontal cortex was significantly increased after a water-rewarded spatial alternation task when compared with that after sham training. The elevated expression of NOS neurons occurred not only in the earlier memory stage, but also in the later memory stage. In addition, the expression location and cell counts of NOS neurons in dentate gyrus and frontal cortex with NADPH-d staining or nNOS immunoreactivity resembled each other, but the cell counts of NADPH-d positive neurons were a little more than those of nNOS immunoreactive neurons. The involvement of NO in the processes of spatial learning and memory is further suggested.
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PMID:Spatial learning and memory induce up-regulation of nitric oxide-producing neurons in rat brain. 972 7

We investigated the pathophysiological role of nitric oxide synthesized by inducible nitric oxide synthase in the brain, by injecting lipopolysaccharide directly into the rat cerebral cortex/hippocampus. The levels of nitric oxide metabolites, nitrite and nitrate, began to increase in a dose-dependent manner with a 3-h lag, and reached approximately seven-fold the basal levels 8 h after the direct injection of lipopolysaccharide (5 microg). The lipopolysaccharide-induced increase in nitrite and nitrate levels was inhibited by treatment with the specific inducible nitric oxide synthase inhibitor aminoguanidine. The protein synthesis inhibitor cycloheximide delayed the onset of the increase in nitric oxide metabolite levels, and reduced the peak levels. Lipopolysaccharide increased Ca2+-independent, but not Ca2+-dependent, nitric oxide synthase activity in the brain. Intense nicotinamide adenine dinucleotide phosphate-diaphorase activity was observed in round cells in the vicinity of the site of injection of lipopolysaccharide 8 h after the injection. Neuronal death was observed seven days after the injection of lipopolysaccharide. Spatial memory, as assessed by performance in a water maze task and spontaneous alternation behavior in a Y-maze, was significantly impaired in rats which had had previous bilateral injections of lipopolysaccharide into the hippocampus. The lipopolysaccharide-induced neuronal death and spatial memory impairments were prevented by aminoguanidine. These results suggest that direct injection of lipopolysaccharide into the brain causes an induction of inducible nitric oxide synthase in vivo. Furthermore, it is suggested that nitric oxide produced by inducible nitric oxide synthase is responsible for the lipopolysaccharide-induced brain dysfunction.
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PMID:Brain dysfunction associated with an induction of nitric oxide synthase following an intracerebral injection of lipopolysaccharide in rats. 1005 Dec 7

The modulating effect of thymoquinone (TQ) on benzo(a)pyrene (BP)-induced forestomach tumours was investigated in female Swiss albino mice, receiving oral administration of BP at a dose of 1 mg twice weekly for 4 weeks. Administration of 0.01% of TQ in drinking water 1 week before, during and after BP treatment until the end of the experiment resulted in significant suppression of BP-induced tumourigenesis when compared with the group receiving BP alone. TQ inhibited both BP-induced forestomach tumour incidence and multiplicity by 70% and 67%, respectively. Lipid peroxide accumulation and decreased glutathione (GSH) content and glutathione-S-transferase (GST) and DT diaphorase activities were observed in the liver of BP-treated tumour-bearing mice. TQ alone showed a significant induction in the enzyme activities of hepatic GST and DT diaphorase. Mice treated with TQ along with BP showed almost normal hepatic lipid peroxides and GSH levels, and normal enzyme activities compared to the control group. The present data may indicate the potential of TQ, the main constituent of the volatile oil of Nigella sativa seed, as a powerful chemopreventive agent against BP-induced forestomach tumours in mice. The possible modes of action of TQ may be through its antioxidant and anti-inflammatory activities, coupled with enhancement of detoxification processes.
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PMID:Inhibition of benzo(a)pyrene-induced forestomach carcinogenesis in mice by thymoquinone. 1054 99

Freshwater clams Corbicula fluminea were exposed in aquariums to four doses of trichloroethylene-TCE-(1.56 up to 100 mg/1) or toluene-TOL-(7.5 up to 60 mg/1) for 5 days. At the end of exposure, components of (de)toxification metabolism of phases I and II, parameters related to oxidative stress and propionylcholinesterase activity were assayed. Determination of TCE and TOL concentrations in water revealed an important evaporative loss during the experiment, characteristic of acute and occasional contaminations by such products occurring in the environment. Appropriate statistical methods such as ANOVA, Tukey test and discriminant analysis underlined the relevance of cytochromes P450 and P418, NADH-cytochrome c reductase, catalase, peroxided and peroxidizable lipids and net peroxidation as biomarkers of exposure to these solvents in C. fluminea. This experiment emphasised the importance of a multi-biomarker approach in environmental surveys and will be completed further by mesocosm studies.
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PMID:Potential biomarkers of trichloroethylene and toluene exposure in Corbicula fluminea. 1116 53

The flavoprotein Escherichia coli methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate) to 5-methyltetrahydrofolate (CH(3)-H(4)folate). The X-ray crystal structure of the enzyme has revealed the amino acids at the flavin active site that are likely to be relevant to catalysis. Here, we have focused on two conserved residues, Asp 120 and Glu 28. The presence of an acidic residue (Asp 120) near the N1-C2=O position of the flavin distinguishes MTHFR from all other known flavin oxidoreductases and suggests an important function for this residue in modulating the flavin reactivity. Modeling of the CH(3)-H(4)folate product into the enzyme active site also suggests roles for Asp 120 in binding of folate and in electrostatic stabilization of the putative 5-iminium cation intermediate during catalysis. In the NADH-menadione oxidoreductase assay and in the isolated reductive half-reaction, the Asp120Asn mutant enzyme is reduced by NADH 30% more rapidly than the wild-type enzyme, which is consistent with a measured increase in the flavin midpoint potential. Compared to the wild-type enzyme, the mutant showed 150-fold decreased activity in the physiological NADH-CH(2)-H(4)folate oxidoreductase reaction and in the oxidative half-reaction involving CH(2)-H(4)folate, but the apparent K(d) for CH(2)-H(4)folate was relatively unchanged. Our results support a role for Asp 120 in catalysis of folate reduction and perhaps in stabilization of the 5-iminium cation. By analogy to thymidylate synthase, which also uses CH(2)-H(4)folate as a substrate, Glu 28 may serve directly or via water as a general acid catalyst to aid in 5-iminium cation formation. Consistent with this role, the Glu28Gln mutant was unable to catalyze the reduction of CH(2)-H(4)folate and was inactive in the physiological oxidoreductase reaction. The mutant enzyme was able to bind CH(3)-H(4)folate, but reduction of the FAD cofactor was not observed. In the NADH-menadione oxidoreductase assay, the mutant demonstrated a 240-fold decrease in activity.
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PMID:Folate activation and catalysis in methylenetetrahydrofolate reductase from Escherichia coli: roles for aspartate 120 and glutamate 28. 1137 Nov 82


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