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)

Phosphophoryns (PPs) are phosphoproteins specific to the dentin matrix and are the major noncollagenous matrix proteins in rat incisor dentin. It has been hypothesized that their phosphate groups are important in dentin mineralization. PPs have many sequences which are substrates for membrane-associated endogenous messenger-independent kinases. The objective of this study was to localize the protein kinases involved in phosphorylating the PPs. Osteoblast-like ROS 17/2.8 cells, which secrete extracellular matrix phosphoproteins, were lysed. After removal of the nuclei and mitochondria by low-speed centrifugation, the membrane associated organelles were isolated at higher speed from the cytosol. The Golgi vesicle and rough microsome fractions were collected from 29-43.7% sucrose density gradients. Each fraction was tested for casein kinase II (CKII) activity using an in vitro phosphorylation assay with PPs as substrate. To characterize and confirm the nature of the components of the sucrose gradient fractions, the activities of specific enzymes such as N-acetylglucosamine galactosyltransferase and cytochrome c reductase, which are exclusively associated with the Golgi and rough microsomes, respectively, were determined. Electron microscopy of the isolated fractions confirmed the enzyme assay characterizations. CKII activity capable of phosphorylating the PP was found in the rough microsome fraction but not in the Golgi. Thus, phosphorylation of the secreted phosphoproteins would appear to take place in the endoplasmic reticulum as a cotranslational event.
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PMID:Casein kinase localization in the endoplasmic reticulum of the ROS 17/2.8 cell line. 761 Sep 32

As is known from literature, iprodione, a dicarboximide fungicide, has a highly specific action, with a capacity to cause oxidative damage through production of free oxygen radicals (ROS), but it does not appear to be species selective. Since this substance is able to diffuse in water, evaluation of its capacity to induce oxidative damage in an aquatic organism such as the rainbow trout (Oncorhynchus mykiss) was considered of particular interest. A study was, therefore, undertaken to investigate the effect of iprodione on free radicals (ROS) and malondialdehyde (MDA) production, reduced glutathione (GSH) content and catalase activity (CAT), in primary cultured trout hepatocytes, following treatment with 0.2, 0.3 and 0.4 mM concentrations for a 24-h period. The iprodione 0.3 and 0.4 mM concentrations increased both ROS and MDA production and decreased GSH content and CAT activity. These results suggest that iprodione is able to produce oxidative damage in primary cultured fish hepatocytes, thus confirming that its action is specific, but not species selective. It is also well known that ROS production in fungi is due to interaction with the flavin enzyme NADPH cytochrome c reductase to the extent that the normal electron flow from NADPH to cytochrome c is blocked. In contrast, we observed that, in primary cultured trout hepatocytes, iprodione appears to have no effect on NADPH cytochrome c reductase activity. It is, therefore, possible to presume that the mechanism of oxidative damage in trout hepatocytes differs from that observed in fungi. Moreover, our experiments also demonstrate that iprodione is able to induce "in vitro" CYP1A1, leading to the conclusion that the production of ROS is due to this phenomenon.
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PMID:Effect of iprodione, a dicarboximide fungicide, on primary cultured rainbow trout (Oncorhynchus mykiss) hepatocytes. 1145 25

1. The enzymes responsible for the reductive activation of NFT are not known. We have now shown that under aerobic conditions, inhibitors of cytochrome P450 or P450 reductase but not DT diaphorase prevented NFT induced cytotoxicity and reactive oxygen species ("ROS") formation. This suggests that NFT was reductively activated by reduced cytochrome P450 and/or P450 reductase. 2. The subcellular organelle oxidative stress effects leading to cytotoxicity are not known. Hepatocyte mitochondrial membrane potential was only slightly decreased by NFT before cytotoxicity ensued. However NFT induced lysosomal damage and hepatocyte protease activation. Endocytosis inhibitors, lysosomotropic agents or lysosomal protease inhibitors also prevented NFT induced cytotoxicity. 3. Lipid peroxidation also preceded cytotoxicity. Furthermore desferoxamine (a ferric chelator), antioxidants or ROS scavengers (catalase, mannitol, TEMPOL or dimethylsulfoxide) prevented NFT cytotoxicity. 4. It is concluded that H2O2 reacts with lysosomal Fe(+2) to form "ROS" which causes lysosomal lipid peroxidation, membrane disruption, protease release and cell death.
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PMID:Lysosomal oxidative stress cytotoxicity induced by nitrofurantoin redox cycling in hepatocytes. 1176 51

Ubiquinone is inhomogenously distributed in subcellular biomembranes. Apart from mitochondria, where ubiquinone has bioenergetic and pathophysiological functions, unusually high levels of ubiquinone have also been reported in Golgi vesicles and lysosomes. In lysosomes, the interior differs from other organelles in its low pH value which is important to ensure optimal activity of hydrolytic enzymes. Since redox-cycling of ubiquinone is associated with the acceptance and release of protons, we assumed that ubiquinone is part of a redox chain contributing to unilateral proton distribution. A similar function of ubiquinone was earlier suggested by Crane to operate in Golgi vesicles. Support for the involvement of ubiquinone in a presumed couple of redox carriers came from our observation that almost 70% of total lysosomal ubiquinone was in the divalently reduced state. Further reduction was seen in the presence of external NADH. Analysis of the components involved in the transfer of reducing equivalents from cytosolic NADH to ubiquinone revealed the existence of an FAD-containing NADH dehydrogenase. The latter was found to reduce ubiquinone by means of a b-type cytochrome. Proton translocation into the interior was linked to the activity of the novel lysosomal redox chain. Oxygen was found to be the terminal electron acceptor, thereby also regulating acidification of the lysosomal matrix. In contrast to mitochondrial respiration, oxygen was only trivalently reduced giving rise to the release of HO radicals. The role of this novel proton-pumping redox chain and the significance of the associated ROS formation has to be elucidated.
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PMID:Lysosomal ROS formation. 1625 87

1. One hundred and sixty 1-d-old Arbor Acre male broiler chicks were fed with maize-soybean based diets for 6 weeks in a 2 x 2 factorial experiment. The factors were CoQ10 supplementation (0 or 40 mg/kg) and Escherichia coli lipopolysaccharide (LPS) challenge (LPS or saline). 2. CoQ10 was supplemented from d 1. From d 18, the chickens received three weekly i.p. injections of LPS (1.0 mg/kg BW) or an equivalent amount of sterile saline as control. From d 10 on, all chickens were exposed to low ambient temperature (12 to 15 degrees C) to induce ascites. 3. The blood packed cell volume and ascites heart index of broiler chickens were reduced by dietary CoQ10 supplementation. Mitochondrial State 3 and State 4 respiration, respiratory control ratio and phosphate oxygen ratio were not changed, but H+/site stoichiometry of complex II + III was elevated by dietary CoQ10 supplementation. 4. Cytochrome c oxidase and H+-ATPase activity were increased by CoQ10 supplementation, whereas NADH cytochrome c reductase and succinate cytochrome c reductase were not influenced. Mitochondrial anti-ROS capability was increased and malondialdehyde content was decreased by CoQ10 supplementation. 5. The work suggested that dietary CoQ10 supplementation could reduce broiler chickens' susceptibility to ascites, which might be the result of improving hepatic mitochondrial function, some respiratory chain-related enzymes activities and mitochondrial antioxidative capability.
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PMID:Effects of dietary coenzyme Q10 supplementation on hepatic mitochondrial function and the activities of respiratory chain-related enzymes in ascitic broiler chickens. 1635 19

The aim of this study was to identify of radiosusceptibility proteins in tissues with different radiosensitivity. C3H/HeJ mice were exposed to 10 Gy. The tissues were processed for proteins extraction and were analyzed by 2-dimensional electrophoresis. The proteins were identified by matrix-assisted laser desorption ionizing time-of-flight mass spectrometry and validated by immunohistochemical staining and Western blotting. The peaks of apoptosis levels were 35.3 +/- 1.7% and 0.6 +/- 0.2% in the spleen and the liver, respectively, after ionizing radiation. Analysis of liver tissue showed that the expression level of ROS related proteins such as cytochrome c, glutathione S transferase, NADH dehydrogenase and peroxiredoxin VI increased after radiation. The expression level of cytochrome c increased to 3-fold after ionizing radiation in both tissues. However in spleen tissue, the expression level of various kinds of apoptosis regulating proteins increased after radiation. These involved iodothyronine, CD 59A glycoprotein precursor, fas antigen and tumor necrosis factor -inducible protein TSG-6n precursor after radiation. The difference in the apoptosis index between the liver and spleen tissues is closely associated with the expression of various kinds of apoptosis-related proteins. The result suggests that the expression of apoptosis-related protein and redox proteins play important roles in this radiosusceptibility.
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PMID:Proteomics analysis of apoptosis-regulating proteins in tissues with different radiosensitivity. 1681 41

Ischemic preconditioning (IPC) strongly protects against ischemia-reperfusion injury; however, its effect on subsequent myocardial oxygenation is unknown. Therefore, we determine in an in vivo mouse model of regional ischemia and reperfusion (I/R) if IPC attenuates postischemic myocardial hyperoxygenation and decreases formation of reactive oxygen/nitrogen species (ROS/RNS), with preservation of mitochondrial function. The following five groups of mice were studied: sham, control (I/R), ischemic preconditioning (IPC + I/R, 3 cycles of 5 min coronary occlusion/5 min reperfusion) and IPC + I/R N(G)-nitro-L-arginine methyl ester treated, and IPC + I/R eNOS knockout mice. I/R and IPC + I/R mice were subjected to 30 min regional ischemia followed by 60 min reperfusion. Myocardial Po(2) and redox state were monitored by electron paramagnetic resonance spectroscopy. In the IPC + I/R, but not the I/R group, regional blood flow was increased after reperfusion. Po(2) upon reperfusion increased significantly above preischemic values in I/R but not in IPC + I/R mice. Tissue redox state was measured from the reduction rate of a spin probe, and this rate was 60% higher in IPC than in non-IPC hearts. Activities of NADH dehydrogenase (NADH-DH) and cytochrome c oxidase (CcO) were reduced in I/R mice after 60 min reperfusion but conserved in IPC + I/R mice compared with sham. There were no differences in NADH-DH and CcO expression in I/R and IPC + I/R groups compared with sham. After 60 min reperfusion, strong nitrotyrosine formation was observed in I/R mice, but only weak staining was observed in IPC + I/R mice. Thus IPC markedly attenuates postischemic myocardial hyperoxygenation with less ROS/RNS generation and preservation of mitochondrial O(2) metabolism because of conserved NADH-DH and CcO activities.
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PMID:Ischemic preconditioning prevents in vivo hyperoxygenation in postischemic myocardium with preservation of mitochondrial oxygen consumption. 1751 95

The mechanism of free radical production by complex I deficiency is ill-defined, although it is of significant contemporary interest. This study studied the ROS production and antioxidant defenses in children with mitochondrial NADH dehydrogenase deficiency. ROS production has remained significantly elevated in patients compared to controls. The expression of all antioxidant enzymes significantly increased at mRNA level. However, the enzyme activities did not correlate with high mRNA or protein expression. Only the activity of superoxide dismutase (SOD) was found to correlate with higher mRNA expression in patient derived cell lines. The activities of the enzymes such as glutathione peroxidase (GPx), Catalase (CAT) and glutathione-S-transferase (GST) were significantly reduced in patients (p<0.05 or p<0.01). Glutathione reductase (GR) activity and intracellular glutathione (GSH) levels were not changed. Decreased enzyme activities could be due to post-translational or oxidative modification of ROS scavenging enzymes. The information on the status of ROS and marking the alteration of ROS scavenging enzymes in peripheral lymphocytes or lymphoblast cell lines will provide a better way to design antioxidant therapies for such disorders.
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PMID:Analysis of reactive oxygen species and antioxidant defenses in complex I deficient patients revealed a specific increase in superoxide dismutase activity. 1855 9

The present study was designed with an aim to evaluate the effects of chronic aluminium exposure (10 mg/kg b.wt, intragastrically for 12 weeks) on mitochondrial energy metabolism in different regions of rat brain in vivo. Mitochondrial preparations from aluminium treated rats revealed significant decrease in the activity of various electron transport complexes viz. cytochrome oxidase, NADH cytochrome c reductase and succinic dehydrogenase as well, in the hippocampus region. The decrease in the activity of these respiratory complexes was also seen in the other two regions viz. corpus striatum and cerebral cortex, but to a lesser extent. This decrease in the activities of electron transport complexes in turn affected the ATP synthesis and ATP levels adversely in the mitochondria isolated from aluminium treated rat brain regions. We also studied the spectral properties of the mitochondrial cytochromes viz. cyt a, cyt b, cyt c1, and cyt c in both control and treated rat brains. The various cytochrome levels were found to be decreased following 12 weeks of aluminium exposure. Further, these impairments in mitochondrial functions may also be responsible for the production of reactive oxygen species and impaired antioxidant defense system as observed in our study. The electron micrographs of neuronal cells depicted morphological changes in mitochondria as well as nucleus only from hippocampus and corpus striatum regions following 12 weeks exposure to aluminium. The present study thus highlights the significance of altered mitochondrial energy metabolism and increased ROS production as a result of chronic aluminium exposure in different regions of the rat brain.
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PMID:Impairment of mitochondrial energy metabolism in different regions of rat brain following chronic exposure to aluminium. 1869 61

In this work, we investigated the biochemical mechanism of acetaminophen (APAP) induced toxicity in SK-MEL-28 melanoma cells using tyrosinase enzyme as a molecular cancer therapeutic target. Our results showed that APAP was metabolized 87% by tyrosinase at 2 h incubation. AA and NADH, quinone reducing agents, were significantly depleted during APAP oxidation by tyrosinase. The IC(50) (48 h) of APAP towards SK-MEL-28, MeWo, SK-MEL-5, B16-F0, and B16-F10 melanoma cells was 100 microM whereas it showed no significant toxicity towards BJ, Saos-2, SW-620, and PC-3 nonmelanoma cells, demonstrating selective toxicity towards melanoma cells. Dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depleting agent, enhanced APAP toxicity towards SK-MEL-28 cells. AA and GSH were effective in preventing APAP induced melanoma cell toxicity. Trifluoperazine and cyclosporin A, inhibitors of permeability transition pore in mitochondria, significantly prevented APAP melanoma cell toxicity. APAP caused time and dose-dependent decline in intracellular GSH content in SK-MEL-28, which preceded cell toxicity. APAP led to ROS formation in SK-MEL-28 cells which was exacerbated by dicoumarol and 1-bromoheptane whereas cyslosporin A and trifluoperazine prevented it. Our investigation suggests that APAP is a tyrosinase substrate, and that intracellular GSH depletion, ROS formation and induced mitochondrial toxicity contributed towards APAP's selective toxicity in SK-MEL-28 cells.
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PMID:Biochemical mechanism of acetaminophen (APAP) induced toxicity in melanoma cell lines. 1875 48


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