Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

In this work we have employed a new extraction buffer for isolation of cardiac lysosomes from control and ischemia canine tissue. Compared to previous techniques, this buffer (0.6 M KCl, 0.25 M sucrose) enabled a 300% increase in the yield of particulate cardiac lysosomes and allowed acceptable levels of specific activity to be maintained. It also permitted great enrichment of a membrane-bound enzyme localized to a microsomal fraction, rotenone-insensitive NADH-cytochrome c reductase (RINCR). Ischemia was produced by ligation of the left anterior descending coronary artery for 2 hr, and myocardial blood flow (MBF) was measured using 85Sr labeled microspheres (15 microns). Enzymatic changes were measured in endocardial tissue of the ischemic left ventricular wall for comparison with control nonischemic samples. N-Acetyl-beta-glucosaminidase (NAG) was the most reliable lysosomal marker enzyme; this was depleted significantly (P < 0.001) in particulate lysosomal fractions; significant increases in the supernatant (P < 0.001) were found in areas of ischemia that were less than 25% of the control MBF. Lysosomal latency also diminished significantly during ischemia. Loss of total activity of RINCR in the microsomal fraction was highly significant (P < 0.001) in the areas of profound ischemia. The above data were compared with those from animals in which methylprednisolone (30 mg/kg) was administered 30 min prior to ligation of the coronary artery. Between the two groups (untreated versus methylprednisolone-treated animals) no significant differences could be found in total losses of NAG and RINCR or the rate at which these enzymatic changes ocurred. Lysosomal latency studies also revealed lack of significant changes between both groups. Thus, no significant "protective" effects of methylprednisolone could be found after 2 hr of ischemia.
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PMID:Lysosomal changes in an animal model of myocardial ischemia. Treatment with methylprednisolone. 742 51

The effects of cerebral metabolism-improving drugs on NADPH diaphorase activity in the mouse brain were studied, and we found that diaphorase activity in the post-mitochondrial fraction of brain homogenate was enhanced by idebenone in a concentration-dependent manner. Histochemical studies also indicated that diaphorase staining was intensified by idebenone at the same concentration. These results suggest that idebenone may stimulate the production of nitric oxide, probably through its direct action on nitric oxide synthase, thus producing its protective action on neurological disorders due to cerebral hypoxia or ischemia as a consequence of dilating the cerebral blood vessels.
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PMID:Biochemical and histochemical studies of the effects of cerebral metabolism-improving drugs on NADPH diaphorase activity in mouse brain. 752 86

Nitric oxide can act as a neurotransmitter and a retrograde modulator of synaptic transmission, but uncontrolled nitric oxide synthase activity has been associated with neural degeneration. Although earlier studies using immunohistochemistry, in situ hybridization, and NADPH-diaphorase staining had suggested that nitric oxide synthase is not expressed in the CA1 neurons of the hippocampus, we have recently demonstrated that NADPH-diaphorase activity can be detected in CA1 neurons of the hippocampus. To confirm that this diaphorase activity reflects nitric oxide synthase, we have developed a more sensitive in situ hybridization procedure, and an RNase protection assay to detect message for constitutive nitric oxide synthase, the form constitutively expressed in many neurons. Message for constitutive nitric oxide synthase is expressed in the hippocampus, and it is localized to neural cell layers CA1, CA3, the dentate gyrus and some displaced neurons, but not to CA2. Expression of constitutive nitric oxide synthase message in the CA1 region was lost when pyramidal neurons died due to transient forebrain ischemia, supporting the conclusion that CA1 pyramidal cells express constitutive nitric oxide synthase. Although constitutive nitric oxide synthase message is strongly expressed in CA3 and the dentate gyrus, there is little diaphorase activity in these cells, suggesting that there may be post-transcriptional controls that limit constitutive nitric oxide synthase expression in some cells. Message for constitutive nitric oxide synthase is also present in a number of other regions, including the amygdala, several hypothalamic nuclei, the cerebellum, the olfactory bulb, two distinct regions of the perirhinal cortex, the subthalamic nuclei, a neuronal layer in the retrosplenial granular cortex, the lateral geniculate nucleus, the presubiculum, the inferior colliculus, the superior colliculus, the pedunculopontine tegmental nucleus, and scattered individual neurons in the cortex, hippocampus and brainstem. These studies support a role for nitric oxide in multiple regions of the central nervous system. In particular, nitric oxide synthase, the enzyme responsible for the synthesis of nitric oxide, is expressed in the CA1 region of the hippocampus, where there is evidence that nitric oxide may play a major role in long-term potentiation. CA1 hippocampal neurons are an example of a population of neurons that express constitutive nitric oxide synthase but are very sensitive to excitotoxicity and ischemic insults.
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PMID:Expression of the neural form of nitric oxide synthase by CA1 hippocampal neurons and other central nervous system neurons. 753 83

The status of glutathione (GSH) and protein thiol homeostasis was examined in rat brain regions during reperfusion after moderate and severe cerebral ischemia. GSH levels were decreased in brain regions during reperfusion for 1 hr after moderate or severe ischemia for 0.5 hr. Maximal loss of GSH (50-66%) was observed in the striatum and hippocampus. The GSH lost from the brain regions was essentially recovered as protein-glutathione mixed disulfide (PrSSG) with concomitant loss of protein thiols (PrSH). The activities of enzymes such as Na+K+ ATPase, NADH dehydrogenase and glutathione reductase were also inhibited but were restored after incubation of the brain homogenate with dithiothreitol. The depletion of GSH was also accompanied by an increase in the levels of malondialdehyde and reactive oxygen species. The total GSH recovered as sum of GSH and PrSSG was significantly higher than the sham-operated controls in the hippocampus and striatum after 1 hr of reperfusion, after moderate ischemia for 0.5 hr, and at the end of 24 hr of reperfusion the GSH-protein thiol homeostasis was restored. In contrast after 1 hr of reperfusion after severe ischemia, the GSH recovered as sum of GSH and PrSSG was not significantly different from sham-operated controls and at the end of 24 hr, 7 of 9 animals died. The recuperation of the brain from oxidative stress during reperfusion after moderate ischemia was thus preceded by increased recovery of total GSH essentially in the form of PrSSG. Thus, rapid restoration of thiol homeostasis in the brain during reperfusion may help the brain recover from reperfusion injury.
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PMID:Glutathione and protein thiol homeostasis in brain during reperfusion after cerebral ischemia. 756 84

Trimetazidine (TMZ) is an anti-ischemic compound whose precise mode of action is unknown, although several studies have suggested a metabolic effect, and there have been reports of protection of mitochondria against oxidative stress damage. Using a Langendorff rat heart model, we examined the effects of TMZ on the mitochondrial damage following 30 minutes of ischemia and 5 minutes of reperfusion. Mitochondrial respiration with succinate, glutamate-malate and ascorbate-N,N,N',N'-tetramethylphenylenediamine (TMPD) as substrates was significantly decreased following ischemia-reperfusion. Preperfusion with 10(-5) M TMZ had no effect on these rates in normoxic or ischemic hearts. However, 10(-3) M TMZ significantly decreased the glutamate-malate rate in mitochondria from normoxic hearts, and this rate was not further decreased following ischemia-reperfusion, and 10(-3) M TMZ also partially protected ascorbate-TMPD activity. The effect on glutamate-malate was probably due to an inhibition of complex I by TMZ, which specifically inhibited reduced nicotinamide-adenine-dinucleotide-cytochrome c reductase and complex I in lysed mitochondria. We also studied the effects of TMZ on the activity of pyruvate dehydrogenase (PDH) in normoxic and ischemic hearts perfused with 0.5 mM palmitate, which caused the enzyme to be almost completely inactivated. After short periods of ischemia (10-20 minutes) the PDH inactivation by palmitate was progressively lost. Preperfusion with 10(-5) M TMZ had a tendency to decrease lactate dehydrogenase release, accompanied by a maintenance of the inhibition of PDH by palmitate. This may allow the heart to oxidize fatty acids preferentially during reperfusion, hence removing possible toxic acyl esters.
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PMID:Trimetazidine effects on the damage to mitochondrial functions caused by ischemia and reperfusion. 764 24

Electron transport and production of O2-/H2O2 by the NADH dehydrogenase flavin-semiquinone (FMNH.) and ubisemiquinone (UQH.) were studied in a model of in vivo ischemia-reperfusion in rat kidney. H2O2 production rates were assessed in isolated mitochondria using either succinate, with and without antimycin, or malate-glutamate, with and without rotenone. Respiratory activities of isolated mitochondria and activity of NADH- and succinate-cytochrome c reductase and of NADH- and succinate-dehydrogenase in submitochondrial particles were measured to evaluate the electron flux throughout respiratory carriers. The mitochondrial H2O2 production rate was approximately 1.5- and 4-times increased in ischemic and ischemic-reperfused kidneys, respectively. Ischemia caused a marked decrease in the electron transport throughout the NADH-UQ segment with no significant changes either in the NADH dehydrogenase activity or in the electron flux trough the succinate-cytochrome oxidase segment. Reperfusion did not further affect the NADH-ubiquinone segment but markedly inhibited the succinate-supported oxygen consumption, succinate-cytochrome c reductase and succinate dehydrogenase activity. Our results show a redistribution of the electron flux with an increased rate of superoxide anion/hydrogen peroxide production at NADH dehydrogenase in mitochondria subjected to ischemia only. After 10 min reperfusion an impairment of the electron flow at succinate-cytochrome c segment is established and hydrogen peroxide production by UQH. increases up to maximal values becoming the major source of superoxide anion/hydrogen peroxide.
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PMID:Mitochondrial sites of hydrogen peroxide production in reperfused rat kidney cortex. 772 10

Copper Fenton systems (Cu(II)/H2O2 and Cu(II)/Asc) inactivated the lipoamide reductase and enhanced the diaphorase activity of pig-heart lipoamide dehydrogenase (LADH). Cupric ions alone were less effective. As a result of Cu(II)/H2O2 treatment, the number of titrated thiols in LADH decreased from 6 to 1 per subunit. NADH and ADP (not NAD+ or ATP) enhanced LADH inactivation by Cu(II). NADH also enhanced the effect of Cu(II)/H2O2. Dihydrolipoamide, dihydrolipoic acid, Captopril, acetylcysteine, EDTA, DETAPAC, histidine, bathocuproine, GSSG and trypanothione prevented LADH inactivation. 100 microM GSH, DL-dithiothreitol, N-(2-mercaptopropionylglicine) and penicillamine protected LADH against Cu(II)/Asc and Cu(II), whereas 1.0 mm GSH and DL-dithiothreitol also protected LADH against Cu(II)/H2O2. Allopurinol provided partial protection against Cu(II)/H2O2. Ethanol, mannitol, Na benzoate and superoxide dismutase failed to prevent LADH inactivation by Cu(II)/H2O2 or Cu(II). Catalase (native or denaturated) and bovine serum albumin protected LADH but that protection should be due to Cu binding. LADH inhibited deoxyribose oxidation and benzoate hydroxylation by Cu(II)/H2O2. It is concluded that site-specifically generated HO, radicals were responsible for LADH inactivation by Cu(II) Fenton systems. The latter effect is discussed in the context of ischemia-reoxygenation myocardial injury.
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PMID:Inactivation of heart dihydrolipoamide dehydrogenase by copper Fenton systems. Effect of thiol compounds and metal chelators. 775

Non-synaptosomal and synaptosomal mitochondrial membrane-linked enzymatic activities, NADH-cytochrome c reductase rotenone insensitive (marker of the outer membrane) and cytochrome oxidase (marker of the inner membrane), were measured in rat brain hippocampus and striatum immediately after and 1, 4 and 7 days following the induction of complete transient ischemia (15 min) by the four vessel occlusion method. Furthermore citrate synthetase activity was measured with and without Triton X-100 in order to qualitatively evaluate the membrane permeability. Non-synaptosomal mitochondrial membranes showed reduction of both activities only in the late reperfusion phase: NADH-CCRRi decreased in striatal mitochondria after 4-7 days and only after 7 days in the hippocampus. COX activity decreased only in striatal mitochondria 7 days after ischemia. Non-synaptosomal mitochondrial membrane permeability did not show changes. Synaptosomal mitochondria showed a decrease of NADH-CCRRi only at 7 days of reperfusion both in hippocampus and striatum, while COX activity decreased only during ischemia and returned to normal levels in the following days in the two areas considered. In summary, free mitochondria showed insensitiveness to ischemia but they resulted damaged in the late reperfusion phase, while mitochondria from the synaptic terminal showed ischemic damage, partially restored during reperfusion. The striatal mitochondria showed a major susceptibility to ischemia/reperfusion damage, showing changes earlier than the hippocampal ones.
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PMID:Changes in non-synaptosomal and synaptosomal mitochondrial membrane-linked enzymatic activities after transient cerebral ischemia. 787 28

DT diaphorase is a flavoprotein that enzymatically transfers two electrons from quinones as intermediate substrates and has been reported to increase its activity in the liver after exposure to toxicants. In this series of experiments, we tested the hypothesis that DT diaphorase also increases its activity after exposure to oxidants following gradient ischemia in skin. Using dorsal rat flaps, oxidant stress was induced immediately or during a 7-day period of preconditioning as a bipedicle flap before the distal attachment was divided. DT diaphorase activity (delta Abs/min/100 g) or expression of message was measured during the period of preconditioning to determine the relationship between skin survival, enzyme activity, and expression of message. There was 4.7 +/- 0.8 cm of skin necrosis in the distal end of acute flaps while the preconditioned flaps had no skin necrosis after the distal attachment was divided. In the acute flaps, the DT diaphorase activity was equal throughout the flap for the first 6 hr. After 24 hr of ischemia, the DT diaphorase activity was significantly higher in the proximal end of the flap (1.83 +/- 0.21 delta Abs/min/100 g) than that in the distal end (0.005 +/- 0.01 delta Abs/min/100 g), which was significant (P < 0.05). In the preconditioned flaps, enzyme activity did not increase but there was as 50-fold increase in DT diaphorase activity at the distal end 24 hr after they were divided (P < 0.05). Maximal enzyme induction of DT diaphorase activity occurred after 4 days of preconditioning and correlated with the maximal expression of mRNA. These studies provide the first evidence that DT diaphorase enzyme activity is inducible after oxidant stress. The data also suggests that DT activity remains elevated for at least 6 hr of ischemia and may be a potential source of anti-oxidant activity in ischemic skin.
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PMID:dT diaphorase: increased enzyme activity and mRNA expression in oxidant stress of skin. 815 25

Previous in vitro studies have shown that isolated mitochondria can generate oxygen radicals. However, whether a similar phenomenon can also occur in intact organs is unknown. In the present study, we tested the hypothesis that resumption of mitochondrial respiration upon reperfusion might be a mechanism of oxygen radical formation in postischemic hearts, and that treatment with inhibitors of mitochondrial respiration might prevent this phenomenon. Three groups of Langendorff-perfused rabbit hearts were subjected to 30 min of global ischemia at 37 degrees C, followed by reflow. Throughout ischemia and early reperfusion the hearts received, respectively: (a) 5 mM KCl (controls), (b) 5 mM sodium amobarbital (Amytal, which blocks mitochondrial respiration at Site I, at the level of NADH dehydrogenase), and (c) 5 mM potassium cyanide (to block mitochondrial respiration distally, at the level of cytochrome c oxidase). The hearts were then processed to directly evaluate oxygen radical generation by electron paramagnetic resonance spectroscopy, or to measure oxygen radical-induced membrane lipid peroxidation by malonyl dialdehyde (MDA) content of subcellular fractions. Severity of ischemia, as assessed by 31P-nuclear magnetic resonance measurements of cardiac ATP, phosphocreatine, and pH, was similar in all groups. Oxygen-centered free radical concentration averaged 3.84 +/- 0.54 microM in reperfused control hearts, and it was significantly reduced by Amytal treatment (1.98 +/- 0.26; p < 0.05), but not by KCN (2.58 +/- 0.96 microM; p = not significant (NS)), consistent with oxygen radicals being formed in the mitochondrial respiratory chain at Site I. Membrane lipid peroxidation of reperfused hearts was also reduced by treatment with Amytal, but not with KCN. MDA content of the mitochondrial fraction averaged 0.75 +/- 0.06 nM/mg protein in controls, 0.72 +/- 0.06 in KCN-treated hearts, and 0.54 +/- 0.05 in Amytal-treated hearts (p < 0.05 versus both groups). Similarly, MDA content of lysosomal membrane fraction was 0.64 +/- 0.09 nM/mg protein in controls, 0.79 +/- 0.15 in KCN-treated hearts, and 0.43 +/- 0.06 in Amytal-treated hearts (p < 0.05 versus both groups). Since the effects of Amytal are known to be reversible, in a second series of experiments we investigated whether transient mitochondrial inhibition during the initial 10 min of reperfusion was also associated with beneficial effects on subsequent recovery of cardiac function after wash-out of the drug. At the end of the experiment, recovery of left ventricular end-diastolic and of developed pressure was significantly greater in those hearts that had been treated with Amytal during ischemia and early reflow, as compared to untreated hearts.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Evidence that mitochondrial respiration is a source of potentially toxic oxygen free radicals in intact rabbit hearts subjected to ischemia and reflow. 839 7


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