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:1.8.1.4 (diaphorase)
2,754 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Reduction of exogenous lipoic acid to dihydrolipoate is known to occur in several mammalian cells and tissues. Dihydrolipoate is a potent radical scavenger, and may provide significant antioxidant protection. Because lipoic acid appears in the bloodstream after oral administration, we have examined the reduction of exogenous lipoate by human erythrocytes. Normal human erythrocytes reduced lipoate to dihydrolipoate only in the presence of glucose; deoxyglucose did not substitute for glucose, indicating that the reduction of lipoate requires glucose metabolism. Furthermore, the reduction was shown to be NADPH dependent. Erythrocytes isolated from a human subject with a genetic deficiency of glucose-6-phosphate dehydrogenase (and, therefore, deficient in the formation of NADPH) did not reduce lipoate. Dehydroepiandrosterone, a specific inhibitor of glucose-6-phosphate dehydrogenase, inhibited lipoate reduction. Our findings imply that some of the reduction of exogenous lipoic acid is catalysed by glutathione reductase, a flavoprotein dehydrogenase; mitomycin C, an inhibitor of FAD-dependent reductases, inhibited lipoate reduction by erythrocytes, and glutathione reductase purified from human erythrocytes was observed to reduce lipoic acid in a cell-free system. We further explored these findings with erythrocyte ghosts and liposomes. Our results indicate that a transport system exists for alpha-lipoic acid and dihydrolipoate; resealed erythrocyte ghosts, containing trapped lipoamide dehydrogenase and pyridine nucleotides, reduced externally added lipoate. By contrast, liposomes prepared with enzyme and pyridine nucleotides did not catalyze reduction of lipoate. This work indicates that uptake of exogenous lipoate and reduction to dihydrolipoate by normal human erythrocytes may contribute to oxidant protection in the human bloodstream.
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PMID:Reduction and transport of lipoic acid by human erythrocytes. 763 70

1. The standard O2-paradox has been studied in the Langendorff-perfused rat heart. 2. Perfusion of glucose-free saline under anoxia did not cause release of creatine kinase (CK) although, it is suggested, there was a progressive rise in [Ca2+]i. 3. Ca(2+)-depletion after anoxia caused CK release. 4. Prolonged anoxic perfusion (55 min) produced a markedly reduced release of CK on Ca(2+)-depletion because, it is suggested, of the reduction in substrates for the release mechanism. 5. No protection against the O2-paradox was found with oxygen radical scavengers and inhibitors. 6. Lowering [Ca2+]o during reoxygenation to 0.1 mM did not reduce CK release. 7. Neither 1 mM amiloride (Na+/H+ antiporter inhibitor) nor 2 x 10(-6) M 1-(5-isoquinolinesulphonyl) piperazine (protein kinase C inhibitor) reduced CK release, unlike their effects in the Ca(2+)-paradox. 8. An hypothesis for events in the O2-paradox in presented: anoxia causes a loss of Ca(2+)-homeostasis and a rise in [Ca2+]i thereby activating a transmembrane NAD(P) oxido-reductase/diaphorase (stage 1); the return of O2 synergistically activates this molecular complex and causes CK release (stage 2).
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PMID:Biochemical pathways of cell damage during the oxygen paradox of the rat heart. 810 57

Microbiosensors based on carbon and and platinum fibers are described. Carbon fibers were used to construct microelectrodes of 7 microm diameter. Electrochemical operations for pre-electrolysis and measuring were examined for the highly sensitive determination of hydrogen peroxide. A triangular potential (-2 to +2V vs Ag/AgCl) was applied before measuring each pair of double pulses (first pulse: 750 mV; second pulse: 1100 mV). The determination limit was 0.1 microM of hydrogen peroxide. The reproducible determination of hydrogen peroxide is possible even in samples containing albumin protein. The separation of hydrogen peroxide from ascorbic acid is also possible because the oxidation potential of ascorbic acid is different from that of hydrogen peroxide. An acetylcholine microsensor was fabricated by immobilizing acetylcholine esterase and choline oxidase on the carbon fiber by entrapment with poly(vinyl alcohol)-quarternized stilbazole (PVA-SbQ). This sensor gave a linear calibration plot for the range 0.1-1.0 mM with a linear correlation coefficient of 0.9842. Glucose oxidase (GOD) and glucose dehydrogenase (GDH) immobilized cylindrical platinum microelectrodes were fabricated, and their characteristics were evaluated, respectively, by using 1,4-benzoquinone (BQ) and ferricyanide as electron mediators. Each enzyme was immobilized by using PVA-SbQ on a cylindrical microelectrode of 2 microm diameter. A linear range in the calibration curve of the GOD-based glucose microsensor was observed to be wider than that obtained using a disk electrode of 1 mm diameter. The mediated response of the 2 microm glucose sensor was compared with the response resulting from hydrogen peroxide detection. This result showed that a higher response and a wider linear range were observed with highly concentrated mediator. A much higher response of the GDH immobilized 2 microm microelectrode was obtained when not only ferricyanide but also diaphorase was employed to reoxidize the NADH produced by the enzyme reaction of GDH. The GHD-based glucose microsensor was found to be unaffected by the concentration of dissolved oxygen.
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PMID:Microbiosensors for acetylcholine and glucose. 835 77

Islet-like cell clusters (ICCs) were prepared from the fetal porcine pancreas by a culture technique. The ICCs (approximately 500) were implanted under the left renal capsule of nude (nu/nu) C57BL/6J mice. Six weeks, months, 12 months, or 16-24 months later, the animals were anesthetized and the blood flows to the xenogeneic islet graft and the adjacent kidney parenchyma were measured with laser-Doppler flowmetry. After the blood flow measurements, the graft-bearing kidneys were prepared for enzyme and immunohistochemistry. The blood perfusion of the graft was higher than that of the kidney at all times investigated. Intraperitoneal administration of glucose caused only slight and parallel changes in renal and graft blood flows 6 weeks, 6 months, or 12 months after transplantation. However, in all but 1 animal (n=16) transplanted >16 months before the blood flow measurements, glucose caused a marked increase in graft blood flow but did not affect renal blood flow. Injection of 2-deoxy-glucose also increased graft blood perfusion in animals transplanted > 16 months earlier (n=5). Treatment with NG-monomethyl-L-arginine (n=6), an inhibitor of nitric oxide synthase, prevented this glucose-induced flow increase. Nicotinamide adenine dinucleotide phosphate diaphorase histochemistry revealed nitric oxide synthase only in the endothelium and media of graft arterioles in animals in the oldest age group. Thus, with the passage of time after implantation, the grafted xenogeneic ICCs seem to achieve an autonomous blood flow regulation, different from that of the implantation organ. The reactivity to an increment in blood glucose concentration in the graft is similar to that seen in native islets in the pancreas but is not present until >16 months after implantation. The mechanisms for the glucose-induced blood flow increase are obscure but probably depend on local release of nitric oxide within graft arterioles.
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PMID:Blood flow regulation in the transplanted fetal endocrine pancreas. Acquisition of a nitric oxide-dependent glucose-induced increase in blood flow. 860 82

The effect of R, S, and racemic forms of a-lipoic acid was tested on the formation of opacity in normal rat lenses incubated with 55.6 mM glucose, as a model for in vivo diabetic cataractogenesis. Control lenses, incubated 8 days with 5.56 mM glucose, did not develop opacities. Formation of lens opacities in vitro was correlated with lactate dehydrogenase (LDH) leakage into the incubation medium. Opacity formation and LDH leakage, resulting from incubation in medium containing 55.6 mM glucose to model diabetes, were both suppressed by the addition of 1 mM R-lipoic acid. Addition of 1 mM racemic lipoic acid reduces these damaging effects to the lens by one-half, while S-lipoic acid potentiated LDH leakage, consistent with the hypothesis that R-lipoic acid is the active form. Although HPLC analysis demonstrated that both stereoisomers of lipoic acid were reduced to dihydrolipoate at comparable rates by the intact lens, the mitochondrial lipoamide dehydrogenase system is highly specific for reduction of exogenous R-lipoic to dihydrolipoic acid. Therefore, stereospecific protection against this opacity is consistent with specific reduction of R-lipoic acid in mitochondria of the vulnerable cells at the lens equator where the first globular degeneration is seen in glucose cataract.
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PMID:Modelling cortical cataractogenesis 17: in vitro effect of a-lipoic acid on glucose-induced lens membrane damage, a model of diabetic cataractogenesis. 867 20

The principal goal of the present study was to test the hypothesis that cytokines modulate glucose transport in skeletal muscle by increasing nitric oxide production. Cultured L6 skeletal muscle cells were incubated in the presence of tumour necrosis factor-alpha, interferon-gamma or lipopolysaccharide (LPS) alone or in combination for 24 h. Neither cytokines nor LPS alone induced NO production, as measured by nitrite concentrations in the medium. However, when used in combination, the two cytokines significantly stimulated NO production, and this effect was synergistically enhanced by the presence of LPS. Reverse transcriptase-PCR (RT-PCR) analysis revealed that NO release was associated with the induction of inducible (macrophage-type) NO synthase (iNOS). The increase in iNOS expression was confirmed at the protein level by Western-blot analysis and NADPH/diaphorase histochemical staining. Cytokines and LPS markedly increased basal glucose transport in L6 myocytes. Insulin also stimulated basal glucose transport, but significantly less in cells chronically exposed to cytokines/LPS. The sensitivity of L6 muscle cells to insulin-stimulated glucose transport was also significantly decreased by cytokines/LPS treatment. The NOS inhibitor NG-nitro-l-arginine methyl ester (l-NAME) inhibited nitrite production in cytokine/LPS-treated cells, and this prevented the increase in basal glucose transport and restored muscle cell responsiveness to insulin. Cytokines/LPS exposure significantly increased GLUT1 transporter protein levels but decreased GLUT4 expression in L6 cells. l-NAME treatment prevented the increase in GLUT1 protein content but failed to restore GLUT4 transporter levels. These results demonstrate that cytokines and LPS affect glucose transport and insulin action by inducing iNOS expression and NO production in skeletal muscle cells. The data further indicate that cytokines and LPS increase the expression of the GLUT1 transporter protein by an NO-dependent mechanism.
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PMID:Cytokines modulate glucose transport in skeletal muscle by inducing the expression of inducible nitric oxide synthase. 923 Jan 32

Diabetes mellitus leads to micro- and macroangiopathy with endothelial dysfunction. To investigate the direct influence of high glucose on endothelial cell structure and possible pharmacologic effects, seven different experimental protocols were carried out on endothelial cells in culture. There were four control groups with either 5 mM D-glucose alone, 5 mM D-glucose plus 15 mM L-glucose (for osmotic control), 5 mM D-glucose plus 500 nM celiprolol, or 5 mM D-glucose plus 57 nM nitrendipine. Three experimental groups had either 20 mM D-glucose alone, 20 mM D-glucose plus 500 nM celiprolol or 20 mM D-glucose plus 57 nM nitrendipine. Treatment of all groups started at the third passage of the cells and lasted until confluence was reached (5-8 days). The endothelial cells were fixed in paraformaldehyde and stained either with hematoxylin-eosin solution, with nitro blue tetrazolium for nicotinamide adenine dinucleotide phosphate (NADPH)- diaphorase staining, or actin staining with phalloidin was carried out. For quantitative analysis of the histologic specimens, the slides were viewed via a microscope and a videocamera. The pictures were converted digitally and could be analyzed with the videopicture-analyzing system, JAVA. In the four control groups, neither treatment with 15 mM L-glucose nor administration of celiprolol or nitrendipine had an effect on cell, cytoplasm, and nuclear area. The number of giant or polynuclear cells and the histochemical NADPH-diaphorase activity were not altered. Incubation of endothelial cells with 20 mM D-glucose for 5-8 days resulted in a significant increase in total and cytoplasmic area, as well as in the number of giant and polynuclear cells, whereas the nuclear area and the NADPH-diaphorase activity were significantly reduced. Concomitant treatment with celiprolol was able to reverse these alterations in endothelial structure significantly but had only a weak effect on the NADPH-diaphorase. Nitrendipine had no beneficial effect on the high D-glucose-induced cell alterations. The actin staining of the control cells showed the typical actin pattern with most of the actin filaments arranged at the periphery of the cells. Administration of 20 mM D-glucose resulted in a disturbance of the actin pattern, with most of the actin filaments now arranged in the middle of the cells. However, neither celiprolol nor nitrendipine exhibited a significant influence on this altered actin structure. High D-glucose treatment over several days thus leads to severe changes in endothelial cell structure, and celiprolol may have a beneficial effect on these hyperglycemia-induced cell alterations.
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PMID:High D-glucose induces alterations of endothelial cell structure in a cell-culture model. 926 45

Previous studies have shown that nitric oxide synthase (NOS), the enzyme that catalyzes the formation of nitric oxide (NO), is expressed in skeletal muscle. The aim of the present study was to test the hypothesis that NO can modulate glucose metabolism in slow- and fast-twitch skeletal muscles. Calcium-dependent NOS was detected in skeletal muscle, and the enzyme activity was greater in fast-type extensor digitorum longus (EDL) muscles than in slow-type soleus muscles. Both the neuronal-type (nNOS) and endothelial-type (eNOS) enzymes are expressed in resting skeletal muscles. However, nNOS protein was only detected in EDL muscles, whereas eNOS protein contents were comparable in soleus and EDL muscles. NOS expression in muscle cryosections (diaphorase histochemistry) was located in vascular endothelium and in muscle fibers, and the staining was greater in type IIb than in type I and IIa fibers. The macrophage-type inducible NOS (iNOS) was not detected in resting muscle, but endotoxin treatment induced its expression, concomitant with elevated NO production. iNOS induction was associated with impaired insulin-stimulated glucose uptake in isolated rat muscles. In vitro, NOS blockade with specific inhibitors did not affect basal or insulin-stimulated glucose transport in EDL or soleus muscles. In contrast, the NO donors GEA 5024 and sodium nitroprusside induced dose-dependent inhibition (up to 50%) of maximal insulin-stimulated glucose transport in both muscles with minor effects on basal uptake values. GEA 5024 also blunted insulin-stimulated glucose transport and amino acid uptake in cultured L6 muscle cells without affecting insulin binding to its receptor. On the other hand, the permeable cGMP analogue dibutyryl cGMP did not affect muscle glucose transport. These results strongly suggest that NO modulates insulin action in both slow- and fast-type skeletal muscles. This novel autocrine action of NO in muscle appears to be mediated by cGMP-independent pathways.
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PMID:Expression of nitric oxide synthase in skeletal muscle: a novel role for nitric oxide as a modulator of insulin action. 935 14

Lucigenin is most noted for its wide use as a chemiluminescent detector of superoxide anion radical (O2-.) production by biological systems. However, its validity as a O2-.-detecting probe has recently been questioned in view of its ability to undergo redox cycling in several in vitro enzymatic systems, which produce little or no O2-.. Whether and to what extent lucigenin redox cycling occurs in systems that produce significant amounts of O2-. has not been carefully investigated. We examined and correlated three end points, including sensitive measurement of lucigenin-derived chemiluminescence (LDCL), O2 consumption by oxygen polarography, and O2-. production by 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide spin trapping to characterize the potential of lucigenin to undergo redox cycling and as such to act as an additional source of O2-. in various enzymatic and cellular systems. Marked LDCL was elicited at lucigenin concentrations ranging from 1 to 5 microM in all of the O2-.-generating systems examined, including xanthine oxidase (XO)/xanthine, lipoamide dehydrogenase/ NADH, isolated mitochondria, mitochondria in intact cells, and phagocytic NADPH oxidase. These concentrations of lucigenin were far below those that stimulated additional O2 consumption or O2-. production in the above systems. Moreover, a significant linear correlation between LDCL and superoxide dismutase-inhibitable cytochrome c reduction was observed in the XO/ xanthine and phagocytic NADPH oxidase systems. In contrast to the above O2-.-generating systems, no LDCL was observed at non-redox cycling concentrations of lucigenin in the glucose oxidase/glucose and XO/NADH systems, which do not produce a significant amount of O2-.. Thus, LDCL still appears to be a valid probe for detecting O2-. production by enzymatic and cellular sources.
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PMID:Validation of lucigenin (bis-N-methylacridinium) as a chemilumigenic probe for detecting superoxide anion radical production by enzymatic and cellular systems. 944 38

The genes encoding succinate dehydrogenase (sdhCDAB), the specific components of the 2-oxoglutarate dehydrogenase complex (ODH, E1o and E2o; sucAB) and succinyl-CoA synthetase (sucCD) form a cluster containing two promoters at 16.3 min in the chromosome of Escherichia coli: Psdh sdhCDAB-Psuc sucAB-sucCD. The gene encoding the lipoamide dehydrogenase component of both the 2-oxoglutarate and pyruvate dehydrogenase complexes (E3; lpdA) is the distal gene of another cluster containing two promoters located at 2.7 min: Ppdh pdhR-aceEF-Plpd lpdA. The responses of the suc and lpd promoters to different environmental conditions and to regulator defects were investigated with appropriate lacZ fusions, in order to understand how expression of the sucAB genes is co-regulated with other genes in the sdhCDAB-sucABCD cluster and with lpdA expression. Expression from the suc promoter was repressed by IHF and partially activated by sigma 38 but it was not regulated by ArcA, FNR, CRP, FruR or Fis, and not repressed by glucose or anaerobiosis, indicating that the well-established catabolite and anaerobic repression of ODH synthesis is imposed elsewhere. In contrast, the lpd promoter was repressed by both glucose (via a CRP-independent mechanism) and anaerobiosis (mediated by ArcA), and activated by Fis, but it was not regulated by FNR, FruR, IHF or sigma 38. These observations support the view that transcription of the sucABCD genes is primarily initiated and regulated at the upstream sdh promoter, and that the lpd promoter is independently co-regulated with Psdh (primarily by ArcA-mediated repression) rather than with Psuc. Direct evidence for co-transcription of the entire sdhCDAB-sucABCD region from Psdh was obtained by detecting a 10 kb transcript in rnc and rne mutants, but not in the parental strains. Three RNaseIII-specific processing sites, which contribute to the extreme instability of the readthrough transcript, were identified in the sdhCDAB-sucABCD intergenic region. Other sites of endonuclease processing were located by interpreting the patterns of transcript subfragments observed in Northern blotting.
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PMID:Transcription and transcript processing in the sdhCDAB-sucABCD operon of Escherichia coli. 972 32


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