Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.6.3.1 (NADPH oxidase)
 11,281 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The NAD(P)H cytochrome b5 oxidoreductase, Ncb5or (previously named b5+b5R), is widely expressed in human tissues and broadly distributed among the animal kingdom. NCB5OR is the first example of an animal flavohemoprotein containing cytochrome b5 and chrome b5 reductase cytodomains. We initially reported human NCB5OR to be a 487-residue soluble protein that reduces cytochrome c, methemoglobin, ferricyanide, and molecular oxygen in vitro. Bioinformatic analysis of genomic sequences suggested the presence of an upstream start codon. We confirm that endogenous NCB5OR indeed has additional NH2-terminal residues. By performing fractionation of subcellular organelles and confocal microscopy, we show that NCB5OR colocalizes with calreticulin, a marker for endoplasmic reticulum. Recombinant NCB5OR is soluble and has stoichiometric amounts of heme and flavin adenine dinucleotide. Resonance Raman spectroscopy of NCB5OR presents typical signatures of a six-coordinate low-spin heme similar to those found in other cytochrome b5 proteins. Kinetic measurements showed that full-length and truncated NCB5OR reduce cytochrome c actively in vitro. However, both full-length and truncated NCB5OR produce superoxide from oxygen with slow turnover rates: kcat = approximately 0.05 and approximately 1 s(-1), respectively. The redox potential at the heme center of NCB5OR is -108 mV, as determined by potentiometric titrations. Taken together, these data suggest that endogenous NCB5OR is a soluble NAD(P)H reductase preferentially reducing substrate(s) rather than transferring electrons to molecular oxygen and therefore not an NAD(P)H oxidase for superoxide production. The subcellular localization and redox properties of NCB5OR provide important insights into the biology of NCB5OR and the phenotype of the Ncb5or-null mouse.
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PMID:NCB5OR is a novel soluble NAD(P)H reductase localized in the endoplasmic reticulum. 1513 Nov 10

The mechanisms involved in the cytotoxic action of oxysterols in the pathogenesis of atherosclerosis still remain poorly understood. Among the major oxysterols present in oxidized low-density lipoprotein, we show here that 7-ketocholesterol (7-Kchol) induces oxidative stress and/or apoptotic events in human aortic smooth muscle cells (SMCs). This specific effect of 7-Kchol is mediated by a robust upregulation (threefold from the basal level) of Nox-4, a reactive oxygen species (ROS)-generating NAD(P)H oxidase homologue. This effect was highlighted by silencing Nox-4 expression with a specific small interfering RNA, which significantly reduced the 7-Kchol-induced production of ROS and abolished apoptotic events. Furthermore, the 7-Kchol activating pathway included an early triggering of endoplasmic reticulum stress, as assessed by transient intracellular Ca(2+) oscillations, and the induction of the expression of the cell death effector CHOP and of GRP78/Bip chaperone via the activation of IRE-1, all hallmarks of the unfolded protein response (UPR). We also showed that 7-Kchol activated the IRE-1/Jun-NH(2)-terminal kinase (JNK)/AP-1 signaling pathway to promote Nox-4 expression. Silencing of IRE-1 and JNK inhibition downregulated Nox-4 expression and subsequently prevented the UPR-dependent cell death induced by 7-Kchol. These findings demonstrate that Nox-4 plays a key role in 7-Kchol-induced SMC death, which is consistent with the hypothesis that Nox-4/oxysterols are involved in the pathogenesis of atherosclerosis.
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PMID:NAD(P)H oxidase Nox-4 mediates 7-ketocholesterol-induced endoplasmic reticulum stress and apoptosis in human aortic smooth muscle cells. 1557 75

The effects of hypoxia (pO2 approximately 25 mm Hg) on Ca2+ signaling stimulated by extracellular ATP in human saphenous vein endothelial cells were investigated using fluorimetric recordings from Fura-2 loaded cells. In the absence of extracellular Ca2+, ATP-evoked rises of cytosolic Ca2+ concentration ([Ca2+]i) because of mobilization from the endoplasmic reticulum (ER). These responses were reduced by prior exposure to hypoxia but potentiated during hypoxia. Hypoxia itself liberated Ca2+ from the ER, but unlike the effects of ATP this effect was not inhibited by blockade of the inositol trisphosphate receptor. By contrast, ryanodine blocked the effects of hypoxia but not those of ATP. Antioxidants abolished the effects of hypoxia but potentiated the effects of ATP. Inhibition of NADPH oxidase also augmented ATP-evoked responses but was without effect on hypoxia-evoked rises of [Ca2+]i. However, either uncoupling mitochondrial electron transport or inhibiting complex I markedly suppressed the actions of hypoxia yet exerted only small inhibitory effects on ATP-evoked rises of [Ca2+]i. Both hypoxia and ATP were able to activate capacitative Ca2+ entry. Our results indicate that hypoxia regulates intracellular Ca2+ signaling via two distinct pathways. First, it modulates agonist-evoked liberation of Ca2+ from the ER primarily through regulation of reactive oxygen species generation from NADPH oxidase. Second, it liberates Ca2+ from the ER via ryanodine receptors, an effect requiring mitochondrial reactive oxygen species generation. These findings suggest that local O2 tension is a major determinant of Ca2+ signaling in the vascular endothelium, a finding that is likely to be of both physiological and pathophysiological importance.
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PMID:Hypoxic modulation of Ca2+ signaling in human venous endothelial cells. Multiple roles for reactive oxygen species. 1566 29

Duox2 (and probably Duox1) is a glycoflavoprotein involved in thyroid hormone biosynthesis, as the thyroid H2O2 generator functionally associated with Tpo (thyroperoxidase). So far, because of the impairment of maturation and of the targeting process, transfecting DUOX into nonthyroid cell lines has not led to the expression of a functional H2O2-generating system at the plasma membrane. For the first time, we investigated the H2O2-generating activity in the particulate fractions from DUOX2- and DUOX1-transfected HEK293 and Chinese hamster ovary cells. The particulate fractions of these cells stably or transiently transfected with human or porcine DUOX cDNA demonstrate a functional NADPH/Ca2+-dependent H2O2-generating activity. The immature Duox proteins had less activity than pig thyrocyte particulate fractions, and their activity depended on their primary structures. Human Duox2 seemed to be more active than human Duox1 but only half as active as its porcine counterpart. TPO co-transfection produced a slight increase in the enzymatic activity, whereas p22(phox), the 22-kDa subunit of the leukocyte NADPH oxidase, had no effect. In previous studies on the mechanism of H2O2 formation, it was shown that mature thyroid NADPH oxidase does not release O2*- but H2O2. Using a spin-trapping technique combined with electron paramagnetic resonance spectroscopy, we confirmed this result but also demonstrated that the partially glycosylated form of Duox2, located in the endoplasmic reticulum, generates superoxide in a calcium-dependent manner. These results suggest that post-translational modifications during the maturation process of Duox2 could be implicated in the mechanism of H2O2 formation by favoring intramolecular superoxide dismutation.
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PMID:Dual oxidase-2 has an intrinsic Ca2+-dependent H2O2-generating activity. 1597 24

Reactive oxygen species (ROS), including superoxide anion, hydrogen peroxide, and hydroxyl radical, and reactive nitrogen species, such as nitric oxide and peroxynitrite, are biologically relevant O2 derivatives increasingly being recognized as important in vascular biology through their oxidation/reduction (redox) potential. All vascular cell types produce ROS primarily via membrane-associated NAD(P)H oxidase. ROS influence vascular function by modulating contraction/dilation, cell growth, apoptosis/anoikis, migration, inflammation, and fibrosis. An imbalance in redox state where prooxidants overwhelm antioxidant capacity results in oxidative stress. Oxidative excess and associated oxidative damage are mediators of altered vascular tone and structural remodeling in many cardiovascular diseases. ROS elicit these effects by influencing intracellular signaling events. In addition to modulating protein tyrosine kinases, protein phosphatases, mitogen-activated protein kinases, and transcription factors, ROS are important regulators of intracellular Ca2+ homeostasis and RhoA/Rho kinase signaling. ROS increase vascular [Ca2+]i by stimulating inositol trisphosphate-mediated Ca2+ mobilization, by increasing cytosolic Ca2+ accumulation through sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibition, and by stimulating Ca2+ influx through Ca2+ channels. Increased ROS generation enhances Ca2+ signaling and up-regulates RhoA/Rho kinase, thereby altering vascular contractility and tone. The present review discusses the importance of ROS in angiotensin II signaling in vascular biology and focuses specifically on the role of oxidative stress in Ca2+ signaling in the vasculature.
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PMID:Reactive oxygen species as mediators of calcium signaling by angiotensin II: implications in vascular physiology and pathophysiology. 1611 36

Membranous nephropathy (MN) is a common cause of nephrotic syndrome in adults. Active and passive Heymann nephritis (HN) in rats are valuable experimental models because their features so closely resemble human MN. In HN, subepithelial immune deposits form in situ as a result of circulating antibodies. Complement activation leads to assembly of C5b-9 on glomerular epithelial cell (GEC) plasma membranes and is essential for sublethal GEC injury and the onset of proteinuria. This review revisits HN and focuses on areas of substantial progress in recent years. The response of the GEC to sublethal C5b-9 attack is not simply due to disruption of the plasma membrane but is due to the activation of specific signaling pathways. These include activation of protein kinases, phospholipases, cyclooxygenases, transcription factors, growth factors, NADPH oxidase, stress proteins, proteinases, and others. Ultimately, these signals impact on cell metabolic pathways and the structure/function of lipids and key proteins in the cytoskeleton and slit-diaphragm. Some signals affect GEC adversely. Thus C5b-9 induces partial dissolution of the actin cytoskeleton. There is a decline in nephrin expression, reduction in F-actin-bound nephrin, and loss of slit-diaphragm integrity. Other signals, such as endoplasmic reticulum stress, may limit complement-induced injury, or promote recovery. The extent of complement activation and GEC injury is dependent, in part, on complement-regulatory proteins, which act at early or late steps within the complement cascade. Identification of key steps in complement activation, the cellular signaling pathways, and the targets will facilitate therapeutic intervention in reversing GEC injury in human MN.
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PMID:Experimental membranous nephropathy redux. 1615

Some properties of microsomal electron transfer chains, dependent for oxidase activity on addition of NADH or NADPH, duroquinone, and oxygen (L. De Luca et al., 1984, Plant Sci Lett 36: 93-98) are described. Activity is characterized by negatively cooperative kinetics toward reduced pyridine nucleotides, with limiting K(m) of 10 to 50 micromolar at pH 7.0 (increasing at lower pH), as well as toward duroquinone with limiting K(m) of 100 to 400 micromolar regardless of pH. Molecular oxygen is reduced by the enzyme complex with S(0.5) of about 30 micromolar and production of H(2)O and H(2)O(2), without superoxide involvement. The ratio NAD(P)H:O(2) averages 1.35 in the presence of KCN and 1.85 in its absence. The pyridine nucleotide specificity of the dehydrogenases has been investigated by kinetic competition experiments. Some enzyme heterogeneity was established for all preparations. At least two enzymes are detectable in plasma membrane-enriched fractions: a major NAD(P)H dehydrogenase having an acid pH optimum, and an NADPH dehydrogenase active around neutrality. Addition of Triton X-100 strongly enhances the activity over most of the pH scale, but depresses it increasingly at pH values higher than 8.0, to the effect that pH profile shows, under these conditions, a major peak at about pH 5.8 for both NADH and NADPH oxidase. Results with endoplasmic reticulum preparations are similar, except that they suggest the presence of still more activities at and above pH 7. The results are interpreted in terms of different complexes catalyzing electron transfer from NAD(P)H to O(2) without release of intermediates.
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PMID:Kinetic characterization of reduced pyridine nucleotide dehydrogenases (duroquinone-dependent) in cucurbita microsomes. 1666 30

Increased levels of reactive oxygen species (ROS) contribute to many cardiovascular diseases. In neutrophils, ROS are generated by a NADPH oxidase containing p22phox and NOX2. NADPH oxidases are also major sources of vascular ROS. Whereas an active NOX2-containing enzyme has been described in endothelial cells, the contribution of recently identified NOX homologues to endothelial ROS production and proliferation has been controversial. The authors, therefore, compared the role of NOX2 with NOX4 and NOX1 in endothelial EaHy926 and human microvascular endothelial cells. NOX2 and NOX4 were abundantly expressed, whereas NOX1 expression was less prominent. NOX2, NOX4, and NOX1 were simultaneously present in a single cell in a perinuclear compartment. NOX2 and NOX4 co-localized with the endoplasmic reticulum (ER) marker calreticulin. Additionally, NOX2 co-localized with F-actin at the plasma membrane. NOX2 and NOX4, which interacted with p22phox, as was shown by bimolecular fluorescent complementation, contributed equally to endothelial ROS production and proliferation, whereas NOX1 depletion did not alter ROS levels under basal conditions. These data show that endothelial cells simultaneously express NOX2, NOX4, and NOX1. NOX2 and NOX4, but not NOX1, equally contributed to ROS generation and proliferation under basal conditions, indicating that a complex relation between NOX homologues controls endothelial function.
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PMID:NOX2 and NOX4 mediate proliferative response in endothelial cells. 1698 4

Transforming growth factor-beta1 (TGF-beta1) plays a pivotal role in increasing airway smooth muscle mass in severe asthma by inducing proliferation and hypertrophy of human airway smooth muscle. The mechanism(s) for these effects of TGF-beta1 have not been fully elucidated. In this study, we demonstrate that TGF-beta1 is a potent inducer of expression of the nonphagocyte NAD(P)H oxidase catalytic homolog Nox4, diphenylene iodonium-inhibitable reactive oxygen species production, proliferation, and hypertrophy in cultured human airway smooth muscle cells. By confocal microscopy, TGF-beta1-induced Nox4 was localized with the endoplasmic reticulum and the nucleus, implying a role for Nox4 in regulation of both the cell cycle and protein synthesis. Consistent with this hypothesis, TGF-beta1 increased retinoblastoma protein phosphorylation at both Ser807/811 and Ser780. Silencing Nox4 prevented TGF-beta1-mediated retinoblastoma protein phosphorylation, proliferation, and cell hypertrophy. TGF-beta1 also increased phosphorylation of eukaryotic translation initiation factor 4E binding protein-1 at Thr37/46, and this was likewise blocked by silencing Nox4. This is the first report to suggest a functional role for Nox4 in cell cycle transition and to demonstrate that Nox4 influences the pathobiochemistry of asthma by generating reactive oxygen species that promote TGF-beta1-induced proliferation and hypertrophy of human airway smooth muscle.
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PMID:Nox4 mediates TGF-beta1-induced retinoblastoma protein phosphorylation, proliferation, and hypertrophy in human airway smooth muscle cells. 1736 89

The catabolism of heme, generating biliverdin, carbon monoxide, and free iron, is mediated by heme oxygenase (HO). One form of this of this enzyme, heme oxygenase-1, is inducible by numerous agents which promote oxidative stress, and is now known to provide important antioxidant protection, as demonstrated in many rodent models of free radical-mediated pathogenesis, and suggested by epidemiology observing favorable health outcomes in individuals carrying high-expression alleles of the HO-1 gene. The antioxidant impact of HO-1 appears to be mediated by bilirubin, generated rapidly from biliverdin by ubiquitously expressed biliverdin reductase. Bilirubin efficiently scavenges a wide range of physiological oxidants by electron donation. In the process, it is often reconverted to biliverdin, but biliverdin reductase quickly regenerates bilirubin, thereby greatly boosting its antioxidant potential. There is also suggestive evidence that bilirubin inhibits the activity or activation of NADPH oxidase. Increased serum bilirubin is associated with reduced risk for atherogenic disease in epidemiological studies, and more limited data show an inverse correlation between serum bilirubin and cancer risk. Gilbert syndrome, a genetic variant characterized by moderate hyperbilirubinemia attributable to reduced hepatic expression of the UDP-glucuronosyltransferase which conjugates bilirubin, has been associated with a greatly reduced risk for ischemic heart disease and hypertension in a recent study. Feasible strategies for boosting serum bilirubin levels may include administration of HO-1 inducers, supplementation with bilirubin or biliverdin, and administration of drugs which decrease the efficiency of hepatic bilirubin conjugation. The well-tolerated uricosuric drug probenecid achieves non-competitive inhibition of hepatic glucuronidation reactions by inhibiting the transport of UDP-glucuronic acid into endoplasmic reticulum; probenecid therapy is included in the differential diagnosis of hyperbilirubinemia, and presumably could be used to induce an ''iatrogenic Gilbert syndrome''. Other drugs, such as rifampin, can raise serum bilirubin through competitive inhibition of hepatocyte bilirubin uptake--although unfortunately rifampin is not as safe as probenecid. Measures which can safely achieve moderate serum elevations of bilirubin may prove to have value in the prevention and/or treatment of a wide range of disorders in which oxidants play a prominent pathogenic role, including many vascular diseases, cancer, and inflammatory syndromes. Phycobilins, algal biliverdin metabolites that are good substrates for biliverdin reductase, may prove to have clinical antioxidant potential comparable to that of bilirubin.
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PMID:''Iatrogenic Gilbert syndrome''--a strategy for reducing vascular and cancer risk by increasing plasma unconjugated bilirubin. 1782 97


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