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:1.5.1.19 (NOS)
7,285 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transgenic tobacco (Nicotiana tabacum L.) plants carrying a fusion between the nopaline synthase (nos) promoter and chloramphenicol acetyltransferase (CAT) reporter gene (caf) were tested for their response to treatment with H2O2. The nos promoter-driven CAT activity increased significantly by addition of H2O2, reaching the maximum level at 15 mM. Kinetic analysis for CAT activity showed that induction by H2O2 was similar to that of methyl jasmonate (MJ), but was much slower than induction by salicylic acid (SA). Time-course experiments for mRNA level also revealed that the response to H2O2 treatment was similar to that of MJ. The nos promoter displayed a rapid and transient induction of mRNA with SA treatment, with the maximum levels occurring at 3 h, whereas the levels induced by H2O2 or MJ treatment increased continuously during the 11-h experimental period. The antioxidants N-acetyl-L-cysteine and catechol did not alter the SA effect. The responses of the nos promoter to H2O2, MJ, and wounding were significantly reduced by deletions of the CAAT box region and the sequence between -112 and -101. However, these deletions did not significantly alter the SA response. This suggests that H2O2 may have a different mechanism from that of SA for inducing nos promotor activity.
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PMID:Induction of nopaline synthase promoter activity by H2O2 has no direct correlation with salicylic acid. 853 87

Interleukin-1 (IL-1) may be a mediator of beta-cell damage in insulin-dependent diabetes mellitus (IDDM). The IL-1 mechanism of action on insulin-producing cells probably includes activation of the transcription nuclear factor kappa B (NF-kappa B), increased transcription of the inducible form of nitric oxide synthase (iNOS) and the subsequent production of nitric oxide (NO). Reactive oxygen intermediates, particularly H2O2, have been proposed as second messengers for NF-kappa B activation. In the present study, we tested whether ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a glutathione peroxidase mimicking compound, could counteract the effects of IL-1 beta, H2O2 and alloxan in rat pancreatic islets and in the rat insulinoma cell line RINm5F (RIN cells). Some of these experiments were also reproduced in human pancreatic islets. Ebselen (20 microM) prevented the increase in nitrite production by rat islets exposed to IL-1 beta for 6 hr and induced significant protection against the acute inhibitory effects of alloxan or H2O2 exposure, as judged by the preserved glucose oxidation rates. However, ebselen failed to prevent the increase in nitrite production and the decrease in glucose oxidation and insulin release by rat islets exposed to IL-1 beta for 24 hr. Ebselen prevented the increase in nitrite production by human islets exposed for 14 hr to a combination of cytokines (IL-1 beta, tumor necrosis factor-alpha and interferon-gamma). In RIN cells, ebselen counteracted both the expression of iNOS mRNA and the increase in nitrite production induced by 6 hr exposure to IL-beta but failed to block IL-1 beta-induced iNOS expression following 24 hr exposure to the cytokine. Moreover, ebselen did not prevent IL-1 beta-induced NF-kappa B activation. As a whole, these data indicate that ebselen partially counteracts cytokine-induced NOS activation in pancreatic beta-cells, an effect not associated with inhibition of NF-kappa B activation.
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PMID:Ebselen and cytokine-induced nitric oxide synthase expression in insulin-producing cells. 898 32

The small subpopulation of striatal neurons containing nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d, recently identified as nitric oxide synthase, NOS) is selectively spared in Huntington's disease. Previous search for pathogenic mechanisms capable of destroying striatal neurons but sparing NADPH-d(+) cells has identified only NMDA receptor-mediated excitotoxicity. In view of suggestions that neuronal death in Huntington's disease may occur by apoptosis, we examined the vulnerability of NADPH-d(+) neurons to apoptosis. Murine striatal or cortical cultures exposed to serum deprivation developed extensive neuronal apoptosis, but NADPH-d(+) neurons were relatively spared. This sparing was seen when cultures were exposed to several other apoptosis-inducing insults. It was not seen after toxic exposure to H2O2, and it was not blocked by NOS inhibition. The selective resistance of NADPH-d(+) neurons to several forms of apoptosis provides key support for the possibility that apoptosis may contribute to the pathogenesis of Huntington's disease.
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PMID:NADPH diaphorase-containing striatal or cortical neurons are resistant to apoptosis. 917 14

In glomerulonephritides, autacoids such as nitric oxide (NO), reactive oxygen species, and prostanoids are produced in increased amounts in response to cytokines such as interleukin-1 (IL-1). These autacoids influence the expression of glomerular injury by their direct as well as interactive actions. We studied the effect of hydrogen peroxide (H2O2) on NO production in rat mesangial cells. We demonstrate that transient exposure of mesangial cells to H2O2 prior to sustained exposure to IL-1 decreased extracellular accumulation of NO2/NO3 and cellular guanosine 3,'5'-cyclic monophosphate (cGMP) content. H2O2 markedly impaired inducible nitric oxide synthase (iNOS) activity induced by IL-1 directly measured by the conversion of L-[14C]arginine to L-[14C]citrulline. Such impairment in iNOS activity was accompanied by a parallel reduction in iNOS protein abundance but not by a reduced expression of iNOS mRNA. The inhibitory effect of H2O2 on NOS activity was further supported by peroxide-induced impairment in IL-1-driven, NO-dependent synthesis of prostaglandin E2. Our studies thus provide the first direct evidence of a posttranscriptional inhibitory effect of H2O2 on iNOS activity. Additionally, our studies uncover the existence of a previously unrecognized effect of H2O2 on the production of NO that may exert influence on the severity of glomerular injury during glomerular inflammation.
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PMID:Hydrogen peroxide downregulates IL-1-driven mesangial iNOS activity: implications for glomerulonephritis. 922 32

Neuronal nitric oxide synthase (nNOS) catalyzes the oxidation of NG-hydroxy-L-arginine (NHA) by hydrogen peroxide. The amino acid products were characterized by high-performance liquid chromatography/mass spectrometry of the o-phthalaldehyde/2-mercaptoethanol derivatives and identified as citrulline and N delta-cyanoornithine (CN-orn). The assignment of CN-orn was confirmed by independent chemical synthesis and comparison of the properties of the enzyme-derived product with those of synthetic CN-orn. The inorganic products detected in the enzymatic reaction were NO2- and NO3-, presumably from oxidation of NO-. The reaction of H2O2 and NHA with nNOS was at least 10-fold slower than the reaction of NADPH, O2, and NHA (Vmax,app = 49 +/- 2 nmol min-1 mg-1 for the reactions with 10 microM added H4B). The reaction exhibited saturation kinetics with respect to hydrogen peroxide [K(m,app)(H2O2) = 10 +/- 1 mM for the reactions with 10 microM added H4B]. No H2O2-dependent reaction was observed with L-arginine as the amino acid substrate. The different products for the NADPH- and H2O2-dependent transformations of NHA are of mechanistic significance in the NOS reaction.
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PMID:Formation of N delta-cyanoornithine from NG-hydroxy-L-arginine and hydrogen peroxide by neuronal nitric oxide synthase: implications for mechanism. 939 65

Nitric oxide radicals are recognized as important mediators in various physiological and pathophysiological processes. During inflammation, increased amounts of nitric oxide (NO) are produced, but it is unclear whether NO radicals are either protective or harmful. To obtain more insight into the role of NO in glomerular inflammation, we studied the temporal expression of endothelial NO synthase (eNOS) and inducible NOS (iNOS) in conjunction with platelet aggregation, inflammatory cell influx, superoxide anion production cells, and nitrotyrosine formation in an experimental model of anti-myeloperoxidase (MPO) associated necrotizing crescentic glomerulonephritis (NCGN). Brown Norway rats were immunized with MPO in complete Freund's adjuvant (CFA) or CFA alone. After two weeks, the left kidney was perfused with a neutrophil lysosomal extract and H2O2. Rats were sacrificed at 24 hours, four days, and 10 days after perfusion. Kidney sections were stained by immunohistochemistry for eNOS, iNOS, platelets, nitrotyrosines, polymorphonuclear cells (PMN), monocytes, and T-cells. Superoxide anion producing cells were identified by enzyme cytochemistry using diaminobenzidine. Strong staining for eNOS was found in glomerular capillaries and interstitial tubular capillaries and larger vessels from non-perfused kidneys. At 24 hours after perfusion, glomerular and interstitial eNOS staining was greatly reduced, which was associated with massive platelet aggregation. At later time points, eNOS expression was absent in severely damaged glomeruli. Inducible NOS expression was found at all time points in infiltrating inflammatory cells, which by double labeling studies were identified as PMNs and monocytes. The peak in iNOS expression was observed at four days after perfusion but declined thereafter. Superoxide anion and nitrotyrosine generating cells were also found at all time points, but were most abundantly present at four days after perfusion, coinciding with the peak in iNOS expression. Double labeling experiments revealed that most nitrotyrosine generating cells also produced superoxide anions and expressed iNOS. In conclusion, these studies suggest that during the course of anti-MPO associated NCGN, loss of NO production by eNOS in conjunction with NO radical production by iNOS contribute to tissue injury. This is compatible with a protective role for eNOS contrasting with the possibly harmful effects of iNOS in anti-MPO associated NCGN.
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PMID:Expression of iNOS, eNOS, and peroxynitrite-modified proteins in experimental anti-myeloperoxidase associated crescentic glomerulonephritis. 946 Oct 97

We investigated how pH affects rat brain neuronal nitric oxide synthase (nNOS) with regard to spin-state equilibrium and the thiolate ligand bond of the haem group, catalytic activity, and monomerleft and right arrow dimer equilibrium. At neutral pH, nNOS containing 1 equiv. of (6R)-5,6,7,8-tetrahydro-l-biopterin (BH4) per dimer was mostly high-spin (lambdamax at 398 nm), whereas the BH4-free enzyme consisted of a mixture of the high-spin and two low-spin forms (lambdamax at 418 nm, and at 376 and 456 nm respectively). With BH4-free nNOS, an appreciable high-spin fraction was only observed between pH 7 and 8; at pH 6 and 9, the 418 and 376/456 nm low-spin forms predominated respectively. With nNOS containing 1 equiv. of BH4 per dimer, similar observations were made, but these involved only half of the enzyme; the other half, presumably the BH4-containing subunits, remained high-spin. Since the spin state in the BH4-free subunit appeared little affected by the state of the other subunit, we conclude that, in dimeric nNOS, the two haem groups function independently. Low pH destabilized thiolate binding and the interaction between NOS subunits, as indicated by CO-binding studies and gel electrophoresis respectively. Formation of l-citrulline was optimal between pH 7.0 and 7.5; the decrease in NOS activity at lower pH proved to be due to uncoupling of NADPH oxidation, resulting in increased formation of H2O2. At high pH strict coupling of l-arginine and NADPH oxidation was maintained, even in the absence of exogenous BH4. The possible pathophysiological implications of the uncoupling at low pH are discussed.
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PMID:Effects of pH on the structure and function of neuronal nitric oxide synthase. 956 Mar 7

Reactive oxygen species (ROS) are produced in ischemia and reperfusion. Since endothelial nitric oxide synthase (eNOS) is key to the endothelium-dependent vasodilation, we examined the effects of peroxide on this enzyme. We treated cells cultured from pig coronary artery endothelium with different concentrations of hydrogen peroxide, washed them, solubilized them and measured NOS activity by arginine to citrulline conversion. Hydrogen peroxide inhibited the eNOS activity with an IC50 value of 0.85 +/- 0.39 mM. In another experiment, we perfused arteries with solutions containing 0 or 1 mM hydrogen peroxide, washed them, removed the endothelium using a cotton swab, centrifuged and solubilized the endothelium and monitored its NOS activity. Hydrogen peroxide (1 mM) did not affect the NOS activity significantly (p > 0.05) in this assay. We conclude that the inactivation of eNOS by hydrogen peroxide does not play a major role in the ischemia-reperfusion damage because the peroxide concentrations attained during ischemia-perfusion are much lower than those affecting the eNOS activity.
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PMID:Effects of peroxide on endothelial nitric oxide synthase in coronary arteries. 965 89

Polymorphonuclear leukocytes (PMN) isolated from the oral cavity of healthy human volunteers, spontaneously generated superoxide, nitric oxide (NO) and other reactive oxygen species (ROS) which exhibited strong luminol chemiluminescence (LCL). To understand the physiological roles of oral PMN (OPMN), biochemical properties of the cells were analyzed. Biochemical analysis revealed that OPMN were already primed under physiological conditions. Western blot analysis revealed that they strongly expressed the inducible type of NO synthase (NOS II) and exhibited the activity to catalyze tyrosine phosphorylation of various proteins including a 115 kDa protein (cbl product). OPMN also generated H2O2 and .OH by some superoxide dismutase (SOD)-sensitive mechanism and released myeloperoxidase (MPO). Kinetic analysis using specific inhibitors revealed that OCl- generated by OPMN was predominantly responsible for the enhanced LCL. During the incubation under standard culture conditions, OPMN underwent apoptosis which proceeded more rapidly than that of the circulating PMN (CPMN). Immunochemical analysis revealed that expression of apoptosis-related gene products, such as Bcl-2, Bcl-xL and Bax, was below detectable levels with both cell types. However, caspase-3 but not caspase-1 was markedly activated in OPMN. These results indicate that the primed OPMN spontaneously generate ROS and play an important role in the defense mechanism in the oral cavity and that the generated ROS activate caspase-3 thereby inducing apoptosis of the cells.
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PMID:Biochemical properties of human oral polymorphonuclear leukocytes. 970 29

Nitric oxide (NO) synthases (NOSs), which catalyse the oxidation of L-arginine to L-citrulline and an oxide of nitrogen, possibly NO or nitroxyl (NO-), are subject to autoinhibition by a mechanism that has yet to be fully elucidated. In the present study we investigated the actions of NO and other NOS-derived products as possible autoregulators of enzyme activity. With the use of purified NOS-I, L-arginine turnover was found to operate initially at Vmax (0-15 min, phase I) although, despite the presence of excess substrate and cofactors, prolonged catalysis (15-90 min, phase II) was associated with a rapid decline in L-arginine turnover. Taken together, these observations suggested that one or more NOS products inactivate NOS. Indeed, exogenously applied reactive nitrogen oxide species (RNSs) decreased Vmax during phase I, although with different potencies (NO->NO> ONOO-) and efficacies (NO>NO-=ONOO-). The NO scavengers oxyhaemoglobin (HbO2; 100 microM) and 1H-imidazol-1 - yloxy - 2 - (4-carboxyphenyl) - 4,5 - dihydro - 4,4,5,5 - tetramethyl - 3 -oxide (CPTIO; 10 microM) and the ONOO- scavenger GSH (7 mM) had no effect on NOS activity during phase I, except for an endogenous autoinhibitory influence of NO and ONOO-. However, superoxide dismutase (SOD; 300 units/ml), which is thought either to increase the half-life of NO or to convert NO- to NO, lowered Vmax in an NO-dependent manner because this effect was selectively antagonized by HbO2 (100 microM). This latter observation demonstrated the requirement of SOD to reveal endogenous NO-mediated autoinhibition. Importantly, during phase II of catalysis, NOS became uncoupled and began to form H2O2 because catalase, which metabolizes H2O2, increased enzyme activity. Consistent with this, exogenous H2O2 also inhibited NOS activity during phase I. Thus during catalysis NOS is subject to complex autoinhibition by both enzyme-derived RNS and H2O2, differentially affecting enzyme activity.
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PMID:Autoinhibition of neuronal nitric oxide synthase: distinct effects of reactive nitrogen and oxygen species on enzyme activity. 1035 60


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