Sulfenic acid formation in human serum albumin by hydrogen peroxide and peroxynitrite

Human serum albumin (HSA), the most abundant protein in plasma, has been proposed to have an antioxidant role. The main feature responsible for this property is its only thiol, Cys34, which comprises approximately 80% of the total free thiols in plasma and reacts preferentially with reactive oxygen and nitrogen species. Herein, we show that the thiol in HSA reacted with hydrogen peroxide with a second-order rate constant of 2.26 M(-1) s(-1) at pH 7.4 and 37 degrees C and a 1:1 stoichiometry. The formation of intermolecular disulfide dimers was not observed, suggesting that the thiol was being oxidized beyond the disulfide. With the reagent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl), we were able to detect the formation of sulfenic acid (HSA-SOH) from the UV-vis spectra of its adduct. The formation of sulfenic acid in Cys34 was confirmed by mass spectrometry using 5,5-dimethyl-1,3-cyclohexanedione (dimedone). Sulfenic acid was also formed from exposure of HSA to peroxynitrite, the product of the reaction between nitric oxide and superoxide radicals, in the absence or in the presence of carbon dioxide. The latter suggests that sulfenic acid can also be formed through free radical pathways since following reaction with carbon dioxide, peroxynitrite yields carbonate radical anion and nitrogen dioxide. Sulfenic acid in HSA was remarkably stable, with approximately 15% decaying after 2 h at 37 degrees C under aerobic conditions. The formation of glutathione disulfide and mixed HSA-glutathione disulfide was determined upon reaction of hydrogen peroxide-treated HSA with glutathione. Thus, HSA-SOH is proposed to serve as an intermediate in the formation of low molecular weight disulfides, which are the predominant plasma form of low molecular weight thiols, and in the formation of mixed HSA disulfides, which are present in approximately 25% of circulating HSA.     

Metabolic control analysis of mitochondrial aconitase: influence over respiration and mitochondrial superoxide and hydrogen peroxide production

Abstract

The Fe-S cluster of mitochondrial aconitase is rapidly and selectively inactivated by oxidants, yielding an inactive enzyme that can be reactivated by reductants and iron in vivo. In order to elucidate the metabolic impact of oxidant-dependent aconitase inhibition over the citric acid cycle, the respiratory chain reactions, and reactive species formation, we performed a metabolic analysis using isolated mitochondria from different rat tissues. Titrations with fluorocitrate showed IC₅₀ for aconitase inhibition ranging from 7 to 24 μM. The aconitase inhibition threshold in mitochondrial oxygen consumption was determined to range from 63 to 98%. Of the tissues examined, brain and heart exhibited the highest values in the flux control coefficient (> 0.95). Aconitase-specific activity varied widely among tissues examined from ?60 mU/mg in liver to 321 mU/mg in kidney at 21% O₂. In brain and heart, aconitase-specific activity increased by 42 and 12%, respectively, at 2% O₂ reflecting aconitase inactivation by oxygen-derived oxidants at 21% O₂. Both mitochondrial membrane potential and hydrogen peroxide production significantly decreased upon aconitase inhibition in heart and brain mitochondria. These results indicate that aconitase can exert control over respiration (with tissue specificity) and support the hypothesis that inactivation of aconitase may provide a control mechanism to prevent O₂^●? and H₂O₂ formation by the respiratory chain.     

Possible involvement of hydroxyl radical on the stimulation of tetrahydrobiopterin synthesis by hydrogen peroxide and peroxynitrite in vascular endothelial cells

We recently described that hydrogen peroxide (H2O2) stimulates the synthesis of tetrahydrobiopterin (BH4) through the induction of the rate-limiting enzyme GTP-cyclohydrolase I (GTPCH), and increases tetrahydrobiopterin content in vascular endothelial cells. Tetrahydrobiopterin is easily oxidized by peroxynitrite (ONOO-), but not by hydrogen peroxide. The aim of this study was to determine the effect of hydroxyl radical and peroxynitrite, which are both toxic biological oxidants, on tetrahydrobiopterin synthesis and the regulation of its content in vascular endothelial cells. In the cell-free assay system, tetrahydrobiopterin was rapidly oxidized by the hydroxyl radical and peroxynitrite, but not by hydrogen peroxide. However, the addition of not only hydrogen peroxide but also the hydroxyl radical and peroxynitrite to vascular endothelial cells transiently decreased tetrahydrobiopterin content, and then markedly increased its content. Interestingly, total biopterin content was also decreased by early treatment with oxidants. Moreover, oxidants induced the expression of GTP-cyclohydrolase I, and the increase of the tetrahydrobiopterin content was blocked by the treatment with GTP-cyclohydrolase I inhibitor. Both the hydrogen peroxide- and peroxynitrite-induced increases in tetrahydrobiopterin content and findings suggest that not only hydrogen peroxide but also the hydroxyl radical and peroxynitrite stimulates tetrahydrobiopterin synthesis through GTP-cyclohydrolase I expression, and that the hydroxyl radical plays a central role in the stimulation of tetrahydrobiopterin synthesis. Moreover, the transient decrease in BH4 to tetrahydrobiopterin.     

Differential effects of superoxide,hydrogen peroxide,and hydroxyl radical on intracellular calcium in human endothelial cells

Changes in intracellular Ca²⁺ homeostasis are thought to contribute to cell dysfunction in oxidative stress. The hypoxanthine-xanthine oxidase system (X-XO) mobilizes Ca²⁺ from intracellular stores and induces a marked rise in cytosolic calcium in different cell types. To identify the reactive O₂ species involved in the disruption of calcium homeostasis by X-XO, we studied the effect of X-XO on [Ca²⁺]_i by spectrofluorimetry with fura-2 in human umbilical vein endothelial cells (HUVEC). The [Ca²⁺]_i response to X-XO was essentially diminished by superoxide dismutase (SOD) (200 U/ml) and catalase (CAT) (200 U/ml), which scavenge the superoxide anion, O₂^?, or H₂O₂, respectively. The [Ca²⁺]_i increase stimulated by 10 nmol H₂O₂/ml/min, generated from the glucose-glucose oxidase system, or 10 μM H₂O₂, given as bolus, was about a third of that induced by X-XO (10 nmol O₂^?/ml/min) but was comparable to that induced by X-XO in the presence of SOD. The X-XO—stimulated [Ca²⁺]_i increase was significantly reduced by 100 μM o-phenanthroline, which inhibits the iron-catalysed formation of the hydroxyl radical. On the other hand, the [Ca²⁺]_i response to low dose X-XO (1 nmol O₂^?/ml/min) was markedly enhanced in the presence of 1 μM H₂O₂, which itself had no effect on [Ca²⁺]_i. More than 50% of this synergistic effect was prevented by o-phenanthroline. These results indicate that the effect of X-XO on calcium homeostasis appears to result from an interaction of O₂^? and H₂O₂, which could be explained by the formation of the hydroxyl radical. © 1995 Wiley-Liss, Inc.     

Reaction of phenylaminoethyl selenides with peroxynitrite and hydrogen peroxide

Peroxynitrite, a reactive cytotoxic species generated by the reaction of superoxide with nitric oxide, rapidly oxidizes phenylaminoethyl selenide (PAESe) and its para-substituted derivatives with second-order rate constants ranging from 900 to 3000 M(-1) s(-1) at neutral pH (pH 7.0) and 25 degrees C. These values are approximately 3 x 10(4) times greater than the corresponding rate constants for the reactions of selenides with hydrogen peroxide. The peroxynitrite reaction was also studied at alkaline pH. HPLC analysis confirms that both the peroxynitrite and hydrogen peroxide reactions produced the corresponding phenylaminoethyl selenoxide (PAESeO) as the sole selenium-containing product, with a stoichiometry of 1 mol of PAESe oxidized per 1 mol of PAESeO formed per 1 mol of oxidant reacted. The influence of para-substituents on the rate constants was investigated using Hammett plots; in both cases the data are consistent with an S(N)2-type mechanism, wherein the selenium atom acts as the nucleophile. Our results provide further evidence that organoselenium compounds may play a protective role in the defense against the many reactive oxidizing species produced in cellular metabolism.     

Functional roles of superoxide and hydrogen peroxide generated by mitochondrial DNA mutation in regulating tumorigenicity of HepG2 cells

Mitochondria are a major source of reactive oxygen species (ROS). Recent studies have estimated that mitochondrial DNA mutations inducing the overproduction of ROS are associated with human cancer. However, a substantial challenge in elucidating their diverse roles in regulating tumorigenesis is the lack of methods for probing ROS in living systems with molecular specificity. In this study, we reported the application of two fluorescent probes, 2‐chloro‐1,3‐dibenzothiazolinecyclohexene and naphthofluorescein disulfonate, which showed high selectivity for superoxide (O₂^•−) and hydrogen peroxide (H₂O₂). They were capable of detecting and visualizing O₂^•− and H₂O₂ overproduction caused by a mutation in the gene encoding nicotinamide adenine dinucleotide dehydrogenase subunit 6 (ND6) in HepG2 cells. The levels of O₂^•− and H₂O₂ in mitochondria isolated from HepG2 cells were found to be 0·63 ± 0·07 and 1·13 ± 0·05 μM, respectively. Using assays of tumorigenesis in mouse models, we found that treatment of the mice with different ROS scavengers suppressed tumour growth. These findings suggested that ROS generated by ND6 gene mutation do play an important role in regulating tumorigenesis and H₂O₂ may be a key modulator. Copyright © 2011 John Wiley & Sons, Ltd.     

Reaction of peroxynitrite with reduced nicotinamide nucleotides, the formation of hydrogen peroxide.

NAD(P)H acts as a two-electron reductant in physiological, enzyme-controlled processes. Under nonenzymatic conditions, a couple of one-electron oxidants easily oxidize NADH to the NAD(.) radical. This radical reduces molecular oxygen to the superoxide radical (O-(2)) at a near to the diffusion-controlled rate, thereby subsequently forming hydrogen peroxide (H(2)O(2)). Because peroxynitrite can act as a one-electron oxidant, the reaction of NAD(P)H with both authentic peroxynitrite and the nitric oxide ((. )NO) and O-(2) releasing compound 3-morpholinosydnonimine N-ethylcarbamide (SIN-1) was studied. Authentic peroxynitrite oxidized NADH with an efficiency of approximately 25 and 8% in the absence and presence of bicarbonate/carbon dioxide (HCO(3)(-)/CO(2)), respectively. NADH reacted 5-100 times faster with peroxynitrite than do the known peroxynitrite scavengers glutathione, cysteine, and tryptophan. Furthermore, NADH was found to be highly effective in suppressing peroxynitrite-mediated nitration reactions even in the presence of HCO(3)(-)/CO(2). Reaction of NADH with authentic peroxynitrite resulted in the formation of NAD(+) and O-(2) and, thus, of H(2)O(2) with yields of about 3 and 10% relative to the added amounts of peroxynitrite and NADH, respectively. Peroxynitrite generated in situ from SIN-1 gave virtually the same results; however, two remarkable exceptions were recognized. First, the efficiency of NADH oxidation increased to 60-90% regardless of the presence of HCO(3)(-)/CO(2), along with an increase of H(2)O(2) formation to about 23 and 35% relative to the amounts of added SIN-1 and NADH. Second, and more interesting, the peroxynitrite scavenger glutathione (GSH) was needed in a 75-fold surplus to inhibit the SIN-1-dependent oxidation of NADH half-maximal in the presence of HCO(3)(-)/CO(2). Similar results were obtained with NADPH. Hence, peroxynitrite or radicals derived from it (such as, e.g. the bicarbonate radical or nitrogen dioxide) indeed oxidize NADH, leading to the formation of NAD(+) and, via O-(2), of H(2)O(2). When peroxynitrite is generated in situ in the presence of HCO(3)(-)/CO(2), i.e. under conditions mimicking the in vivo situation, NAD(P)H effectively competes with other known scavengers of peroxynitrite.     

Bovine aortic endothelial cells release hydrogen peroxide.

Endothelial cells grown on microcarriers are able to release H2O2 to the extracellular environment without any added stimulus. The extracellularly released H2O2 can be detected by luminol-amplified chemiluminescence (CL) if horseradish peroxidase is added. The CL response can be reduced by catalase and blocked by superoxide dismutase, indicating that O2- could be a precursor for H2O2. The CL kinetics, i.e., a long lag time followed by a rapid shift to a new level, indicate activation of an O2(-)-producing enzyme. The cells are also able to protect themselves from H2O2 stimulation by both catalase and the glutatione system. Bradykinin stimulates the H2O2 release, but if the effect is directly stimulatory or if it acts by reduction of the protective system is at present unclear. The extracellularly released H2O2 could be a cause of injury to the endothelial cells or to the subendothelial matrix.     

Neutrophil-mediated methemoglobin formation in the erythrocyte. The role of superoxide and hydrogen peroxide

Human neutrophils incubated with phorbol myristate acetate oxidized hemoglobin within the intact erythrocyte by a mechanism dependent on cell-cell contact but independent of phagocytosis. Spectrophotometric examination of the erythrocyte lysates revealed that the major component formed was methemoglobin along with small amounts of a species with spectral characteristics similar to choleglobin. Methemoglobin formation was directly related to the neutrophil concentration and the time of incubation. The addition of superoxide dismutase or catalase modestly inhibited the formation of methemoglobin, while a combination of the enzymes provided the most dramatic protection. Methemoglobin of hydroxyl radical or hypochlorous acid scavengers. Apparently, either O2.- or H2O2 alone was capable of mediating methemoglobin formation in the intact erythrocyte. Maintenance of the intraerythrocytic hemoglobin in its oxygenated state or its derivatization to carbon monoxyhemoglobin markedly inhibited methemoglobin formation. Blockade of the anion channels in the intact erythrocyte with sulfonated stilbenes inhibited O2.- but not H2O2 from oxidizing intracellular hemoglobin. It appears that neutrophil-derived O2.- and H2O2 can cross the erythrocyte membrane through the anion channel or diffuse directly into the intracellular space and react with oxyhemoglobin or deoxyhemoglobin to form a mixture of hemoglobin oxidation products within the intact cell.     

Selective involvement of superoxide anion, but not downstream compounds hydrogen peroxide and peroxynitrite, in tumor necrosis factor-alpha-induced apoptosis of rat mesangial cells

Tumor necrosis factor-alpha (TNF-alpha) induces reactive oxygen species (ROS) that serve as second messengers for intracellular signaling. Currently, precise roles of individual ROS in the actions of TNF-alpha remain to be elucidated. In this report, we investigated the roles of superoxide anion (O-(2)), hydrogen peroxide (H(2)O(2)), and peroxynitrite (ONOO(-)) in TNF-alpha-triggered apoptosis of mesangial cells. Mesangial cells stimulated by TNF-alpha produced O-(2) and underwent apoptosis. The apoptosis was inhibited by transfection with manganese superoxide dismutase or treatment with a pharmacological scavenger of O-(2), Tiron. In contrast, although exogenous H(2)O(2) induced apoptosis, TNF-alpha-triggered apoptosis was not affected either by transfection with catalase cDNA or by treatment with catalase protein or glutathione ethyl ester. Similarly, although ONOO(-) precursor SIN-1 induced apoptosis, treatment with a scavenger of ONOO(-), uric acid, or an inhibitor of nitric oxide synthesis, N(G)-nitro-L-argininemethyl ester hydrochloride, did not affect the TNF-alpha-triggered apoptosis. Like TNF-alpha-induced apoptosis, treatment with a O-(2)-releasing agent, pyrogallol, induced typical apoptosis even in the concurrent presence of scavengers for H(2)O(2) and ONOO(-). These results suggested that, in mesangial cells, TNF-alpha induces apoptosis through selective ROS. O-(2), but not H(2)O(2) or ONOO(-), was identified as the crucial mediator for the TNF-alpha-initiated, apoptotic pathway.     

Effect of hydrogen peroxide and superoxide anions on cytosolic Ca2+: comparison of endothelial cells from large-sized and small-sized arteries

We compared the Ca(2+) responses to reactive oxygen species (ROS) between mouse endothelial cells derived from large-sized arteries, aortas (aortic ECs), and small-sized arteries, mesenteric arteries (MAECs). Application of hydrogen peroxide (H(2)O(2)) caused an increase in cytosolic Ca(2+) levels ([Ca(2+)](i)) in both cell types. The [Ca(2+)](i) rises diminished in the presence of U73122, a phospholipase C inhibitor, or Xestospongin C (XeC), an inhibitor for inositol-1,4,5-trisphosphate (IP(3)) receptors. Removal of Ca(2+) from the bath also decreased the [Ca(2+)](i) rises in response to H(2)O(2). In addition, treatment of endothelial cells with H(2)O(2) reduced the [Ca(2+)](i) responses to subsequent challenge of ATP. The decreased [Ca(2+)](i) responses to ATP were resulted from a pre-depletion of intracellular Ca(2+) stores by H(2)O(2). Interestingly, we also found that Ca(2+) store depletion was more sensitive to H(2)O(2) treatment in endothelial cells of mesenteric arteries than those of aortas. Hypoxanthine-xanthine oxidase (HX-XO) was also found to induce [Ca(2+)](i) rises in both types of endothelial cells, the effect of which was mediated by superoxide anions and H(2)O(2) but not by hydroxyl radical. H(2)O(2) contribution in HX-XO-induced [Ca(2+)](i) rises were more significant in endothelial cells from mesenteric arteries than those from aortas. In summary, H(2)O(2) could induce store Ca(2+) release via phospholipase C-IP(3) pathway in endothelial cells. Resultant emptying of intracellular Ca(2+) stores contributed to the reduced [Ca(2+)](i) responses to subsequent ATP challenge. The [Ca(2+)](i) responses were more sensitive to H(2)O(2) in endothelial cells of small-sized arteries than those of large-sized arteries.     

Oxycytochrome P-450: its breakdown to superoxide for the formation of hydrogen peroxide.

Four different experimental studies are described which were designed to evaluate the role of oxycytochrome P-450 in the formation of superoxide anions and hydrogen peroxide. The use of lipophilic copper chelates with superoxide dismutase like activity revealed that the primary site of interaction of these agents is related to the inhibition of the flavoprotein. NADPH-cytochrome P-450 reductase. Measurements of the proton assisted nucleophilic displacement of superoxide from oxycytochrome P-450 by high concentrations of sodium azide indicated an increase in the rate of hydrogen peroxide formation concomitant with the inhibition of the N-demethylation of ethylmorphine. Studies on the effect of NADH on the rate of hydrogen peroxide formation during NADPH oxidation by liver microsomes failed to reveal a stimulatory or synergistic effect in a manner analogous to results obtained during the cytochrome P-450 dependent oxidation of substrates such as ethylmorphine. These results suggest that hydrogen peroxide formation may not require the reduction of oxycytochrome P-450 to peroxycytochrome P-450. Measurements of the reduction of succinylated cytochrome c using purified cytochrome P-450 and the flavoprotein, NADPH-cytochrome P-450 reductase, directly demonstrate the formation of superoxide anions. It is concluded that oxycytochrome P-450 may decompose to generate hydrogen peroxide.     

Ozone inhibits endothelial cell cyclooxygenase activity through formation of hydrogen peroxide

We have previously demonstrated that a 2H exposure of cultured pulmonary endothelial cells to ozone (0.0-1.0 ppm) in-vitro resulted in a concentration-dependent reduction of endothelial prostacyclin production (90% decrease at the 1.0 ppm level). Ozone-exposed endothelial cells, incubated with 20 uM arachidonate, also demonstrated a significant inhibition of prostacyclin synthesis. To further examine the mechanisms of the inhibition of prostacyclin synthesis, bovine pulmonary endothelial cells were exposed to 1.0 ppm ozone for 2H. A significant decrease in prostacyclin synthesis was found within 5 min of exposure (77 +/- 36% of air-exposed control values, p less than 0.05). Endothelial prostacyclin synthesis returned to baseline levels by 12H after ozone exposure, a time point which was similar to the recovery time of unexposed endothelium treated with 0.5 uM acetylsalicylic acid. Incubation of endothelial cells, previously exposed to 1.0 ppm ozone for 2 hours, with 4 uM PGH2 resulted in restoration of essentially normal prostacyclin synthesis. When endothelial cells were co-incubated with catalase (5 U/ml) during ozone exposure, no inhibition of prostacyclin synthesis was observed. Co-incubation with either heat-inactivated catalase or superoxide dismutase (10 U/ml) did not affect the ozone-induced inhibition of prostacyclin synthesis. These data suggest that H2O2 is a major toxic species produced in endothelial cells during ozone exposure and responsible for the inhibition of endothelial cyclooxygenase activity.     

Ozone inhibits endothelial cell cyclooxygenase activity through formation of hydrogen peroxide

We have previously demonstrated that a 2H exposure of cultured pulmonary endothelial cells to ozone (0.0–1.0 ppm) resulted in a concentration-dependent reduction of endothelial prostacyclin production (90% decrease at the 1.0 ppm level). Ozone-exposed endothelial cells, incubated with 20 uM arachidonate, also demonstrated a significant inhibition of prostacyclin synthesis. To further examine the mechanisms of the inhibition of prostacyclin synthesis, bovine pulmonary endothelial cells were exposedto 1.0 ppm ozone for 2H. A significant decease in protacyclin synthesis was found within 5 min of exposure (77 ± 36% of air-exposed control values, p < 0.05). Endothelial prostacyclin synthesis returned to baseline levels by 12H after ozone exposure, a time point which was similar to the recovery time of unexposed endothelium treated with 0.5 uM acetylsalicylic acid. Incubation of endothelial cells, previously exposed to 1.0 ppm ozone for 2 hours, with 4 uM PGH₂ resulted in restoration of essentially normal prostacyclin synthesis. When endothelial cells were co-incubated with catalase (5U/ml) during ozone exposure, no inhibition of prostacycline synthesis was observed. Co-incubation with either heat-inactivated catalase or superoxide dismutase (10U/ml) did not affect the ozone-induced inhibition of prostacycline synthesis. These data suggest that H₂O₂ is a major toxic species produced in endothelial cells during ozone exposure and responsible for the inhibiton of endothelial cyclooxygenase activity.     

Generation of superoxide anion and hydrogen peroxide at the surface of plant cells

In addition to well-known cell wall peroxidases, there is now evidence for the presence of this enzyme at the plasma membrane of the plant cells (surface peroxidase). Both are able to catalyze, through a chain of reactions involving the superoxide anion, the oxidation of NADH to generate hydrogen peroxide. The latter is oxidized by other wall-bound peroxidases to convert cinnamoyl alcohols into radical forms, which, then polymerize to generate lignin. However, there are other enzymes at the surface of plasma membranes capable of generating hydrogen peroxide (cell wall polyamine oxidase), superoxide anion (plasma membrane Turbo reductase), or both (plasma membrane flavoprotein?). These enzymes utilize NAD(P)H as a substrate. The Turbo reductase and the flavoprotein catalyze the univalent reduction of Fe³⁺ and then of O₂ to produce Fe²⁺ and \(O_2^{\bar \cdot } \) , respectively. The superoxide anion, in the acidic environment of the cell wall, may then dismutate to H₂O₂. These superoxide anion- and hydrogen peroxide-generating systems are discussed in relation to their possible involvement in physiological and pathological processes in the apoplast of plant cells.     

Mechanisms involved in the contraction of endothelial cells by hydrogen peroxide

The importance of endothelial contraction in the genesis of inflammatory edema has been reported. ROS are metabolites synthesized in pathological conditions in that a significant intravascular fluid leak occurs, such as ischemia-reperfusion. Present experiments were designed to test the hypothesis that ROS, particularly H2O2, may elicit the contraction of endothelial cells, and to explore the mechanisms involved. Bovine aortic endothelial cells incubated with H2O2 showed a significant reduction in planar cell surface area (PCSA), and a significant increase in myosin light chain phosphorylation (MLCP), with a time- and dose-dependent pattern, without any significant toxicity. This effect of H2O2 was not blocked by sulotroban (TxA2 antagonist) or BN 52021 (PAF antagonist). Lanthanum chloride (calcium channel blocker) and EGTA partially inhibited the increase in MLCP induced by H2O2. H7 and staurosporine, PKC inhibitors, and PKC down-regulation (phorbol myristate acetate treatment, 24 h) also blocked H2O2-dependent endothelial contraction, measured as PCSA or MLCP. H2O2 increased the intracellular calcium concentration, an effect blunted by EGTA and lanthanum chloride. H2O2 also increased the phosphorylation of an 80 kD polypeptide, probably MARCKS, a PKC substrate. In summary, the present results demonstrate the ROS-dependent contraction of endothelial cells, an effect that could explain the intravascular fluid leak observed in some pathophysiological situations. Calcium and PKC may be involved in the development of this contraction.     

Impact of SOD in eNOS uncoupling: a two-edged sword between hydrogen peroxide and peroxynitrite

In endothelial cell dysfunction, the uncoupling of eNOS results in higher superoxide (O₂^??) and lower NO production and a reduction in NO availability. Superoxide reacts with NO to form a potent oxidizing agent peroxynitrite (ONOO^?) resulting in nitrosative and nitroxidative stresses and dismutates to form hydrogen peroxide. Studies have shown superoxide dismutase (SOD) plays an important role in reduction of O₂^?? and ONOO^? during eNOS uncoupling. However, the administration or over-expression of SOD was ineffective or displayed deleterious effects in some cases. An understanding of interactions of the two enzyme systems eNOS and SOD is important in determining endothelial cell function. We analyzed complex biochemical interactions involving eNOS and SOD in eNOS uncoupling. A computational model of biochemical pathway of the eNOS-related NO and O₂^?? production and downstream reactions involving NO, O₂^??, ONOO^?, H₂O₂ and SOD was developed. The effects of SOD concentration on the concentration profiles of NO, O₂^??, ONOO^? and H₂O₂ in eNOS coupling/uncoupling were investigated. The results include (i) SOD moderately improves NO production and concentration during eNOS uncoupling, (ii) O₂^?? production rate is independent of SOD concentration, (iii) Increase in SOD concentration from 0.1 to 100 μM reduces O₂^?? concentration by 90% at all [BH₄]/[TBP] ratios, (iv) SOD reduces ONOO^? concentration and increases H₂O₂ concentration during eNOS uncoupling, (v) Catalase can reduce H₂O₂ concentration and (vi) Dismutation rate by SOD is the most sensitive parameter during eNOS uncoupling. Thus, SOD plays a dual role in eNOS uncoupling as an attenuator of nitrosative/nitroxidative stress and an augmenter of oxidative stress.     

Antibiotic activities of peptides, hydrogen peroxide and peroxynitrite in plant defence.

Genes encoding plant antibiotic peptides show expression patterns that are consistent with a defence role. Transgenic over-expression of defence peptide genes is potentially useful to engineer resistance of plants to relevant pathogens. Pathogen mutants that are sensitive to plant peptides in vitro have been obtained and a decrease of their virulence in planta has been observed, which is consistent with their hypothetical defence role. A similar approach has been followed to elucidate the potential direct anti-microbial role of hydrogen peroxide. Additionally, a scavenger of peroxynitrite has been used to investigate its involvement in plant defence.