The mechanisms for nitration and nitrotyrosine formation in vitro and in vivo: Impact of diet

The detection of 3-nitro-L-tyrosine residues associated with many disease states, including gastric cancer, has implicated a role for peroxynitrite in vivo, and thus endogenously produced nitric oxide and superoxide. Additionally, dietary nitrate has been suggested to be involved in the pathogenesis of gastric cancer through a mechanism involving reduction to nitrite and subsequent formation of potentially mutagenic nitrosocompounds. Studies have now demonstrated that a multitude of reactive nitrogen species other than peroxynitrite are capable of producing nitrotyrosine. Thus, we have reviewed the evidence that dietary nitrate, amongst other reactive nitrogen species, may contribute to the body burden of nitrotyrosine.     

Book reviews

The action of peroxynitrite in vivo has been proposed to account for the involvement of nitrotyrosine in the pathogenesis of many diseases. However, it has been demonstrated that nitrite under acidic conditions, similar to those in the human stomach, also has the ability to nitrate tyrosine. Dietary nitrate is also implicated in the progression of gastritis and gastric cancer and elevated levels of nitrate are found in many disease states in which nitrotyrosine may play a role. Thus, we investigated whether the dietary nitrate intake might contribute towards the plasma protein-bound levels of nitrotyrosine.

Seven healthy, non-smokers participated in a two-day study consisting of a nitrate-low control day followed by a day during which three nitrate-rich meals were consumed. Maximal urinary excretion was attained 4–6 hours after consumption of a meal and the maximum was proportional to the dose. Plasma nitrate was elevated nine-fold, 1 hour after consumption of a meal containing 128.3 mg nitrate. Plasma nitrated protein levels did not appear to alter significantly from basal 1 hour after supplementation with a nitrate-rich meal. Thus dietary nitrate does not appear to contribute to the levels of plasma nitrated proteins, as determined using a competitive inhibition of binding ELISA assay, but this does not preclude any contribution it may make to the total body burden of nitrotyrosine.     

Lack of influence of dietary nitrate/nitrite on plasma nitrotyrosine levels measured using a competitive inhibition of binding ELISA assay

The action of peroxynitrite in vivo has been proposed to account for the involvement of nitrotyrosine in the pathogenesis of many diseases. However, it has been demonstrated that nitrite under acidic conditions, similar to those in the human stomach, also has the ability to nitrate tyrosine. Dietary nitrate is also implicated in the progression of gastritis and gastric cancer and elevated levels of nitrate are found in many disease states in which nitrotyrosine may play a role. Thus, we investigated whether the dietary nitrate intake might contribute towards the plasma protein-bound levels of nitrotyrosine.

Seven healthy, non-smokers participated in a two-day study consisting of a nitrate-low control day followed by a day during which three nitrate-rich meals were consumed. Maximal urinary excretion was attained 4-6 hours after consumption of a meal and the maximum was proportional to the dose. Plasma nitrate was elevated nine-fold, 1 hour after consumption of a meal containing 128.3 mg nitrate. Plasma nitrated protein levels did not appear to alter significantly from basal 1 hour after supplementation with a nitrate-rich meal. Thus dietary nitrate does not appear to contribute to the levels of plasma nitrated proteins, as determined using a competitive inhibition of binding ELISA assay, but this does not preclude any contribution it may make to the total body burden of nitrotyrosine.     

Nitrate tolerance does not increase production of peroxynitrite in the heart

Clinical studies have suggested that long-term nitrate treatment does not improve and may even worsen cardiovascular mortality, and the possible role of nitrate tolerance has been suspected. Nitrate tolerance has been recently shown to increase vascular superoxide and peroxynitrite production leading to vascular dysfunction. Nevertheless, nitrates exert direct cardiac effects independent from their vascular actions. Therefore, we investigated whether in vivo nitroglycerin treatment leading to vascular nitrate tolerance increases cardiac formation of nitric oxide (NO), reactive oxygen species, and peroxynitrite, thereby leading to cardiac dysfunction. Nitrate tolerance increased bioavailability of NO in the heart without increasing formation of reactive oxygen species. Despite elevated myocardial NO, neither cardiac markers of peroxynitrite formation nor cardiac mechanical function were affected by nitroglycerin treatment. However, serum free nitrotyrosine, a marker for systemic peroxynitrite formation, was significantly elevated in nitroglycerin-treated animals. This is the first demonstration that, although the systemic effects of nitroglycerin may be deleterious due to enhancement of extracardiac peroxynitrite formation, nitroglycerin does not result in oxidative damage in the heart.     

Nitrate tolerance aggravates postischemic myocardial apoptosis and impairs cardiac functional recovery after ischemia

Objectives: This study examined the effects of nitrate tolerance (NT) on myocardial ischemia reperfusion (MI/R) injury and elucidated the potential mechanisms involved. Furthermore, the effects of GSH on postischemic myocardial apoptosis in NT rats were investigated. Methods and results: Male Sprague–Dawley rats were randomized to receive nitroglycerin (60 μg/kg/h) or saline for 12 h followed by 40 min of MI and 4 h of reperfusion. Myocardial apoptosis, infarct size, nitrotyrosine formation, plasma CK and LDH activity, and cardiac function were determined. MI/R resulted in significant apoptotic cell death, which was further increased in animals with NT. In addition, NT further increased plasma CK and LDH activity, enlarged infarct size, and impaired cardiac functional recovery after ischemia. Myocardial nitrotyrosine, a footprint for cytotoxic reactive nitrogen species formation, was further enhanced in the NT heart after MI/R. Treatment of NT animals with exogenous GSH inhibited nitrotyrosine formation, reduced apoptosis, decreased infarct size, and improved cardiac functional recovery. Conclusion: Our results demonstrate that nitrate tolerance markedly enhances MI/R injury and that increased peroxynitrite formation likely plays a role in this pathologic process. In addition, our results suggest that GSH could decrease peroxynitrite formation and reduce MI/R injury in nitrate tolerant hearts.     

Temporal patterns of tyrosine nitration in embryo heart development

Tyrosine nitration is a biomarker for the production of peroxynitrite and other reactive nitrogen species. Nitrotyrosine immunoreactivity is present in many pathological conditions including several cardiac diseases. Because the events observed during heart failure may recapitulate some aspects of development, we tested whether nitrotyrosine is present during normal development of the rat embryo heart and its potential relationship in cardiac remodeling through apoptosis. Nitric oxide production is highly dynamic during development, but whether peroxynitrite and nitrotyrosine are formed during normal embryonic development has received little attention. Rat embryo hearts exhibited strong nitrotyrosine immunoreactivity in endocardial and myocardial cells of the atria and ventricles from E12 to E18. After E18, nitrotyrosine staining faded and disappeared entirely by birth. Tyrosine nitration in the myocardial tissue coincided with elevated protein expression of nitric oxide synthases (eNOS and iNOS). The immunoreactivity for these NOS isoforms remained elevated even after nitrotyrosine had disappeared. Tyrosine nitration did not correlate with cell death or proliferation of cardiac cells. Analysis of tryptic peptides by MALDI-TOF showed that nitration occurs in actin, myosin, and the mitochondrial ATP synthase α chain. These results suggest that reactive nitrogen species are not restricted to pathological conditions but may play a role during normal embryonic development.     

The role of reactive nitrogen species in secondary spinal cord injury: formation of nitric oxide, peroxynitrite, and nitrated protein

To determine whether reactive nitrogen species contribute to secondary damage in CNS injury, the time courses of nitric oxide, peroxynitrite, and nitrotyrosine production were measured following impact injury to the rat spinal cord. The concentration of nitric oxide measured by a nitric oxide-selective electrode dramatically increased immediately following injury and then quickly declined. Nitro-L-arginine reduced nitric oxide production. The extracellular concentration of peroxynitrite, measured by perfusing tyrosine through a microdialysis fiber into the cord and quantifying nitrotyrosine in the microdialysates, significantly increased after injury to 3.5 times the basal level, and superoxide dismutase and nitro-L-arginine completely blocked peroxynitrite production. Tyrosine nitration examined immunohistochemically significantly increased at 12 and 24 h postinjury, but not in sham-control sections. Mn(III) tetrakis(4-benzoic acid)-porphyrin (a novel cell-permeable superoxide dismutase mimetic) and nitro-L-arginine significantly reduced the numbers of nitrotyrosine-positive cells. Protein-bound nitrotyrosine was significantly higher in the injured tissue than in the sham-operated controls. These results demonstrate that traumatic injury increases nitric oxide and peroxynitrite production, thereby nitrating tyrosine, including protein-bound tyrosine. Together with our previous report that trauma increases superoxide, our results suggest that reactive nitrogen species cause secondary damage by nitrating protein through the pathway superoxide + nitric oxide peroxynitrite protein nitration.     

Immunological detection of nitrosative stress-mediated modified Tamm–Horsfall glycoprotein (THP) in calcium oxalate stone formers

Abstract

The generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in hyperoxaluric condition has been proved experimentally. This may result in the formation of the cytotoxic metabolite peroxynitrite, which is capable of causing lipid peroxidation and protein modification. The presence of nitrotyrosine in proteins has been associated with several pathological conditions. The present study investigated the presence of nitrotyrosine in the stone formers Tamm–Horsfall glycoprotein (THP). In vitro nitration of control THP was carried out using peroxynitrite. New Zealand white rabbits were immunized with peroxynitrated THP at 15-day intervals. Antisera collected following the third immunization were assayed for antibody titres using solid-phase ELISA. Antibodies were purified by affinity chromatography. The carbonyl content of control, stone formers and nitrated THP were determined. Western blotting was carried with control, stone formers and nitrated THPs. Immunodiffusion studies demonstrated cross-reaction with nitrated bovine serum albumin. Significant amounts (p<0.001) of carbonyl content were present in both stone formers and nitrated THPs. Western blot analysis confirmed the presence of nitrated amino acid 3-nitrotyrosine in stone formers, which could bring about structural and functional modifications of THP in hyperoxaluric patients. A cross-reaction with nitrated bovine serum albumin confirms that the raised antibody has certain paratopes similar to the epitope of nitrated protein molecules. Detection of 3-nitrotyrosine in stone formers THP indicates that it is one of the key factors influencing the conversion of THP to a structurally and immunologically altered form during calcium oxalate stone formation.     

Significance of ERK nitration in portal hypertensive gastropathy and its therapeutic implications

Portal hypertensive (PHT) gastric mucosa increases susceptibility to injury and delayed mucosal healing. It is possible that nitration of ERK by peroxynitrite might alter MAPK (ERK) signaling in PHT gastric mucosa, leading to delayed mucosal healing, since excessive nitric oxide production is implicated in PHT gastric mucosa and MAPK (ERK) signaling induces cell proliferation and leads to gastric mucosal healing in response to injury. Portal hypertension was produced by staged portal vein ligation, and sham-operation (SO) rats served as controls. Lipid peroxide (LPO) and nitrotyrosine increased significantly in PHT gastric mucosa compared with SO rats. ERK activation was impaired in PHT gastric mucosa in response to ethanol injury, whereas no significant difference in the phosphorylation of MEK, an upstream molecule of ERK, was seen between the two groups. The nitration of ERK by peroxynitrite, as detected by the coimmunoprecipitation of ERK and nitrotyrosine, was significantly enhanced in PHT gastric mucosa. Administration of rebamipide, a gastroprotective drug that acts as an oxygen-derived free radical scavenger, significantly decreased LPO and nitrotyrosine as well as the nitration of ERK by peroxynitrite in PHT gastric mucosa, therefore normalizing ERK activation and restoring the gastric mucosal healing response to ethanol injury. Enhanced nitration of ERK by peroxynitrite is involved in the impaired MAPK (ERK) signaling in PHT gastric mucosa. These findings demonstrate a new molecular mechanism in which PHT gastric mucosa is predisposed to injury and impaired healing.     

Nitrotyrosine formation and its role in various pathological conditions

The formation of peroxynitrite and nitrotyrosine was examined in a variety of in vitro and in vivo animal models and its relation to cell or tissue damage was examined. In polymorphonuclear leukocyte (PMN)-induced injury to cardiac myocytes or endothelial cells, activated PMN produced peroxynitrite. Peroxynitrite appears to be responsible for the injury but it was not a major mediator of endothelial cell injury. In the experiment of ischemia-reperfusion injury of the rat brain nitrotyrosine was formed in the peri-infarct and core-of infarct regions. The degradation curve of nitrotyrosine revealed that its t1/2 was about 2.2 hours. In the radiation-induced lung injury of rats, nitrotyrosine was also formed but it was not the sole mechanism for the injury. Levels of nitrotyrosine correlated with the severity of myocardial dysfunction in the canine model of cytokine-induced cardiac injury. Inhibition of NO generation abolished the formation of peroxynitrite and nitrotyrosine in all experiments. In conclusion; although nitrotyrosine is formed in a variety of pathological conditions where the generation of NO is increased, its presence does not always correlate with the severity of injury.     

Nitric oxide and peroxynitrite. The ugly,the uglier and the not so good

Nitric oxide, a gaseous free radical, is poorly reactive with most biomolecules but highly reactive with other free radicals. Its ability to scavenge peroxyl and other damaging radicals may make it an important antioxidant in vivo, particular in the cardiovascular system, although this ability has been somewhat eclipsed in the literature by a focus on the toxicity of peroxynitrite, generated by reaction of O^·-₂ with NO^· (or of NO^- with O₂). On balance, experimental and theoretical data support the view that ONOO^- can lead to hydroxyl radical (OH^·) generation at pH 7.4, but it seems unlikely that OH^· contributes much to the cytotoxicity of ONOO^-. The cytotoxicity of ONOO^- may have been over-emphasized: its formation and rapid reaction with antioxidants may provide a mechanism of using NO^· to dispose of excess O^·-₂, or even of using O^·-₂ to dispose of excess NO^·, in order to maintain the correct balance between these radicals in vivo. Injection or instillation of “bolus” ONOO^- into animals has produced tissue injury, however, although more experiments generating ONOO^- at steady rates in vivo are required. The presence of 3-nitrotyrosine in tissues is still frequently taken as evidence of ONOO^- generation in vivo, but abundant evidence now exists to support the view that it is a biomarker of several “reactive nitrogen species”. Another under-addressed problem is the reliability of assays used to detect and measure 3-nitrotyrosine in tissues and body fluids: immunostaining results vary between laboratories and simple HPLC methods are susceptible to artefacts. Exposure of biological material to low pH (e.g. during acidic hydrolysis to liberate nitrotyrosine from proteins) or to H₂O₂ might cause artefactual generation of nitrotyrosine from NO^-₂ in the samples. This may be the origin of some of the very large values for tissue nitrotyrosine levels quoted in the literature. Nitrous acid causes not only tyrosine nitration but also DNA base deamination at low pH: these events are relevant to the human stomach since saliva and many foods are rich in nitrite. Several plant phenolics inhibit nitration and deamination in vitro, an effect that could conceivably contribute to their protective effects against gastric cancer development.     

Superoxide reacts with nitric oxide to nitrate tyrosine at physiological pH via peroxynitrite

Tyrosine nitration is a widely used marker of peroxynitrite (ONOO(-)) produced from the reaction of nitric oxide with superoxide. Pfeiffer and Mayer (Pfeiffer, S., and Mayer, B. (1998) J. Biol. Chem. 273, 27280-27285) reported that superoxide produced from hypoxanthine plus xanthine oxidase in combination with nitric oxide produced from spermine NONOate did not nitrate tyrosine at neutral pH. They suggested that nitric oxide and superoxide at neutral pH form a less reactive intermediate distinct from preformed alkaline peroxynitrite that does not nitrate tyrosine. Using a stopped-flow spectrophotometer to rapidly mix potassium superoxide with nitric oxide at pH 7.4, we report that an intermediate spectrally and kinetically identical to preformed alkaline cis-peroxynitrite was formed in 100% yield. Furthermore, this intermediate nitrated tyrosine in the same yield and at the same rate as preformed peroxynitrite. Equivalent concentrations of nitric oxide under aerobic conditions in the absence of superoxide did not produce detectable concentrations of nitrotyrosine. Carbon dioxide increased the efficiency of nitration by nitric oxide plus superoxide to the same extent as peroxynitrite. In experiments using xanthine oxidase as a source of superoxide, tyrosine nitration was substantially inhibited by urate formed from hypoxanthine oxidation, which was sufficient to account for the lack of tyrosine nitration previously reported. We conclude that peroxynitrite formed from the reaction of nitric oxide with superoxide at physiological pH remains an important species responsible for tyrosine nitration in vivo.     

Peroxynitrite affects exocytosis and SNARE complex formation and induces tyrosine nitration of synaptic proteins

The reactive species peroxynitrite, formed via the near diffusion-limited reaction of nitric oxide and superoxide anion, is a potent oxidant that contributes to tissue damage in neurodegenerative disorders. Peroxynitrite readily nitrates tyrosine residues in proteins, producing a permanent modification that can be immunologically detected. We have previously demonstrated that in the nerve terminal, nitrotyrosine immunoreactivity is primarily associated with synaptophysin. Here we identify two other presynaptic proteins nitrated by peroxynitrite, Munc-18 and SNAP25, both of which are involved in sequential steps leading to vesicle exocytosis. To investigate whether peroxynitrite affects vesicle exocytosis, we used the fluorescent dye FM1-43 to label a recycling population of secretory vesicles within the synaptosomes. Bolus addition of peroxynitrite stimulated exocytosis and glutamate release. Notably, these effects were strongly reduced in the presence of NaHCO(3), indicating that peroxynitrite acts mainly intracellularly. Furthermore, peroxynitrite enhanced the formation of the sodium dodecyl sulfate-resistant SNARE complex in a dose-dependent manner (100-1000 microm) and induced the formation of 3-nitrotyrosine in proteins of SNARE complex. These data suggest that modification(s) of synaptic vesicle proteins induced by peroxynitrite may affect protein-protein interactions in the docking/fusion steps, thus promoting exocytosis, and that, under excessive production of superoxide and nitric oxide, neurons may up-regulate neuronal signaling.     

Mercaptoethylguanidine attenuates inflammation in bacterial meningitis in rabbits

Reactive oxygen and nitrogen species participate in the inflammatory process during meningitis. Among them, superoxide, nitric oxide (NO), and their reaction product peroxynitrite exert cytotoxic effects. Mercaptoethylguanidine (MEG) exerts beneficial effects in in vivo inflammatory conditions by scavenging peroxynitrite and inhibiting the inducible NO synthase. This study was designed to investigate whether MEG may attenuate inflammation and brain injury in experimental meningitis. Meningitis increased nitrite/nitrate, and protein content in the cerebrospinal fluid (CSF). In the brain tissue high levels of malondialdehyde and formation of nitrotyrosine indicated lipid peroxidation and nitrosative stress, respectively. Myeloperoxidase activity was increased indicating accumulation of neutrophils into the brain parenchyma. Treatment with MEG decreased nitrite/nitrate levels whereas it did not affect the bacterial clearance from the CSF. Furthermore, treatment with MEG markedly reduced brain tissue levels of myeloperoxidase and malondialdehyde. These data demonstrate that MEG could have a therapeutic role in meningitis.     

Cirrhotic patients with minimal hepatic encephalopathy have increased capacity to eliminate superoxide and peroxynitrite in lymphocytes,associated with cognitive impairment

Patients with minimal hepatic encephalopathy (MHE) show increased oxidative stress in blood. We aimed to assess whether MHE patients show alterations in different types of blood cells in (a) basal reactive oxygen and nitrogen species levels; (b) capacity to metabolise these species. To assess the mechanisms involved in the altered capacity to metabolise these species we also analysed: (c) peroxynitrite formation and d) peroxynitrite reaction with biological molecules. Levels of reactive oxygen and nitrogen species were measured by flow cytometry in blood cell populations from cirrhotic patients with and without MHE and controls, under basal conditions and after adding generators of superoxide (plumbagin) or nitric oxide (NOR-1) to assess the capacity to eliminate them. Under basal conditions, MHE patients show reduced superoxide and peroxynitrite levels and increased nitric oxide (NO) and nitrotyrosine levels. In patients without MHE plumbagin strongly increases cellular superoxide, moderately peroxynitrite and reduces NO levels. In MHE patients, plumbagin increases slightly superoxide and strongly peroxynitrite levels and affects slightly NO levels. NOR-1 increases NO levels much less in patients with than without MHE. These data show that the mechanisms and the capacity to eliminate cellular superoxide, NO and peroxynitrite are enhanced in MHE patients. Superoxide elimination is enhanced through reaction with NO to form peroxynitrite which, in turn, is eliminated by enhanced reaction with biological molecules, which could contribute to cognitive impairment in MHE. The data show that basal free radical levels do not reflect the oxidative stress status in MHE.     

Nitrative DNA damage in inflammation and its possible role in carcinogenesis.

Chronic inflammation has long been recognized as a risk factor for human cancer at various sites. Examples include Helicobacter pylori-induced gastritis for gastric cancer, inflammatory bowel disease (ulcerative colitis and Crohn's disease) for colorectal cancer and chronic viral hepatitis for liver cancer. Here we review the role in carcinogenesis of nitrative damage to nucleic acids, DNA and RNA, which occurs during inflammation through the generation of reactive nitrogen species, such as peroxynitrite, nitroxyl, and nitrogen dioxide. Enhanced formation of 8-nitroguanine, representative of nitrative damage to nucleobases, has been detected in various inflammatory conditions. The biochemical nature of DNA damage mediated by reactive nitrogen species is discussed in relation to its possible involvement in mutations, genetic instability, and cell death. Better understanding of the mechanisms and role of such nitrative damage in chronic inflammation-associated human cancer is a necessary basis to develop new strategies for cancer prevention by modulating the process of inflammation.     

Reactive oxygen and nitrogen metabolites modulate fibronectin-induced fibroblast migration in vitro

Nitration of proteins by peroxynitrite may alter protein function. We hypothesized that reactive nitrogen species modulate fibronectin-induced fibroblast migration. To test this hypothesis, we evaluated fibroblast migration induced by fibronectin incubated with and without peroxynitrite. Peroxynitrite attenuated fibronectin-induced fibroblast migration in a dose-dependent manner but did not attenuate complement-activated serum-induced fibroblast migration. The reducing agents, deferoxamine and dithiothreitol (DTT), and L-tyrosine reversed the inhibition by peroxynitrite. PAPA-NONOate, a nitric oxide (NO) donor, and superoxide generated by the action of xanthine oxidase on lumazine or xanthine, also showed an inhibitory effect on fibroblast migration. The peroxynitrite generator, 3-morpholinosydnonimine (SIN-1), caused a concentration-dependent inhibition of fibroblast migration. Peroxynitrite reduced fibronectin binding to fibroblasts and resulted in nitrotyrosine formation. These findings are consistent with nitration of tyrosine by peroxynitrite with subsequent inhibition of fibronectin binding to fibroblasts and suggest that peroxynitrite may play a role in regulation of fibroblast migration.     

Increased expression of inducible nitric oxide synthase and peroxynitrite in Helicobacter pylori gastric ulcer.

The role of nitric oxide in ulcer formation remains unknown. Accordingly, we assessed local expression of inducible nitric oxide synthase (NOS) and nitration of tyrosine as an indicator of peroxynitrite formation in patients with Helicobacter pylori (HP)-associated gastric ulcers compared with HP-negative ulcers. Biopsy specimens were taken from the ulcer margin and from an area remote from the ulcer portion. Inducible NOS, nitrotyrosine, and macrophage immunoreactivity were assessed immunohistochemically using a labeled streptavidin-biotin method. In HP-positive gastric ulcers, inducible NOS and nitrotyrosine immunoreactivity was frequently observed at active ulcer margins, sometimes in surface epithelial cells as well as in the lamina propria. Occasionally, inducible NOS and nitrotyrosine reactivity were found in areas remote from the lesion in cases of HP-positive ulcer and HP-related gastritis. Macrophages accumulated significantly in the margin of HP-positive ulcers. In HP-negative gastric ulcers, inducible NOS and nitrotyrosine immunoreactivity also were frequent at the ulcer margin, but no significant immunoreactivity was observed at a distance. HP eradication caused significant attenuation in inducible NOS and macrophage immunoreactivity. In conclusion, nitric oxide and peroxynitrite formation is increased in HP-infected gastric mucosa, suggesting that HP promotes nitric oxide stress.     

Immunological detection of nitrosative stress-mediated modified Tamm-Horsfall glycoprotein (THP) in calcium oxalate stone formers.

The generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in hyperoxaluric condition has been proved experimentally. This may result in the formation of the cytotoxic metabolite peroxynitrite, which is capable of causing lipid peroxidation and protein modification. The presence of nitrotyrosine in proteins has been associated with several pathological conditions. The present study investigated the presence of nitrotyrosine in the stone formers Tamm-Horsfall glycoprotein (THP). In vitro nitration of control THP was carried out using peroxynitrite. New Zealand white rabbits were immunized with peroxynitrated THP at 15-day intervals. Antisera collected following the third immunization were assayed for antibody titres using solid-phase ELISA. Antibodies were purified by affinity chromatography. The carbonyl content of control, stone formers and nitrated THP were determined. Western blotting was carried with control, stone formers and nitrated THPs. Immunodiffusion studies demonstrated cross-reaction with nitrated bovine serum albumin. Significant amounts (p < 0.001) of carbonyl content were present in both stone formers and nitrated THPs. Western blot analysis confirmed the presence of nitrated amino acid 3-nitrotyrosine in stone formers, which could bring about structural and functional modifications of THP in hyperoxaluric patients. A cross-reaction with nitrated bovine serum albumin confirms that the raised antibody has certain paratopes similar to the epitope of nitrated protein molecules. Detection of 3-nitrotyrosine in stone formers THP indicates that it is one of the key factors influencing the conversion of THP to a structurally and immunologically altered form during calcium oxalate stone formation.     

Mechanical wounding induces a nitrosative stress by down-regulation of GSNO reductase and an increase in S-nitrosothiols in sunflower (Helianthus annuus) seedlings

Nitric oxide (NO) and related molecules such as peroxynitrite, S-nitrosoglutathione (GSNO), and nitrotyrosine, among others, are involved in physiological processes as well in the mechanisms of response to stress conditions. In sunflower seedlings exposed to five different adverse environmental conditions (low temperature, mechanical wounding, high light intensity, continuous light, and continuous darkness), key components of the metabolism of reactive nitrogen species (RNS) and reactive oxygen species (ROS), including the enzyme activities L-arginine-dependent nitric oxide synthase (NOS), S-nitrosogluthathione reductase (GSNOR), nitrate reductase (NR), catalase, and superoxide dismutase, the content of lipid hydroperoxide, hydrogen peroxide, S-nitrosothiols (SNOs), the cellular level of NO, GSNO, and GSNOR, and protein tyrosine nitration [nitrotyrosine (NO(2)-Tyr)] were analysed. Among the stress conditions studied, mechanical wounding was the only one that caused a down-regulation of NOS and GSNOR activities, which in turn provoked an accumulation of SNOs. The analyses of the cellular content of NO, GSNO, GSNOR, and NO(2)-Tyr by confocal laser scanning microscopy confirmed these biochemical data. Therefore, it is proposed that mechanical wounding triggers the accumulation of SNOs, specifically GSNO, due to a down-regulation of GSNOR activity, while NO(2)-Tyr increases. Consequently a process of nitrosative stress is induced in sunflower seedlings and SNOs constitute a new wound signal in plants.