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Nitrite has emerged as an endogenous signaling molecule with potential therapeutic implications for cardiovascular disease. Steady-state levels of nitrite are derived in part from dietary sources; therefore, we investigated the effects of dietary nitrite and nitrate supplementation and deficiency on NO homeostasis and on the severity of myocardial ischemia-reperfusion (MI/R) injury. Mice fed a standard diet with supplementation of nitrite (50 mg/liter) in their drinking water for 7 days exhibited significantly higher plasma levels of nitrite, exhibited significantly higher myocardial levels of nitrite, nitroso, and nitrosyl-heme, and displayed a 48% reduction in infarct size (Inf) after MI/R. Supplemental nitrate (1 g/liter) in the drinking water for 7 days also increased blood and tissue NO products and significantly reduced Inf. A time course of ischemia-reperfusion revealed that nitrite was consumed during the ischemic phase, with an increase in nitroso/nitrosyl products in the heart. Mice fed a diet deficient in nitrite and nitrate for 7 days exhibited significantly diminished plasma and heart levels of nitrite and NO metabolites and a 59% increase in Inf after MI/R. Supplementation of nitrite in the drinking water for 7 days reversed the effects of nitrite deficiency. These data demonstrate the significant influence of dietary nitrite and nitrate intake on the maintenance of steady-state tissue nitrite/nitroso levels and illustrate the consequences of nitrite deficiency on the pathophysiology of MI/R injury. Therefore, nitrite and nitrate may serve as essential nutrients for optimal cardiovascular health and may provide a treatment modality for cardiovascular disease.

Protein S-nitrosylation, the covalent binding of nitric oxide (NO) to protein cysteine residues, is one of the main mechanisms of NO signaling in plant and animal cells. Using a combination of the biotin switch assay and label-free LC-MS/MS analysis, we revealed the S-nitroso-proteome of the woody model plant Populus x canescens. Under normal conditions, constitutively S-nitrosylated proteins in poplar leaves and calli comprise all aspects of primary and secondary metabolism. Acute ozone fumigation was applied to elicit ROS-mediated changes of the S-nitroso-proteome. This treatment changed the total nitrite and nitrosothiol contents of poplar leaves and affected the homeostasis of 32 S-nitrosylated proteins. Multivariate data analysis revealed that ozone exposure negatively affected the S-nitrosylation status of leaf proteins: 23 proteins were de-nitrosylated and 9 proteins had increased S-nitrosylation content compared to the control. Phenylalanine ammonia-lyase 2 (log2[ozone/control] = -3.6) and caffeic acid O-methyltransferase (-3.4), key enzymes catalyzing important steps in the phenylpropanoid and subsequent lignin biosynthetic pathways, respectively, were de-nitrosylated upon ozone stress. Measuring the in vivo and in vitro phenylalanine ammonia-lyase activity indicated that the increase of the phenylalanine ammonia-lyase activity in response to acute ozone is partly regulated by de-nitrosylation, which might favor a higher metabolic flux through the phenylpropanoid pathway within minutes after ozone exposure.

Changes in intestinal function, notably impaired transit, following ischemia/reperfusion (I/R) injury are likely to derive, at least in part, from damage to the enteric nervous system. Currently, there is a lack of quantitative data and methods on which to base quantitation of changes that occur in enteric neurons. In the present work, we have investigated quantifiable changes in response to ischemia of the mouse small intestine followed by reperfusion from 1 h to 7 days. I/R caused distortion of nitric oxide synthase (NOS)-containing neurons, the appearance of a TUNEL reaction in neurons, protein nitrosylation and translocation of Hu protein. Protein nitrosylation was detected after 1 h and was detectable in 10% of neurons by 6 h in the ischemic region, indicating that reactive peroxynitrites are rapidly produced and can interact with proteins soon after reperfusion. Apoptosis, revealed by TUNEL staining, was apparent at 6 h. The profile sizes of NOS neurons were increased by 60% at 2 days and neurons were still swollen at 7 days, both in the ischemic region and proximal to the ischemia. The distribution of the enteric neuron marker and oligonucleotide binding protein, Hu, was significantly changed in both regions. Hu protein translocation to the nucleus was apparent by 3 h and persisted for up to 7 days. Particulate Hu immunoreactivity was observed in the ganglia 3 h after I/R but was never observed in control. Our observations indicate that effects of I/R injury can be detected after 1 h and that neuronal changes persist to at least 7 days. Involvement of NO and reactive oxygen species in the changes is indicated by the accumulation of nitrosylated protein aggregates and the swelling and distortion of nitrergic neurons. It is concluded that damage to the enteric nervous system, which is likely to contribute to functional deficits following ischemia and re-oxygenation in the intestine, can be quantified by Hu protein translocation, protein nitrosylation, swelling of nitrergic neurons and apoptosis.

Studies with the use of highly sensitive enzymatic sensor have shown the presence of various forms of nitrosyl iron complexes, including those undetectable by other methods, in living tissues. All these complexes are long-living compounds and constitute the major part of nitroso compounds in the blood, muscles, liquor, and amniotic fluid.

The covalent addition of nitric oxide (NO) to protein thiols, a posttranslational modification termed S-nitrosation, is a ubiquitous event that modulates diverse cellular processes. The in vivo addition of NO to protein amines (N-nitrosation) has also been described and may similarly modify protein structure and function. While mass spectrometry has been employed for identification of nitrosoproteins, little is known about how S- and N-nitrosopeptides fragment. Such knowledge is important for its potential to inform on sites of protein nitrosation. Here we used electrospray tandem mass spectrometry to elucidate collision-induced dissociation (CID) features of S- and N-nitrosopeptide ions. We show that S- and N-nitrosopeptide ions readily lose NO, giving rise to species that contain thiyl and aminyl radicals, respectively. Fragmentation (MS 3) of these radical peptide ions revealed an atypical pattern, characterized by the cleavage of select αC C and N αC bonds, rather than the more usual cleavage of amide bonds that result in b- and y-ions. These unanticipated fragmentation patterns are reconciled by radical-mediated abstraction of hydrogen from β-carbon followed by β-fragmentation. For thiyl radical peptides, we also observed dominant loss of SH and CH 2SH from the Cys side-chain. Our findings provide new insights into the gas-phase chemistry of NO-modified peptide ions and suggest an unusual fragmentation pattern that may aid in future MS-based attempts to define the nitrosoproteome.

Purpose The objective of this study was to investigate the effect of welding as well as the impact of smoking and protection measures on biological effect markers in exhaled breath condensate. Additionally, biomonitoring of chromium, aluminium and nickel in urine was performed to quantify internal exposure. Methods Exhaled breath condensate (EBC) and urine samples of 45 male welders and 24 male non-exposed control subjects were collected on Friday pre-shift and after 8 h of work post-shift. In EBC, biological effect markers such as malondialdehyde, nitrite, nitrate, 3-nitrotyrosine, tyrosine, hydroxyproline, proline, H 2 O 2 and pH-value were measured while aluminium, nickel, and chromium were measured in the urine samples. Results Although internal exposure to aluminium, nickel and chromium in this study was low, welders showed significantly increased concentrations of all these parameters at baseline compared to non-exposed controls. Moreover, welders had higher nitrate concentrations in EBC at baseline and after shift. Nitrate concentration was considerably lower after shift if personal protection equipment was used. H 2 O 2 was increased only when subjects smoked during shift. Conclusion It has been shown that welding-associated long-term and short-term health effects could be detected in a population of welders. The results also showed that using personal protection equipment is of high importance and H 2 O 2 may be an effect marker associated with smoking rather than with welding fumes, while nitrate in EBC seems to be sensitive to welding fume exposure.

Experiments were conducted to investigate the hypothesis that N-nitrosodimethylamine (NDMA) is a potential disinfection by-product. NDMA was formed by the reaction of dimethylamine (DMA) with monochloramine and also with free chlorine in the presence of ammonia. We proposed a mechanism for NDMA formation which does not require the presence of nitrite as in N-nitrosation. The critical NDMA formation reactions consist of i) the formation of monochloramine by combination of free chlorine with ammonia, ii) the formation of 1,1-dimethylhydrazine (UDMH) intermediate from the reaction of DMA with monochloramine followed by, iii) the oxidation of UDMH by monochloramine to NDMA, and iv) the reversible chlorine transfer reaction between free chlorine/monochloramine and DMA which is parallel with i) and ii). A kinetic model was developed to validate the proposed mechanism.

Studies with the use of a highly specific enzymatic sensor demonstrated that, contrary to the common opinion, normally nitrate is in fact not present in the most important physiological fluids. NO metabolites in the amniotic fluid and semen are mainly presented by NO donor compounds. Therefore, the intensity of NO synthesis can be evaluated by the total content of all its metabolites, but not by the widely used summary nitrite+nitrate content.