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The reactions of nitric oxide (*NO) during inflammatory conditions must be revealed in order to gain insight into the metabolic fates of *NO in viva In a cell line expressing physiological levels of 15lipoxygenase (LOX), rates of linoleate-stimulated *NO consumption, 7-fold higher than rates of *NO production by activated rodent macrophages, were seen. This *NO consumption did not occur in l3galactosidase-transfected controls, indicating that cellular 15-LOX has the potential to antagonize *NO signalling. Inhibition of ISlipoxygenases by *NO was previously proposed to result from ironnitrosyl complex formation between *NO and the active site ferrous iron. However, at biological *NO concentrations, no reaction with either native (Bred), or ferric lipoxygenase to form nitrosyl complexes was found. Rather, *NO consumption resulted from reaction with an enzyme-substrate Intermediate formed during the dioxygenase cycle, most probably the enzyme-bound lipid peroxyl radical, BredLOO*. During *NO consumption, partial reversible inhibition of 13(S)HPODE generation occurred, but lipid product profile was unchanged. Kinetic modelling of diene conjugation and *NO consumption rates revealed a rapid reaction of *NO with BredLOO*. Since co-generation of *NO and lS-lipoxygenase products occurs in inflammatory conditions such as atherosclerosis, interactions between these pathways will result in modulation of both *NO bioavailability and extents of production of lipid signalling mediators.

We have studied a 17-year-old girl with lactic acidosis (3-18 mEq/liter) and progressive muscle weakness since 9 years of age. Morphological findings in muscle were of a typical ragged red myopathy with multiple collections of bizarre mitochondria, some containing paracrystalline inclusions. The carnitine content of serum and muscle was normal, as were the activities of carnitine palmitoyltransferase, carnitine octanoyltransferase, and carnitine acetyltransferase in the patient's muscle. Measurement of the enzymes of oxidative phosphorylation in both crude muscle homogenates and mitochondrial fractions showed close to normal activities of cytochrome c oxidase, succinate dehydrogenase, and ATPase. In contrast, succinate cytochrome c reductase activity was greatly reduced in the patient, being 0.035 mumol/min/g tissue in whole muscle (controls 1.16 +/- 0.47 mumol/min/g tissue) and 8 nmol/min/mg protein in the mitochondria (control, 340 nmol/min/mg protein). Rotenonesensitive NADH-cytochrome c reductase was also undetectable in the patient's mitochondria. Spectral analysis of cytochromes showed decrease of reducible cytochrome b to 16% of the control. These results indicate a defect of ubiquinol-cytochrome c reductase or the cytochrome bc1 segment (complex III) of the electron transport chain. Antibody-binding studies of the individual components of complex III showed additional deficiencies of core proteins I and II and peptide VI, indicating a more widespread defect of complex III than was evident from spectral analysis and enzyme activity measurements alone. Urine organic acid analysis after fasting and following a medium chain triglyceride load showed unusually high levels of lactate and 3-hydroxybutyrate, lower than expected levels of acetoacetate and dicarboxylic acids, and the presence of several other metabolites suggesting a disturbed citric acid cycle and redox state.(ABSTRACT TRUNCATED AT 250 WORDS)

The bioenergetic capacity of skeletal muscle in a 17-year-old patient with a severe defect in complex III of the electron transport chain has been examined by 31P NMR measurements of the molar ratio of phosphocreatine to inorganic phosphate (PCr/Pi). Resting ratios were 1.3-2.5, which can be compared with roughly 8.6 for a young, normal female control at rest. Quantitative evaluation of the activity of oxidative metabolism was afforded by the rate of recovery of PCr/Pi from exercise and was found to be 2.5% of normal. After administration of menadione and ascorbate, we found a 21-fold increase of the recovery rate relative to the pretherapy value, to within 56% of the recovery rate of the young female control. Thus, NMR examinations of skeletal muscle at rest and in recovery from activity document marked improvement to specific drug therapy in the electron transport capabilities and the ATP synthesis rate of a patient with a deficiency in a cytochrome b-containing complex III. Improvements in functional ability, although not as dramatic as biochemical changes, are also apparent.

To clarify the difference in the protein composition of pancreatic juice between patients with pancreatic cancer and normal controls, the proteins of pure pancreatic juice from three cases of cancer of the head of the pancreas and six apparently healthy adults were analyzed by two-dimensional gel electrophoresis (two-DE) followed by silver staining. Two minor proteins of Mr 59,000 and Mr 78,000 present in all the three patients were not detected in the six controls. We have identified the minor proteins with Mr 59,000 and Mr 78,000 as alpha 1-antitrypsin and transferrin, respectively, using Western blotting with anti-alpha 1-antitrypsin and anti-transferrin antibodies. In addition, serum albumin also identified by antibody binding was abundantly present in patients compared to controls. The increase in the amount of serum albumin in the patient was also confirmed by a quantitative analysis using SDS-PAGE and gel densitometry. The data indicate that not only serum albumin but also alpha 1-antitrypsin and transferrin are increased in the pancreatic juice of the patients with pancreatic cancer, as compared with apparently healthy adults. The data also suggests that the analysis of pancreatic juice proteins by two-DE with silver staining is useful for the diagnosis of pancreatic diseases.

1. 1. Cytochrome c oxidases from a number of eukaryotic organisms have been isolated and show spectral properties similar to those of the bovine enzyme. All possess both Cu and Fe atoms. 2. 2. Ultracentrifuge studies reveal that the camel oxidase is monomeric yet exhibits similar properties to the bovine enzyme. 3. 3. Investigations of the subunit composition have shown 7 subunits for all the species examined and similarities are suggested on the basis of the 203 Hg labelling of the subunits. 4. 4. Steady state kinetic measurements show two kinetically functional cytochrome c binding sites on all the oxidases examined. The pH optima of the monomeric camel enzyme (pH 7.2) is significantly different to the dimeric bovine enzyme (pH 6.2). 5. 5. Pre-steady state measurements show a fast initial electron entry from ferrocytochrome c to ferricytochrome a followed by a slower second phase. These results are similar for all the oxidases studied and comparable to those already established for the bovine enzyme. 6. 6. A complete cycle of full reduction followed by oxidation by molecular oxygen leads to an activated (“pulsed”) form of cytochrome oxidase. This property was exhibited by the enzymes from all the species examined.

Fast reaction kinetic experiments on the electron transfer reaction between azurin and cytochrome c 551 isolated from Pseudomonas aeruginosa confirmed the existence of two redox forms of reduced azurin previously reported. The pH dependence of the amplitudes of the relaxation processes observed in temperature jump experiments indicate that these two redox forms are in pH dependent equilibrium. The pH independence of the overall equilibrium constant indicates that redox active and inactive forms of cytochrome c 551 may also exist. Evidence that reduced cytochrome c 551 undergoes a pH transition is given by optical spectrophotometry. The nature of the transition is discussed in the context of recent nmr studies and in terms of the Marcus theory of electron transfer. The metabolic consequences of these transitions are also discussed.

We have expanded our studies of a patient with a mitochondrial myopathy caused by a defect at the level of complex III of the respiratory chain. Using activity measurements, electron microscopy, protein synthesis in the presence of emetine, and antibody binding, we have demonstrated that the defect is not expressed in cultured skin fibroblasts from this patient. Electron microscopy of peripheral blood leukocytes and activity measurements in transformed lymphoid cells indicated that the defect was not expressed in these cells either. These results imply that there are either isoforms of complex III components which show differential tissue expression or that independent segregation and assortment of defective mitochondria has occurred during development.

The ischaemic myocardium shows a number of distinct oxygen dependent responses to reperfusion. In the case of myocardial stunning and reperfusion arrhythmias there appears to be a beneficial effect of scavenging radicals in the extracellular space. This result is supported by the finding that free radicals can be detected extracellularly after reperfusion. The source of these oxidants and site of action is as yet unclear. In contrast hypoxic myocytes shown an oxygen dependent Ca2+ uptake on reoxygenation which is not affected by externally applied antioxidants. This Ca2+ uptake may in turn lead to the cell lysis characteristic of the oxygen paradox in the perfused heart. As yet there is no compelling evidence to suggest that this aspect of reperfusion damage is due to oxidative stress. It appears more likely that mitochondrial electron transport and ion movement play a central role. In the open chested dog model of ischaemia reperfusion, in which the volume of infarcted tissue is measured, considerable evidence suggests that oxidants derived from activated neutrophils contribute to cell death. This is not however the sole mechanism for cell damage in this model since an inhibitor of mitochondrial Ca2+ uptake, ruthenium red, can improve recovery after reperfusion. We conclude that a number of mechanisms may contribute to the observed oxygen dependent dysfunctions which occur on reperfusion of ischaemic tissue.

Modification of beef heart cytochrome c oxidase with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) or 1-ethyl-3-[3-(trimethylamino)propyl]carbodiimide (CH3EDC) has been found to significantly inhibit the high-affinity phase of the reaction of this enzyme with cytochrome c. Reaction conditions leading to a 70% inhibition of Vmax resulted in a 16-fold increase in the Km for cytochrome c. The loss in activity was accompanied by modification of subunit II to form a new species, II', which migrated somewhat more rapidly than the unmodified subunit during sodium dodecyl sulfate (NaDodSO4) gel electrophoresis. This new species was the major site of radiolabeling when cytochrome c oxidase was treated with [14C]CH3EDC, indicating covalent incorporation of the carbodiimide. Equimolar concentrations of cytochrome c dramatically protected cytochrome c oxidase from inhibition by the carbodiimide and in approximately the same proportion shielded subunit II from modification to the labeled II' species. In addition, cytochrome c was cross-linked to subunit II to form a new species migrating somewhat faster than subunit I during NaDodSO4 gel electrophoresis. This cross-linked species was shown to contain subunit II by using subunit-specific antibodies. We propose that EDC or CH3EDC reacts with one or more partially buried carboxyl groups on subunit II to form a positively charged N-acylurea which inhibits cytochrome c binding. In the presence of cytochrome c, EDC promotes formation of amide cross-links between lysine amino groups on cytochrome c and their complementary carboxyl groups on cytochrome c oxidase.

The cysteine residues in beef cytochrome c oxidase (E.C.1.9.3.1) which act as ligands to a redox site have been located in the C-terminal portion of subunit II.

Using the small amounts of muscle available from biopsy (approximately 100 mg), from both normal controls and a patient with a previously identified defect of the mitochondrial electron transfer protein complex III, we analyzed both structural and mitochondrial proteins. The myosin light chains were found to be unchanged with respect to charge or size between patient and control. Two prominent proteins detected after two dimensional gel electrophoresis were present in the patient's total homogenised muscle protein but were not detected in the controls. One protein was positively identified as cytochrome c oxidase subunit II and the other tentatively as a component of the ATP synthetase. We suggest that the increased amounts of these proteins represents a response of the patients muscle cells to the ATP deficiency caused by the primary lesion in complex III.

The production of superoxide and nitric oxide individually has been associated with the development of several diseases but only recently has it been realised that interactions between them may also be important in disease pathology. The central hypothesis which is emerging is that the balance between nitric oxide and superoxide generation is a critical determinant in the aetiology of many human diseases including atherosclerosis, neurodegenerative disease, ischaemia-reperfusion and cancer. These ideas are discussed in this short overview and placed in the context of the current and future status of therapies which could modulate the balance between nitric oxide and superoxide.

Thionitrobenzoate-modified yeast cytochrome c was shown to react with both monomeric and dimeric forms of beef heart cytochrome c oxidase through subunit III. This cytochrome c derivative was found to inhibit electron transfer in the dimer but not in the monomer. These results are interpreted to show that the high affinity binding site for cytochrome c is a cleft at the interface between monomers in the cytochrome c oxidase dimer.

Protein thiolation by glutathione is a reversible and regulated post-translational modification that is increased in response to oxidants and nitric oxide. Because many mitochondrial enzymes contain critical thiol residues, it has been hypothesized that thiolation reactions regulate cell metabolism and survival. However, it has been difficult to differentiate the biological effects due to protein thiolation from other oxidative protein modifications. In this study, we used diamide to titrate protein glutathiolation and examined its impact on glycolysis, mitochondrial function, and cell death in rat aortic smooth muscle cells. Treatment of cells with diamide increased protein glutathiolation in a concentration-dependent manner and had comparably little effect on protein–protein disulfide formation. Diamide increased mitochondrial proton leak and decreased ATP-linked mitochondrial oxygen consumption and cellular bioenergetic reserve capacity. Concentrations of diamide above 200 μM promoted acute bioenergetic failure and caused cell death, whereas lower concentrations of diamide led to a prolonged increase in glycolytic flux and were not associated with loss of cell viability. Depletion of glutathione using buthionine sulfoximine had no effect on basal protein thiolation or cellular bioenergetics but decreased diamide-induced protein glutathiolation and sensitized the cells to bioenergetic dysfunction and death. The effects of diamide on cell metabolism and viability were fully reversible upon addition of dithiothreitol. These data suggest that protein thiolation modulates key metabolic processes in both the mitochondria and cytosol.

The post-translational modification of serine and threonine residues of nuclear and cytoplasmic proteins by the O-linked attachment of the monosaccharide β-N-acetyl-glucosamine (O-GlcNAc) is emerging as an important mechanism for the regulation of numerous biological processes critical for normal cell function. Active synthesis of O-GlcNAc is essential for cell viability and acute activation of pathways resulting in increased protein O-GlcNAc levels improves the tolerance of cells to a wide range of stress stimuli. Conversely sustained increases in O-GlcNAc levels have been implicated in numerous chronic disease states, especially as a pathogenic contributor to diabetic complications. There has been increasing interest in the role of O-GlcNAc in the heart and vascular system and acute activation of O-GlcNAc levels have been shown to reduce ischemia/reperfusion injury, attenuate vascular injury responses as well mediate some of the detrimental effects of diabetes and hypertension on cardiac and vascular function. Here we provide an overview of our current understanding of pathways regulating protein O-GlcNAcylation, summarize the different methodologies for identifying and characterizing O-GlcNAcylated proteins and subsequently focus on two emerging areas: 1) the role of O-GlcNAc as a potential regulator of cardiac metabolism and 2) the cross talk between O-GlcNAc and reactive oxygen species. This article is part of a Special Section entitled “Post-translational Modification.”

The principal antioxidant in human LDL, alpha-tocopherol, is converted to the alpha-tocopheroxyl radical after reaction with peroxyl radicals or Cu2+, and, if it does not terminate with peroxyl radicals, could initiate lipid peroxidation; a phenomenon called 'tocopherol mediated peroxidation'. Only in the presence of Cu2+ and low levels of lipid hydroperoxides was an alpha-tocopherol dependent decrease in the resistance of LDL to oxidation detected. This suggests that tocopherol mediated peroxidation will probably not contribute significantly as a pro-oxidant process in those individuals most at risk of developing atherosclerosis through an oxidative mechanism.

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The polypeptide subunits of cytochrome c oxidase (E.C. 1.9.3.1.) isolated from beef heart which react with 203 [Hg]Cl 2 have been identified. A rapid and simple method for identifying and numbering the subunits is described which is independent of the type of sodium dodecyl sulphate-polyacrylamide gel system employed to separate them.

Langendorff perfused rat hearts were subjected to 30 min hypoxia followed by 20 min reoxygenation and the levels of the oxidised and reduced forms of glutathione measured. No change in the concentration of oxidised glutathione was detected in reoxygenated hearts when compared to normoxic controls. In contrast hearts exposed to oxidative stress in the form of H2O2 showed elevated levels of both oxidised glutathione (GSSG) and the glutathione-protein mixed disulphide. These results suggest that if oxidants do contribute to cell damage on reoxygenation of the hypoxic myocardium then their action is local and not through overwhelming of the cells antioxidant defences.

The skeletal muscle of a patient with a mitochondrial myopathy was examined. A defect in the electron transport chain was identified at the position of complex III by activity measurements and the low levels of reducible cytochrome b. The polypeptide composition of complex III in the patient's mitochondria was determined by antibody binding experiments. The method allowed detection of individual polypeptides at a lower limit of 10-40 ng of protein. Characterization of protein composition is thus possible by using a biopsy sample of 1 g of tissue. The level of core proteins, FeS protein, and subunit VI was greatly diminished in the patient's mitochondria. Cytochrome c1 polypeptide was found at normal levels but was sensitive to proteolysis by trypsin. These results show that complex III is not assembled in the patient's mitochondria. The possible role of cytochrome b as the site of the primary lesion is discussed.

1. A low-pH lithium dodecyl sulphate/polyacrylamide-gradient slab-gel system, suitable for electrophoresis, is described, and the migration properties of standard proteins are compared on this and conventional high-pH gels. 2. Cytochrome oxidase may be partially resolved into its component polypeptides. The order of migration of these is, however, dependent on the pH of the gel system.

We report our studies on the metabolic defects which caused a newborn infant to present with a severe lactic acidemia (25 mM) and to die on the 3rd d after birth despite intensive supportive measures. The mitochondrial fractions prepared from skeletal muscle and liver oxidised NAD+-linked substrates and succinate slowly. Spectrophotometric assays for complexes I, II, and HI of the respiratory chain demonstrate a specific defect of complex III in the skeletal muscle and liver mitochondrial fractions. The concentrations of cytochrome b were 75% lower in the skeletal muscle and heart mitochondria than in control preparations. The amount of non-heme iron sulphur protein of complex III was low in skeletal muscle, liver, and heart. This case differs from previous reports of complex III deficiency in three respects: the patient presented in the neonatal period, the defect was expressed in several tissues, and it was fatal.

Reoxygenation of isolated rat cardiac myocytes following a period of hypoxia and substrate deprivation resulted in a 1.5-2-fold increase in the total Ca2+ content which could be inhibited by 1 microM antimycin A or ruthenium red (50% inhibition at 2.5 microM). This increase in Ca2+ content was not accompanied by any release of creatine kinase into the medium. Treatment of reoxygenated cells with digitonin also resulted in an antimycin A-sensitive increase in Ca2+ but this was inhibited by a lower concentration of ruthenium red (50% inhibition at 0.25 microM) and was associated with a substantial release of creatine kinase from the cells. It is concluded that the reoxygenation-stimulated increase in Ca2+ is dependent on functioning mitochondria and does not occur as a result of physical damage to the sarcolemma. In a parallel series of experiments, the effects of antimycin A and ruthenium red on the reoxygenation-induced increase in Ca2+ and release of cytosolic contents in the perfused heart (the oxygen paradox) were also investigated. As was observed with the isolated myocytes, each of the compounds significantly reduced the magnitude of the Ca2+ increase that occurred on reoxygenation: the compounds also reduced the extent of release of cell contents in the perfused heart. The implications of these results for the series of events occurring on reoxygenation of the hypoxic myocardium are discussed.

The arrangement of subunit IV in beef heart cytochrome c oxidase has been explored by chemical labeling and protease digestion studies. This subunit has been purified from four samples of cytochrome c oxidase that had been reacted with N-(4-azido-2-nitrophenyl)-2-aminoethyl[35S]-sulfonate (NAP-taurine), diazobenzene[35S]sulfonate, 1-myristoyl-2-[12-[(4-azido-2-nitrophenyl)amino]lauroyl]-sn-glycero-3- [14C]phosphocholine (I), and 1-palmitoyl-2-(2-azido-4-nitrobenzoyl)-sn-glycero-3-[3H]phosphocholine (II), respectively. The labeled polypeptide was then fragmented by cyanogen bromide, at arginyl side chains with trypsin (after maleylation), and the distribution of the labeling within the sequence was analyzed. The N-terminal part of subunit IV (residues 1-71) was shown to be heavily labeled by water-soluble, lipid-insoluble reagents but not by the phospholipid derivatives. These latter reagents labeled only in the region of residues 62-122, containing the long hydrophobic and putative membrane-spanning stretch. Trypsin cleavage of native cytochrome c oxidase complex at pH 8.2 was shown to clip the first seven amino acids from subunit IV. This cleavage was found to occur in submitochondrial particles but not in mitochondria or mitoplasts. These results are interpreted to show that subunit IV is oriented with its N terminus on the matrix side of the mitochondrial inner membrane and spans the membrane with the extended sequence of hydrophobic lipid residues 79-98 buried in the bilayer.

Oxidation of low density lipoprotein (LDL) in the artery wall leads to the formation of cholesterol oxidation products that may result in cytotoxicity. Different mechanisms could contribute to LDL oxidation in vivo resulting in characteristic and specific modification of the cholesterol molecule. Alternatively, attack on cholesterol by chain propagating peroxyl radicals could result in the same distribution of oxidation products irrespective of the initial pro-oxidant mechanism. To distinguish between these possibilities we have monitored the formation of nine oxysterols during LDL oxidation, promoted by copper, myoglobin, peroxynitrite, or azo bis amidino propane. Regardless of the oxidant used, the pattern of oxysterol formation was essentially the same. The yields of products identified decreased in the order 7-oxocholesterol > 7 beta-hydroxycholesterol > 7 alpha-hydroxycholesterol > 5,6 beta-epoxycholesterol > 5,6 alpha-epoxycholesterol except in the case of peroxynitrite in which case a higher yield of 5, 6 beta-epoxycholesterol relative to 7-oxocholesterol was found. No formation of cholestane 3 beta, 5 alpha, 6 beta-triol, or the 24-,25-,27-hydroxycholesterols was seen. Concentration of 7-oxocholesterol levels in LDL was positively correlated with the degree of protein modification. Endogenous alpha-tocopherol in LDL or supplementation with butylated hydroxytoluene prevented oxysterol formation. Taken together these data indicate that the oxidation of cholesterol and protein in LDL occur as secondary oxidation events consequent on the attack of fatty acid peroxyl/alkoxyl radicals on the 7-position of cholesterol, and with amino acids on apoB. Furthermore, oxidant processes with atherogenic potential, such as peroxynitrite, copper, and myoglobin are capable of producing oxidized LDL containing cytotoxic mediators.

Background It has been suggested that reendothelialization of damaged blood vessels protects against the vascular injury response. The goal of the present study was to determine whether estrogen restores endothelial cell function in balloon-injured rat carotid arteries. Methods and Results Ten-week-old male and female Sprague-Dawley rats with intact gonads underwent balloon injury to the right common carotid artery. Female rats were randomized to receive either daily subcutaneous injections of 17β-estradiol (17β-E 2 ; 20 μg · kg −1 · d −1 ) or vehicle over the course of the study. Vessel morphology was assessed 2 weeks after injury. Significant neointima formation was observed in vehicle-treated males. This response was blunted in vehicle-treated and 17β-E 2 –supplemented females. Intima-to-media ratios were 1.28±0.23 (males), 0.72±0.07 (vehicle-treated females), and 0.49±0.07 (17β-E 2 –supplemented females). To test whether reductions in neointimal lesion formation were related to the functional reendothelialization of the damaged vessel, endothelium-dependent relaxation was tested in isolated ring segments from the three experimental groups. Vessels were precontracted with phenylephrine followed by cumulative administration of acetylcholine, an endothelium-dependent vasodilator. Maximum relaxation to acetylcholine was 8.13±1.70% (males), 22.06±4.36% (vehicle-treated females), and 46.47±3.48% (17β-E 2 –supplemented females). The enhanced endothelium-dependent relaxation of rings from 17β-E 2 –supplemented females correlated with reduced neointimal proliferation in this group. The concentration of nitric oxide metabolites in plasma correlated positively with plasma 17β-E 2 concentration. Conclusions These results suggest that estrogen protects against neointimal injury in the balloon-injured rat, at least in part, by facilitating the reendothelialization of the damaged vessel.

Dietary polyphenolics such as those in soy or red wine can have beneficial effects on the development of chronic human diseases. The mechanisms of action of isoflavones have been diverse and include their roles as weak estrogens, inhibitors of tyrosine kinase-dependent signal transduction processes and as antioxidants. Recent insights into the oxidative stress model of atherosclerosis suggest an interesting synthesis of these concepts. Sites of inflammation are associated with the formation of complex mixtures of reactive oxygen, nitrogen and halogenating species capable of modifying both endogenous biomolecules and polyphenolics. Of particular significance are the halogenation reactions mediated by myeloperoxidase that can modify key amino acids such as arginine and polyphenolics such as genistein. Hypochlorite, the reaction product of myeloperoxidase can halogenate polyphenolics to form stable derivatives with modified biological activity. Thus the in situ metabolism at sites of inflammation is unique and generates novel pharmacophores with potentially distinct modes of action from the parent compounds.

Many of the biological and pathological effects of nitric oxide (NO) are mediated through cell signaling pathways that are initiated by NO reacting with metalloproteins. More recently, it has been recognized that the reaction of NO with free radicals such as superoxide and the lipid peroxyl radical also has the potential to modulate redox signaling. Although it is clear that NO can exert both cytotoxic and cytoprotective actions, the focus of this overview are those reactions that could lead to protection of the cell against oxidative stress in the vasculature. This will include the induction of antioxidant defenses such as glutathione, activation of mitogen-activated protein kinases in response to blood flow, and modulation of mitochondrial function and its impact on apoptosis. Models are presented that show the increased synthesis of glutathione in response to shear stress and inhibition of cytochrome c release from mitochondria. It appears that in the vasculature NO-dependent signaling pathways are of three types: (i) those involving NO itself, leading to modulation of mitochondrial respiration and soluble guanylate cyclase; (ii) those that involve S-nitrosation, including inhibition of caspases; and (iii) autocrine signaling that involves the intracellular formation of peroxynitrite and the activation of the mitogen-activated protein kinases. Taken together, NO plays a major role in the modulation of redox cell signaling through a number of distinct pathways in a cellular setting.

Peroxynitrite (ONOO-), formed from the rapid reaction of superoxide (O2-•) with NO, is known to generate stable compounds capable of donating NO on reaction with thiols and molecules containing hydroxy groups. Using glycerol as a model compound for the reactions of ONOO- with biomolecules containing hydroxy groups, we separated the products and identified them by HPLC/MS. It was shown that both glyceryl mononitrate and glyceryl mononitrite were formed and released NO on incubation with copper and L-cysteine. The compounds were stable over a period of 4 h when shielded from light and kept on ice. Slow spontaneous decomposition occurred in the buffer used for the bioassay, but this was not sufficient to explain the vasorelaxing properties of these NO donors. It is concluded that the stable organic nitrate and nitrite have the capacity to be metabolized by vascular tissues, resulting in vasorelaxation.

An important aspect of the risk of cancer is the involvement of the inflammatory response. Currently, antiinflammatory agents are used in chemopreventive strategies. For example, aspirin is recommended for the prevention of colon cancer as well as breast and other cancers. The inflammatory response involves the production of cytokines and proinflammatory oxidants such as hypochlorous acid (HOCI) and peroxynitrite (ONO 2 -) produced by neutrophils and macrophages, respectively. These oxidants react with phenolic tyrosine residues on proteins to form chloro- and nitrotyrosine. Diets rich in polyphenols (green tea catechins, soy isoflavones) have also been shown to be chemopreventive. The aromatic nature of polyphenols makes them potential targets of HOCI and ONO 2 -. These reactions may create novel pharmacophores at the site of inflammation. Previous studies in the neutrophil-like cell line, differentiated HL-60 cells, demonstrated the formation of chlorinated and nitrated isoflavones. In this study we have examined whether similar reactions occur in freshly isolated human neutrophils. After induction of a respiratory burst with a phorbol ester, isoflavones and their metabolites were identified by liquid chromatography-tandem mass spectrometry and then quantitatively measured by LC-mass spectrometry using multiple-reaction ion monitoring. The data obtained indicate that both chlorinated and nitrated genistein are formed by human neutrophils. The extent of chlorination of genistein was markedly increased by the phorbol ester whereas the low level of nitration of genistein was constitutive and unaffected. These data imply a potential role for modified forms of genistein that would be produced in the inflammatory environment in and around a tumor.

The oxidation of lipids is an autocatalytic process consisting of a number of well-defined interrelated chemical reactions. Its importance has long been recognized in the food and polymer industry, and recent advances in the understanding of vascular diseases have shown that lipid peroxidation also contributes to human disease. The various chemical stages of the reaction offer several therapeutic targets for inhibition, and from the structural characteristics of phytoestrogens it is anticipated that they should exhibit antioxidant properties. Alone, it is not sufficient for compounds such as the phytoestrogens to exhibit biological activity as antioxidants; the criteria that should be satisfied for this mechanism to be relevant biologically are discussed.

Nitric oxide (NO) and peroxynitrite both inhibit respiration by brain submitochondrial particles, the former reversibly at cytochrome c oxidase, the latter irreversibly at complexes I–III. Both GSH (IC50 = 10 μM) and glucose (IC50 = 8 mM) prevented inhibition of respiration by peroxynitrite (ONOO-), but neither glucose (100 mM) nor GSH (100 μM) affected that by NO. Thus, unless ONOO- is formed within mitochondria it is unlikely to inhibit respiration in cells directly, because of reactions with cellular thiols and carbohydrates. However, the reversible inhibition of respiration at cytochrome c oxidase by NO is likely to occur (e.g. in the brain during ischaemia) and could be responsible for cytotoxicity.