Myeloperoxidase has directly-opposed effects on nitration reaction--study on myeloperoxidase-deficient patient and myeloperoxidase-knockout mice

Myeloperoxidase (MPO) catalyzes a nitration reaction to form nitrotyrosine in the presence of high nitrite, the metabolite of NO. Human leukocyte was shown to cause phenolic nitration using released MPO as a catalyst in the presence of nitrite. It opposes our previous finding that inhibition of MPO was essential for phenol nitration in human leukocyte study. To clarify the role of MPO, we utilized MPO-deficient human leukocytes and MPO-knockout mice. Even in the absence of exogenously added nitrite, high nitration product was observed in MPO-deficient leukocytes. In liver subjected to ischemia/reperfusion injury, a significantly higher amount of nitrotyrosine was produced in MPO-knockout mice than in normal mice. These results clearly demonstrate that MPO inhibits the accumulation of nitration products in vivo . Further experiments showed that MPO could degrade nitrotyrosine in the presence of glutathione. Thus, MPO-induced degradation of nitration products may cause the underestimation of the nitration product generated in vivo . We conclude that MPO may act predominantly to scavenge nitrotyrosine under physiological nitrite condition, and protect against injurious effect of nitrotyrosine.     

A tale of two controversies: defining both the role of peroxidases in nitrotyrosine formation in vivo using eosinophil peroxidase and myeloperoxidase-deficient mice, and the nature of peroxidase-generated reactive nitrogen species

Nitrotyrosine is widely used as a marker of post-translational modification by the nitric oxide ((.)NO, nitrogen monoxide)-derived oxidant peroxynitrite (ONOO(-)). However, since the discovery that myeloperoxidase (MPO) and eosinophil peroxidase (EPO) can generate nitrotyrosine via oxidation of nitrite (NO(2)(-)), several questions have arisen. First, the relative contribution of peroxidases to nitrotyrosine formation in vivo is unknown. Further, although evidence suggests that the one-electron oxidation product, nitrogen dioxide ((*)NO(2)), is the primary species formed, neither a direct demonstration that peroxidases form this gas nor studies designed to test for the possible concomitant formation of the two-electron oxidation product, ONOO(-), have been reported. Using multiple distinct models of acute inflammation with EPO- and MPO-knockout mice, we now demonstrate that leukocyte peroxidases participate in nitrotyrosine formation in vivo. In some models, MPO and EPO played a dominant role, accounting for the majority of nitrotyrosine formed. However, in other leukocyte-rich acute inflammatory models, no contribution for either MPO or EPO to nitrotyrosine formation could be demonstrated. Head-space gas analysis of helium-swept reaction mixtures provides direct evidence that leukocyte peroxidases catalytically generate (*)NO(2) formation using H(2)O(2) and NO(2)(-) as substrates. However, formation of an additional oxidant was suggested since both enzymes promote NO(2)(-)-dependent hydroxylation of targets under acidic conditions, a chemical reactivity shared with ONOO(-) but not (*)NO(2). Collectively, our results demonstrate that: 1) MPO and EPO contribute to tyrosine nitration in vivo; 2) the major reactive nitrogen species formed by leukocyte peroxidase-catalyzed oxidation of NO(2)(-) is the one-electron oxidation product, (*)NO(2); 3) as a minor reaction, peroxidases may also catalyze the two-electron oxidation of NO(2)(-), producing a ONOO(-)-like product. We speculate that the latter reaction generates a labile Fe-ONOO complex, which may be released following protonation under acidic conditions such as might exist at sites of inflammation.     

Modulation of arachidonic and linoleic acid metabolites in myeloperoxidase-deficient mice during acute inflammation

Acute inflammation is a common feature of many life-threatening pathologies, including septic shock. One hallmark of acute inflammation is the peroxidation of polyunsaturated fatty acids forming bioactive products that regulate inflammation. Myeloperoxidase (MPO) is an abundant phagocyte-derived hemoprotein released during phagocyte activation. Here, we investigated the role of MPO in modulating biologically active arachidonic acid (AA) and linoleic acid (LA) metabolites during acute inflammation. Wild-type and MPO-knockout (KO) mice were exposed to intraperitoneally injected endotoxin for 24 h, and plasma LA and AA oxidation products were comprehensively analyzed using a liquid chromatography–mass spectrometry method. Compared to wild-type mice, MPO-KO mice had significantly lower plasma levels of LA epoxides and corresponding LA- and AA-derived fatty acid diols. AA and LA hydroxy intermediates (hydroxyeicosatetraenoic and hydroxyoctadecadienoic acids) were also significantly lower in MPO-KO mice. Conversely, MPO-deficient mice had significantly higher plasma levels of cysteinyl-leukotrienes with well-known proinflammatory properties. In vitro experiments revealed significantly lower amounts of AA and LA epoxides, LA- and AA-derived fatty acid diols, and AA and LA hydroxy intermediates in stimulated polymorphonuclear neutrophils isolated from MPO-KO mice. Our results demonstrate that MPO modulates the balance of pro- and anti-inflammatory lipid mediators during acute inflammation and, in this way, may control acute inflammatory diseases.     

Oxidative modification of low-density lipoprotein: lipid peroxidation by myeloperoxidase in the presence of nitrite

Oxidative modification of low-density lipoprotein (LDL) is a pivotal process in early atherogenesis and can be brought about by myeloperoxidase (MPO), which is capable of reacting with nitrite, a NO metabolite. We studied MPO-mediated formation of conjugated dienes in isolated human LDL in dependence on the concentrations of nitrite and chloride. This reaction was strongly stimulated by low concentrations (5-50 microM) of nitrite which corresponds to the reported concentration in the arterial vessel wall. Under these conditions no protein tyrosine nitration occurred; this reaction required much higher nitrite concentrations (100 microM-1 mM). Chloride neither supported lipid peroxidation alone nor was its presence mandatory for the effect of nitrite. We propose a prominent role of lipid peroxidation for the proatherogenic action of the MPO/nitrite system, whereas peroxynitrite may be competent for protein tyrosine nitration of LDL. Monomeric and oligomeric flavan-3-ols present in cocoa products effectively counteracted, at micromolar concentrations, the MPO/nitrite-mediated lipid peroxidation of LDL. Flavan-3-ols also suppressed protein tyrosine nitration induced by MPO/nitrite or peroxynitrite as well as Cu2+-mediated lipid peroxidation of LDL. This multi-site protection by (-)-epicatechin or other flavan-3-ols against proatherogenic modification of LDL may contribute to the purported beneficial effects of dietary flavan-3-ols for the cardiovascular system.     

Hemoglobin can nitrate itself and other proteins.

Incubation of human hemoglobin with nitrite and hydrogen peroxide was found to induce autonitration and nitration of another protein (bovine serum albumin), as demonstrated by detection of nitrotyrosine residues in Western blots of separated membrane proteins. Inhibition of nitration by conversion of hemoglobin into the cyanmet form demonstrates that nitration is due to the pseudoperoxidase activity of hemoglobin. Incubation of whole erythrocytes with nitrite and hydrogen peroxide induces nitration of erythrocyte membrane proteins, much stronger when cellular catalase was inhibited with azide. These results suggest that hemoglobin and other hemoproteins may contribute to the tyrosine nitration in vivo.     

Nitrate reductase alters 3-nitrotyrosine accumulation and cell cycle progression in LPS + IFN-gamma-stimulated RAW 264.7 cells.

Nitrite (NO2-), an end product of nitrogen radical metabolism, has recently been shown to increase tyrosine nitration by activated leukocytes indicating that nitrite modulates the immune response. We investigated the hypothesis that nitrite may increase nitration of molecular targets within activated cells leading to altered cell cycle progression. Intracellular nitrite was increased by transfection of murine macrophage-like RAW 264.7 cells with the nitrate reductase gene obtained from barley. Nitrate reductase facilitates the conversion of nitrate to nitrite; thus when extracellular nitrate is present, intracellular nitrite will be increased. Results show that addition of KNO3 increases NO2- production and intracellular nitrotyrosine accumulation in the transfectant but not the parent. Inhibition of nitric oxide synthesis with L-NAME during activation with IFN-gamma + LPS reduced NO2- production to the same extent in both cell lines; however, cellular accumulation of nitrotyrosine was reduced by only 25% in the transfectant (P = 0.21) and 49% in the parent cell line (P = 0.007), suggesting that intracellular nitrite increased nitrotyrosine accumulation through a pathway not requiring NO synthesis, i.e., myeloperoxidase system. Approximately 15% of the transfected cells had 4n DNA content 24 h postactivation compared to < 1% of the parent cells. Increased DNA copy number was correlated to nitrotyrosine accumulation. These findings show that intracellular nitrite can increase accumulation of nitrotyrosine and that nitration is linked to cell cycle perturbation.     

Eosinophil peroxidase nitrates protein tyrosyl residues. Implications for oxidative damage by nitrating intermediates in eosinophilic inflammatory disorders.

Eosinophil peroxidase (EPO) has been implicated in promoting oxidative tissue injury in conditions ranging from asthma and other allergic inflammatory disorders to cancer and parasitic/helminthic infections. Studies thus far on this unique peroxidase have primarily focused on its unusual substrate preference for bromide (Br(-)) and the pseudohalide thiocyanate (SCN(-)) forming potent hypohalous acids as cytotoxic oxidants. However, the ability of EPO to generate reactive nitrogen species has not yet been reported. We now demonstrate that EPO readily uses nitrite (NO(2)(-)), a major end-product of nitric oxide ((.)NO) metabolism, as substrate to generate a reactive intermediate that nitrates protein tyrosyl residues in high yield. EPO-catalyzed nitration of tyrosine occurred more readily than bromination at neutral pH, plasma levels of halides, and pathophysiologically relevant concentrations of NO(2)(-). Furthermore, EPO was significantly more effective than MPO at promoting tyrosine nitration in the presence of plasma levels of halides. Whereas recent studies suggest that MPO can also promote protein nitration through indirect oxidation of NO(2)(-) with HOCl, we found no evidence that EPO can indirectly mediate protein nitration by a similar reaction between HOBr and NO(2)(-). EPO-dependent nitration of tyrosine was modulated over a physiologically relevant range of SCN(-) concentrations and was accompanied by formation of tyrosyl radical addition products (e.g. o,o'-dityrosine, pulcherosine, trityrosine). The potential role of specific antioxidants and nucleophilic scavengers on yields of tyrosine nitration and bromination by EPO are examined. Thus, EPO may contribute to nitrotyrosine formation in inflammatory conditions characterized by recruitment and activation of eosinophils.     

Protein nitration in rat lungs during hyperoxia exposure: a possible role of myeloperoxidase

Several studies have suggested that exposure to hyperoxia causes lung injury through increased generation of reactive oxygen and nitrogen species. The present study was aimed to investigate the effects of hyperoxia exposure on protein nitration in lungs. Rats were exposed to hyperoxia (>95%) for 48, 60, and 72 h. Histopathological analysis showed a dramatic change in the severity of lung injury in terms of edema and hemorrhage between 48- and 60-h exposure times. Western blot for nitrotyrosine showed that several proteins with molecular masses of 29-66 kDa were nitrated in hyperoxic lung tissues. Immunohistochemical analyses indicate nitrotyrosine staining of alveolar epithelial and interstitial regions. Furthermore, immunoprecipitation followed by Western blot revealed the nitration of surfactant protein A and t1alpha, proteins specific for alveolar epithelial type II and type I cells, respectively. The increased myeloperoxidase (MPO) activity and total nitrite levels in bronchoalveolar lavage and lung tissue homogenates were observed in hyperoxic lungs. Neutrophils and macrophages isolated from the hyperoxia-exposed rats, when cocultured with a rat lung epithelial L2 cell line, caused a significant protein nitration in L2 cells. Inclusion of nitrite further increased the protein nitration. These studies suggest that protein nitration during hyperoxia may be mediated in part by MPO generated from activated phagocytic cells, and such protein modifications may contribute to hyperoxia-mediated lung injury.     

Myeloperoxidase functions as a major enzymatic catalyst for initiation of lipid peroxidation at sites of inflammation

Initiation of lipid peroxidation and the formation of bioactive eicosanoids are pivotal processes in inflammation and atherosclerosis. Currently, lipoxygenases, cyclooxygenases, and cytochrome P450 monooxygenases are considered the primary enzymatic participants in these events. Myeloperoxidase (MPO), a heme protein secreted by activated leukocytes, generates reactive intermediates that promote lipid peroxidation in vitro. For example, MPO catalyzes oxidation of tyrosine and nitrite to form tyrosyl radical and nitrogen dioxide ((.)NO(2)), respectively, reactive intermediates capable of initiating oxidation of lipids in plasma. Neither the ability of MPO to initiate lipid peroxidation in vivo nor its role in generating bioactive eicosanoids during inflammation has been reported. Using a model of inflammation (peritonitis) with MPO knockout mice (MPO(-/-)), we examined the role for MPO in the formation of bioactive lipid oxidation products and promoting oxidant stress in vivo. Electrospray ionization tandem mass spectrometry was used to simultaneously quantify individual molecular species of hydroxy- and hydroperoxy-eicosatetraenoic acids (H(P)ETEs), F(2)-isoprostanes, hydroxy- and hydroperoxy-octadecadienoic acids (H(P)ODEs), and their precursors, arachidonic acid and linoleic acid. Peritonitis-triggered formation of F(2)-isoprostanes, a marker of oxidant stress in vivo, was reduced by 85% in the MPO(-/-) mice. Similarly, formation of all molecular species of H(P)ETEs and H(P)ODEs monitored were significantly reduced (by at least 50%) in the MPO(-/-) group during inflammation. Parallel analyses of peritoneal lavage proteins for protein dityrosine and nitrotyrosine, molecular markers for oxidative modification by tyrosyl radical and (.)NO(2), respectively, revealed marked reductions in the content of nitrotyrosine, but not dityrosine, in MPO(-/-) samples. Thus, MPO serves as a major enzymatic catalyst of lipid peroxidation at sites of inflammation. Moreover, MPO-dependent formation of (.)NO-derived oxidants, and not tyrosyl radical, appears to serve as a preferred pathway for initiating lipid peroxidation and promoting oxidant stress in vivo.     

Induction of myeloperoxidase and nitrotyrosine formation in a human eosinophilic leukemia cell line, EoL-1

The human eosinophilic leukemia cell line, EoL-1, differentiated with butyrate as an eosinophilic cellular model was evaluated for peroxidase-dependent tyrosine nitration. Butyrate suppressed cell growth and induced eosinophilic granules in EoL-1 cells after 9 days of culture. Peroxidase activity was detected biochemically and histochemically from 3-day cultures and it increased in a time dependent manner. This peroxidase activity was inhibited by cyanide. Nitrotyrosine formation catalysed by peroxidase using hydrogen peroxide and nitrite was detected at a high level similar to that of mature eosinophils. However, no expression of eosinophil peroxidase (EPO) was detected by RT-PCR or immunocytochemistry. In contrast, the induction of myeloperoxidase (MPO) by butyrate was clearly detected by RT-PCR, Northern blot, and immunocytochemical staining. These results suggest that butyrate induces MPO rather than EPO in EoL-1 cells and that the formation of nitrotyrosine in butyrate-induced cells is dependent on MPO.     

Characterization of nitric oxide consumption pathways by normal,chronic granulomatous disease and myeloperoxidase-deficient human neutrophils

The detailed mechanisms by which acutely activated leukocytes metabolize NO and regulate its bioactivity are unknown. Therefore, healthy, chronic granulomatous disease (CGD) or myeloperoxidase (MPO)-deficient human neutrophils were examined for their ability to consume NO and attenuate its signaling. fMLP or PMA activation of healthy neutrophils caused NO consumption that was fully blocked by NADPH oxidase inhibition, and was absent in CGD neutrophils. Studies using MPO-deficient neutrophils, enzyme inhibitors, and reconstituted NADPH oxidase ruled out additional potential NO-consuming pathways, including Fenton chemistry, PGH synthase, lipoxygenase, or MPO. In particular, the inability of MPO to consume NO resulted from lack of H(2)O(2) substrate since all superoxide (O(2)(-.) reacted to form peroxynitrite. For healthy or MPO-deficient cells, NO consumption rates were 2- to 4-fold greater than O(2)(-.) generation, significantly faster than expected from 1:1 termination of NO with O(2)(-.). Finally, fMLP or PMA-stimulated NO consumption fully blocked NO-dependent neutrophil cGMP synthesis. These data reveal NADPH oxidase as the central regulator of NO signaling in human leukocytes. In addition, they demonstrate an important functional difference between CGD and either normal or MPO-deficient human neutrophils, namely their inability to metabolize NO which will alter their ability to adhere and migrate in vivo.     

Role of myeloperoxidase in the neutrophil-induced oxidation of low density lipoprotein as studied by myeloperoxidase-knockout mouse

Low density lipoprotein was oxidized by neutrophils derived from either C57BL/6 mice or myeloperoxidase (MPO)-knockout mice. The generation of superoxide from neutrophils of MPO-knockout mice was about 70% of that from wild-type mice. The extent of the oxidation of human low density lipoprotein (LDL) by phorbol myristate acetate (PMA)-activated neutrophils of wild-type and MPO-knockout mice was assessed by measuring consumption of a-tocopherol and formation of phosphatidylcholine hydroperoxide (PCOOH) and cholesteryl ester hydroperoxide (CEOOH). Little consumption of a-tocopherol was observed in both oxidations. It was found, however, that lipid hydroperoxides were accumulated with time in both oxidations and that the rates of formation of PCOOH and CEOOH in the oxidation by MPO-knockout neutrophils were about 66 and 44% of those by wild-type neutrophils, respectively. The lipid peroxidation was completely inhibited by adding superoxide dismutase (SOD) in both cases. The addition of L-tyrosine and SOD enhanced lipid peroxidation of LDL induced by wild-type neutrophils but not by MPO-knockout ones. These results suggest that, regardless of their MPO activity, neutrophils induce lipid peroxidation of LDL by a superoxide-dependent pathway, and that MPO-catalyzed lipid peroxidation is enhanced by the presence of an appropriate amount of free tyrosine and further enhanced by SOD.     

pH profile of cytochrome c-catalyzed tyrosine nitration

In the present study, we investigated how cytochrome c catalyzed the nitration of tyrosine at various pHs. The cytochrome c-catalyzed nitration of tyrosine occurred in proportion to the concentration of hydrogen peroxide, nitrite or cytochrome c. The cytochromec-catalyzed nitration of tyrosine was inhibited by catalase, sodium azide, cystein, and uric acid. These results show that the cytochrome c-catalyzed nitrotyrosine formation was due to peroxidase activity. The rate constant between cytochrome c and hydrogen peroxide within the pH range of 3-8 was the largest at pH 6 (37 degrees C). The amount of nitrotyrosine formed was the greatest at pH 5. At pH 3, only cytochromec-independent nitration of tyrosine occurred in the presence of nitrite. At this pH, the UV as well as visible spectrum of cytochrome c was changed by nitrite, even in the presence of hydrogen peroxide, probably via the formation of a heme iron-nitric oxide complex. Due to this change, the peroxidase activity of cytochrome c was lost.     

Role of interleukin-6 in a non-septic shock model induced by zymosan.

In the present study, we used IL-6 knock-out mice (IL-6KO) to evaluate a possible role of IL-6 in the pathogenesis of non-septic shock induced by peritoneal injection of zymosan. A severe inflammatory response characterized by peritoneal exudation, high peritoneal levels of nitrate/nitrite, and leukocyte infiltration into peritoneal exudate was induced by zymosan administration in wild-type control (WT) mice. This inflammatory process coincided with the damage to the lung and small intestine, as assessed by histological examination. Lung, small intestine and liver myeloperoxidase (MPO) activity, indicative of neutrophil infiltration and lipid peroxidation, were significantly increased in zymosan-treated WT mice. Peritoneal administration of zymosan in the WT mice also induced a significant increase in the plasma levels of nitrite/nitrate and in the levels of peroxynitrite, 18 hours after zymosan challenge. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine in the lung of zymosan-treated WT mice. Zymosan-treated IL-6KO showed significantly decreased mortality and inhibition of the development of peritonitis. In addition, IL-6KO mice showed significant protection from the development of organ failure, since tissue injury and MPO was reduced in the lung, small intestine and liver. Furthermore, a significant reduction of suppression of mitochondrial respiration, DNA strand breakage and reduction of cellular levels of NAD+ was observed in ex vivo macrophages harvested from the peritoneal cavity of IL-6KO mice subjected to zymosan-induced non-septic shock. In vivo treatment with anti-IL-6 (5,000 ng/day per mouse, 24 and 1 hour before zymosan administration) significantly reduced the inflammatory process. Taken together, the present study clearly demonstrates that IL-6 exerts a role in zymosan-induced non-septic shock.     

Myeloperoxidase plays critical roles in killing Klebsiella pneumoniae and inactivating neutrophil elastase: effects on host defense

Activated neutrophils use myeloperoxidase (MPO) to generate an array of potent toxic oxidants. In the current studies we used genetically altered mice deficient in MPO to investigate the role of the enzyme in host defense against the Gram-negative bacterium Klebsiella pneumoniae, an important human pathogen. For comparison, we used mice deficient in the antimicrobial molecule, neutrophil elastase (NE). When challenged i.p., mice deficient in either MPO or NE were markedly more susceptible to bacterial infection and death. In vitro studies suggested that MPO impairs the morphology of bacteria in a distinctive way. Of importance, our in vitro studies found that MPO mediated oxidative inactivation of NE, an enzyme that has been widely implicated in the pathogenesis of various tissue-destructive diseases. This pathway of oxidative inactivation may be physiologically relevant, because activated neutrophils isolated from MPO-deficient mice exhibited increased elastase activity. Our observations provide strong evidence that MPO, like NE, is a key player in the killing of K. pneumoniae bacteria. They also suggest that MPO may modulate NE to protect the host from the tissue-degrading activity of this proteinase.     

Factors determining the selectivity of protein tyrosine nitration.

Tyrosine nitration is a covalent posttranslational protein modification derived from the reaction of proteins with nitrating agents. Protein nitration appears to be a selective process since not all tyrosine residues in proteins or all proteins are nitrated in vivo. To investigate factors that may determine the biological selectivity of protein tyrosine nitration, we developed an in vitro model consisting of three proteins with similar size but different three-dimensional structure and tyrosine content. Exposure of ribonuclease A to putative in vivo nitrating agents revealed preferential nitration of tyrosine residue Y(115). Tyrosine residue Y(23) and to a lesser extent residue Y(20) were preferentially nitrated in lysozyme, whereas tyrosine Y(102) was the only residue modified by nitration in phospholipase A(2). Tyrosine Y(115) was the residue modified by nitration after exposure of ribonuclease A to different nitrating agents: chemically synthesized peroxynitrite, nitric oxide, and superoxide generated by SIN-1 or myeloperoxidase (MPO)/H(2)O(2) plus nitrite (NO(-2)) in the presence of bicarbonate/CO(2). The nature of the nitrating agent determined in part the protein that would be predominantly modified by nitration in a mixture of all three proteins. Ribonuclease A was preferentially nitrated upon exposure to MPO/H(2)O(2)/NO(-2), whereas phospholipase A(2) was the primary target for nitration upon exposure to peroxynitrite. The data also suggest that the exposure of the aromatic ring to the surface of the protein, the location of the tyrosine on a loop structure, and its association with a neighboring negative charge are some of the factors determining the selectivity of tyrosine nitration in proteins.     

Endogenously nitrated proteins in mouse brain: links to neurodegenerative disease

Increased abundance of nitrotyrosine modifications of proteins have been documented in multiple pathologies in a variety of tissue types and play a role in the redox regulation of normal metabolism. To identify proteins sensitive to nitrating conditions in vivo, a comprehensive proteomic data set identifying 7792 proteins from a whole mouse brain, generated by LC/LC-MS/MS analyses, was used to identify nitrated proteins. This analysis resulted in the identification of 31 unique nitrotyrosine sites within 29 different proteins. More than half of the nitrated proteins that have been identified are involved in Parkinson's disease, Alzheimer's disease, or other neurodegenerative disorders. Similarly, nitrotyrosine immunoblots of whole brain homogenates show that treatment of mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an experimental model of Parkinson's disease, induces an increased level of nitration of the same protein bands observed to be nitrated in brains of untreated animals. Comparing sequences and available high-resolution structures around nitrated tyrosines with those of unmodified sites indicates a preference of nitration in vivo for surface accessible tyrosines in loops, a characteristic consistent with peroxynitrite-induced tyrosine modification. In addition, most sequences contain cysteines or methionines proximal to nitrotyrosines, contrary to suggestions that these amino acid side chains prevent tyrosine nitration. More striking is the presence of a positively charged moiety near the sites of nitration, which is not observed for non-nitrated tyrosines. Together, these observations suggest a predictive tool of functionally important sites of nitration and that cellular nitrating conditions play a role in neurodegenerative changes in the brain.     

Enhanced killing of penicillin-treated gram-positive cocci by human granulocytes: role of bacterial autolysins, catalase, and granulocyte oxidative pathways

Staphylococci pretreated with subminimal inhibitory concentrations (subMIC) of cell-wall active antibiotics exhibit increased susceptibility to killing by human polymorphonuclear leukocytes (PMNs), even when phagosome information is impaired by the mold metabolite, cytochalasin B. To investigate the role of specific bacterial factors in the process, studies were carried out with organisms lacking catalase (streptococci) or cell-wall autolytic enzymes and compared to findings with Staphylococcus aureus 502A. Neutrophil factors were studied using inhibitors, oxygen radical scavengers, myeloperoxidase (MPO)-deficient PMNs, or PMNs from a patient with chronic granulomatous disease (CGD). Documentation of the enhanced susceptibility of the streptococcal strains to killing by PMNs following subMIC penicillin pretreatment required the use of cytochalasin B. Enhancement of killing occurred independent of the presence or absence of bacterial autolysins or catalase. SubMIC penicillin pretreatment of S. pneumoniae R36A specifically promoted the susceptibility of these organisms to killing by myeloperoxidase (MPO)-mediated mechanisms (enhancement lost using MPO-deficient or azide-treated cells). Factors other than MPO or toxic oxygen products generated by the PMN respiratory burst are responsible for enhanced killing of penicillin-pretreated S. aureus 502A (enhancement preserved using MPO-deficient, azide-treated, or chronic granulomatous disease patient cells). These studies define methods to study the interaction of antimicrobial agents and PMNs in the killing of microorganisms. They also demonstrate that penicillin treatment can change the susceptibility of gram-positive cocci to the action of specific PMN microbicidal mechanisms. The mechanism of the enhancement appears to be bacterial strain-dependent and not predictable by bacterial autolysin or catalase activity.     

Role of copper,zinc-superoxide dismutase in catalyzing nitrotyrosine formation in murine liver

The only known function of Cu,Zn-superoxide dismutase (SOD1) is to catalyze the dismutation of superoxide anion into hydrogen peroxide. Our objective was to determine if SOD1 catalyzes murine liver protein nitration induced by acetaminophen (APAP) and lipopolysaccharide (LPS). Liver and plasma samples were collected from young adult SOD1 knockout mice (SOD1(-/-)) and wild-type (WT) mice at 5 or 6 h after an ip injection of saline, APAP, or LPS. Hepatic nitrotyrosine formation was induced by APAP and LPS only in the WT mice. The diminished hepatic protein nitration in the SOD1(-/-) mice was not directly related to plasma nitrite and nitrate concentrations. Similar genotype differences were seen in liver homogenates treated with a bolus of peroxynitrite. Adding only the holo-, and not the apo-, SOD1 enzyme into the liver homogenates enhanced the reaction in an activity-dependent fashion and nearly eliminated the genotype difference at the high doses. Mass spectrometry showed four more nitrotyrosine residues in bovine serum albumin and 10 more nitrated protein candidates in the SOD1(-/-) liver homogenates by peroxynitrite with added SOD1. In conclusion, the diminished hepatic protein nitration mediated by APAP or LPS in the SOD1(-/-) mice is due to the lack of SOD1 activity per se.