1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) damages the nigrostriatal dopaminergic pathway as seen in Parkinson's disease (PD), a common neurodegenerative disorder with no effective protective treatment. Consistent with a role of glial cells in PD neurodegeneration, here we show that minocycline, an approved tetracycline derivative that inhibits microglial activation independently of its antimicrobial properties, mitigates both the demise of nigrostriatal dopaminergic neurons and the formation of nitrotyrosine produced by MPTP. In addition, we show that minocycline not only prevents MPTP-induced activation of microglia but also the formation of mature interleukin-1beta and the activation of NADPH-oxidase and inducible nitric oxide synthase (iNOS), three key microglial-derived cytotoxic mediators. Previously, we demonstrated that ablation of iNOS attenuates MPTP-induced neurotoxicity. Now, we demonstrate that iNOS is not the only microglial-related culprit implicated in MPTP-induced toxicity because mutant iNOS-deficient mice treated with minocycline are more resistant to this neurotoxin than iNOS-deficient mice not treated with minocycline. This study demonstrates that microglial-related inflammatory events play a significant role in the MPTP neurotoxic process and suggests that minocycline may be a valuable neuroprotective agent for the treatment of PD.

Formation of nitric oxide-derived oxidants has been linked to development of atherosclerosis and associated thrombotic complications. Although systemic levels of protein nitrotyrosine predict risk for coronary artery disease, neither specific proteins targeted for modification nor functional consequences that might contribute to disease pathogenesis have been defined. Here we report a selective increase in circulating levels of nitrated fibrinogen in patients with coronary artery disease. Exposure of fibrinogen to nitrating oxidants, including those produced by the myeloperoxidase-hydrogen peroxide-nitrite system, significantly accelerates clot formation and factor XIII cross-linking, whereas exposure of fibrinogen to non-nitrating oxidants decelerates clot formation. Clots formed with fibrinogen exposed to nitrating oxidants are composed of large bundles made from twisted thin fibrin fibers with increased permeation and a decrease in storage modulus G value, suggesting that these clots could be easily deformed by mechanical stresses. In contrast, clots formed with fibrinogen exposed to non-nitrating oxidants showed decreased permeation with normal architecture. Fibrinogen modified by exposure to physiologic nitration systems demonstrated no difference in the rate of plasmin-induced clot lysis, platelet aggregation, or binding. Thus, increased levels of fibrinogen nitration may lead to a pro-thrombotic state via acceleration in formation of fibrin clots. The present results may account, in part, for the association between nitrative stress and risk for coronary artery disease.Epidemiological studies have indicated that increased levels of circulating fibrinogen is an independent predictor of coronary heart disease and in some cases of premature death from cardiovascular disease, although a causative relationship between high levels of fibrinogen and cardiovascular disease has not been firmly established (1-4). Fibrinogen is a multifunctional protein essential for hemostasis. It is a 340-kDa glycoprotein, consisting of three non-identical peptide chains A␣, B␤, and ␥, which are linked together by 29 disulfide bonds (5). During coagulation, the soluble fibrinogen is converted to insoluble fibrin polymers. The process is initiated by thrombin, a serine protease, which catalyzes the cleavage first of two fibrinopeptides from the amino termini of the A␣ chains and then two fibrinopeptides from the amino termini B␤ chains. Upon release of the fibrinopeptides, the remaining fibrin monomers aggregate spontaneously to form ordered fibrin polymers (5). The clot is stabilized by the formation of covalent bonds introduced by the action of a transglutaminase, factor XIII (6). Under physiological conditions, fibrinolysis is dependent on the binding of circulating plasminogen and tissue-type plasminogen activator (tPA) 1 to fibrin clots. Urokinase and tPA convert plasminogen to the active protease plasmin, which then cleaves fibrin polymers to soluble fragments completing the coagulation and clot resolution cycle.A major cause...

Nitrotyrosine is widely used as a marker of post-translational modification by the nitric oxide ( ⅐ 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 ( ⅐ 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 heliumswept reaction mixtures provides direct evidence that leukocyte peroxidases catalytically generate ⅐ 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 ⅐ 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, ⅐ NO 2 ; 3) as a minor reaction, peroxidases may also catalyze the two-electron oxidation of NO 2 ؊