• Archives of Pharmacal Research
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• v.28 no.3
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• pp.358-363
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• 2005

We have studied the interaction of 3-nitrotyrosine with peroxynitrite using three different methods; chemiluminescence, spectrophotometry and HPLC. Peroxynitrite-induced luminol or lucigenin chemiluminescence were significantly decreased by 3-nitrotyrosine, in concentration­dependent manners. The intensity of the peroxynitrite spectrum was also markedly reduced in the presence of 3-nitrotyrosine in the spectrophometric assay. However, there was no attenuation of the 3-nitrotyrosine signal in the HPLC assay after mixing with peroxynitrite. The interaction of 3-nitrotyrosine and hypochlorous acid (HOCI) was also studied via the chemilumines-cence assay, where the HOCI-induced responses were markedly inhibited by 3-nitrotyrosine. These results suggest that caution should be taken when studying the levels or interactions of 3-nitrotyrosine.

• Journal of Veterinary Clinics
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• v.36 no.2
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• pp.102-105
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• 2019

Nitrotyrosine was found to be dependent on the severity of myxomatous mitral valve disease (MMVD). However, a correlation of serum nitrotyrosine concentration in dogs with MMVD and the progression of the disease has not been investigated. This study compared changes in serum nitrotyrosine concentration with the progression of MMVD. Nine client-owned dogs were recruited for the study. Dogs were classified by measuring the amount of regurgitation using echocardiography into mild, moderate, or severe MMVD groups. Serum nitrotyrosine concentration was measured by an enzyme-linked immunosorbent assay test. Serum nitrotyrosine concentration was significantly higher at 180 days than at 0 day (P < 0.05). However, serum nitrotyrosine concentration at 360 days was lower than that at 180 days (P < 0.05). Serum nitrotyrosine concentration at 540 days was lower than at 180 days (P < 0.05). There was no correlation between serum nitrotyrosine and left atrial to aortic root diameter ratio (LA/Ao ratio) (n = 33, $R^2=0.003$, P = 0.759). Also, there was no correlation between serum nitrotyrosine and vertebral heart score (VHS) (n = 33, $R^2=0.026$, P = 0.368) and left ventricular end-diastolic diameter, normalized for body weight by the formula (LVEDDN) (n = 33, $R^2=0.053$, P = 0.196). The results of the study suggest that the progression of MMVD is correlated with changes in serum nitrotyrosine concentration, which shows potential for use as a cardiac biomarker which can be used to analyze the progression of disease in MMVD.

• Korean Journal of Veterinary Research
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• v.57 no.1
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• pp.17-21
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• 2017

The aim of this study was to compare serum nitrotyrosine concentrations in healthy dogs with those in dogs with myxomatous mitral valve disease (MMVD). Fifty client-owned dogs were included in this study. Based on echocardiographic results, dogs were categorized into healthy (control), mild-, moderate-, and severe-MMVD groups. Serum nitrotyrosine concentrations were determined from enzyme-linked immunosorbent assays. No significant difference between control dogs and dogs with mild MMVD was detected (p = 0.31). However, dogs with moderate MMVD had significantly higher serum concentrations of nitrotyrosine (p = 0.04) than that in controls, and dogs with severe MMVD had significantly lower serum concentrations of nitrotyrosine (p = 0.03) than that in moderate MMVD dogs. There were negative correlations in the association of serum nitrotyrosine with age (n = 30, $R^2=0.067$, p = 0.27), left atrial-to-aortic root diameter ratio (n = 30, $R^2=0.02$, p = 0.57), and platelet count (n = 30, $R^2=0.39$, p = 0.003); however, only the platelet correlation was significant. Among dogs with MMVD, there was no significant difference in serum nitrotyrosine concentration between males and females. The results of this study suggest that tyrosine nitration end-products might be potential biomarkers for the detection of MMVD in dogs.

• BMB Reports
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• v.39 no.2
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• pp.189-196
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• 2006

Alterations in the amino acid structure or sequence can generate neo-epitopes from self-proteins causing autoaggressive immune attack. Reactive nitrogen species are an important factor that induces post-translational modification of proteins by cellular reduction and oxidation mechanism; cysteinyl-nitrosylation or tyrosine nitration leading to potentially pathogenic pathways. It was thought of interest to investigate the immunogenicity of nitrated poly L-tyrosine vis-$\`{a}$-vis its possible role in the induction of antibodies in systemic lupus erythematosus (SLE). Commercially available poly L-tyrosine was exposed to nitrating species and the damage was monitored by UV spectroscopy and alkaline gel electrophoresis. The results indicated the formation of 3-nitrotyrosine. Nitrated poly L-tyrosine induced higher titre antibodies as compared to the native form. Nitrated poly L-tyrosine was recognized by the autoantibodies present in the sera of patients suffering from SLE by enzyme immunoassays and band shift assay. The possible role of nitrated self-proteins has been discussed in the production of circulating anti-DNA antibodies in SLE.

• BMB Reports
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• v.38 no.5
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• pp.577-583
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• 2005

Subtilisin QK, which is newly identified as a fibrinolytic enzyme from Bacillus subtilis QK02, has the ability of preventing nitrotyrosine formation in bovine serum albumin induced by nitrite, hydrogen peroxide and hemoglobin in vitro verified by ELISA, Western-blot and spectrophotometer assay. Subtilisin QK also attenuates the fluorescence emission spectra of bovine serum albumin in the course of oxidation caused by nitrite, hydrogen peroxide and hemoglobin. Furthermore, subtilisin QK could suppress the transformation of oxy-hemoglobin to met-hemoglobin caused by sodium nitrite, but not the heat-treated subtilisn QK. Compared with some other fibrinolytic enzymes and inactivated subtilisin QK treated by phenylmethylsulfonylfluoride, the ability of inhibiting met-hemoglobin formation of subtilisin QK reveals that the anti-oxidative ability of subtilisin QK is not concerned with its fibrinolytic function. Additionally, nitrotyrosine formation in proteins from brain, heart, liver, kidney, and muscle of mice that is intramuscular injected the mixture of nitrite, hydrogen peroxide and hemoglobin is attenuated by subtilisin QK. Subtilisin QK can also protect Human umbilical vein endothelial cell (ECV-304) from the damage caused by nitrite and hydrogen peroxide.

• The Korean Journal of Physiology and Pharmacology
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• v.5 no.3
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• pp.205-212
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• 2001

It has been proposed that nitirc oxide is involved in the pathogenesis of cerebral ischemia-reperfusion. Because superoxide production is also enhanced during reperfusion, the cytotoxic oxidant peroxynitrite could be formed, but it is not known if this occurs following global forebrain ischemia-reperfusion. We examined whether peroxynitrite generation is increased in the vulnerable regions after forebrain ischemia-reperfusion. Transient forebrain ischemia was produced in the conscious rat by four-vessel occlusion. Rats were subjected to 10 or 15 min of forebrain ischemia. Immunohistochemical method was used to detect 3-nitrotyrosine, a marker of peroxynitrite production. 3-Nitrotyrosine immunoreactivity was enhanced in the hippocampal CA1 area 3 days after reperfusion. Furthermore, in rats subjected to ischemia for 15 min, this change was also observed in the lateral striatal region and the lateral septal nucleus $2{\sim}3$ days after reperfusion. The cresyl violet staining of adjacent sections showed that neuronal cell death was induced in parallel with the nitrotyrosine immunoreactivity in the hippocampal CA1 area and the lateral striatal region. Our findings suggest that oxygen free radical accumulation and consequent peroxynitrite production play a role in neuronal death caused by cerebral ischemia-reperfusion.

• IMMUNE NETWORK
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• v.4 no.2
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• pp.108-115
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• 2004

Background: Production of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in the pathology of autoimmune disease. It is unknown whether iNOS expression is increased within testes and whether iNOS and NO have essential roles in the pathogenesis of EAO. Methods: EAO was induced in guinea pig testes at 17 days after secondary immunization by administration of crude extract (CE) and purified glycoprotein 1 (GP1) from normal guinea pig testes. iNOS gene expression was assessed by RT-PCR and Northern blot analysis in testes. Localization of iNOS and Mac-1 and the indicator of NO-mediated tissue injury, nitrotyrosine, were detected in the testicular lesion by immunohistochemistry. Results: In control testes, inflammation and iNOS gene expression were not detected, whereas, in CE- and GP1-injected testes, inflammation and marked iNOS gene expression were evident at day 17 after secondary immunization. Immunohistochemistry of Mac-1 showed the colocalization with iNOS protein and nitrotyrosyl proteins in intertubules, suggesting that NO produced by infiltrated macrophages may be involved in inflammatory lesions of intertubules. Intraperitoneal administration of aminoguanidine significantly prevented EAO with reduction of inflammation, iNOS expression and nitrotyrosine formation. Conclusion: These results suggest that NO production by macrophages may be important in the pathogenesis of CE- and GP1-induced EAO. Furthermore, this study demonstrated the therapeutic potential of iNOS inhibitor in the treatment of inflammatory and autoimmune mediated-diseases.

• Bulletin of the Korean Chemical Society
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• v.31 no.9
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• pp.2581-2587
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• 2010

Nitration of tyrosine and tryptophan residues is common in cells under nitrative stress. However, physiological consequences of protein nitration are not well characterized on a molecular level due to limited availability of the 3D structures of nitrated proteins. Molecular dynamics (MD) simulation can be an alternative tool to probe the structural perturbations induced by nitration. In this study we developed molecular mechanics parameters for 3-nitrotyrosine (NIY) and 6-nitrotryptophan (NIW) that are compatible with the AMBER-99 force field. Partial atomic charges were derived by using a multi-conformational restrained electrostatic potential (RESP) methodology that included the geometry optimized structures of both $\alpha$- and $\beta$-conformers of a capped tripeptide ACE-NIY-NME or ACE-NIW-NME. Force constants for bonds and angles were adopted from the generalized AMBER force field. Torsional force constants for the proper dihedral C-C-N-O and improper dihedral C-O-N-O of the nitro group in NIY were determined by fitting the torsional energy profiles obtained from quantum mechanical (QM) geometry optimization with those from molecular mechanical (MM) energy minimization. Force field parameters obtained for NIY were transferable to NIW so that they reproduced the QM torsional energy profiles of ACE-NIW-NME accurately. Moreover, the QM optimized structures of the tripeptides containing NIY and NIW were almost identical to the corresponding structures obtained from MM energy minimization, attesting the validity of the current parameter set. Molecular dynamics simulations of thioredoxin nitrated at the single tyrosine and tryptophan yielded well-behaved trajectories suggesting that the parameters are suitable for molecular dynamics simulations of a nitrated protein.

• Safety and Health at Work
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• v.5 no.2
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• pp.91-96
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• 2014

Background: Chronic obstructive pulmonary disease (COPD) is an important cause of occupational mortality in miners exposed to coal mine dust. Although the inflammatory mediators involved in COPD have not been defined, many studies have shown that inflammatory mediators such as reactive oxygen and nitrogen species are involved in orchestrating the complex inflammatory process in COPD. Methods: To investigate the relevance of exhaled biomarkers of oxidative and nitrosative stress in participants with COPD, we determined the levels of hydrogen peroxide, malondialdehyde (MDA), and 3-nitrotyrosine (3-NT) in exhaled breath condensate (EBC) in 90 retired elderly coal miners (53 non-COPD and 37 COPD participants). Results: Mean levels of MDA (4.64 nMvs. 6.46 nM, p = 0.005) and 3-NT (3.51 nMvs. 5.50 nM, p = 0.039) in EBC were significantly higher in participants with COPD. The median level of MDA did show statistical difference among the COPD severities (p = 0.017), and the area under the receiver operating characteristic curve forMDA (0.67) for the diagnostic discrimination of COPD indicated the biomarker. The optimal cutoff values were 5.34 nM (64.9% sensitivity and 64.2% specificity) and 5.58 nM (62.2% sensitivity and 62.3% specificity) forMDA and 3-NT, respectively. The results suggest that high levels ofMDA and 3-NT in EBC are associated with COPD in retired elderly miners. Conclusion: These results showed that the elevated levels of EBC MDA and EBC 3-NT in individuals with COPD are biomarkers of oxidative or nitrosative stress.

• The Korean Journal of Physiology and Pharmacology
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• v.8 no.1
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• pp.1-5
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• 2004

Transient forebrain ischemia results in the delayed neuronal death in the CA1 area of the hippo-campus. The present study was performed to determine effects of aminoguanidine, a selective iNOS inhibitor, on the generation of peroxynitrite and delayed neuronal death occurring in the hippocampus following transient forebrain ischemia. Transient forebrain ischemia was produced in the conscious rats by four-vessel occlusion for 10 min. Treatment with aminoguanidine (100 mg/kg or 200 mg/kg, i.p.) or saline (0.4 ml/100 g, i.p.) was started 30 min following ischemia-reperfusion and the animals were then injected twice daily until 12 h before sacrifice. Immunohistochemical method was used to detect 3-nitrotyrosine, a marker of peroxynitrite production. Posttreatment of aminoguanidine (200 mg/kg) significantly attenuated the neuronal death in the hippocampal CA1 area 3 days, but not 7 days, after ischemia-reperfusion. 3-Nitrotyrosine immunoreactivity was enhanced in the hippocampal CA1 area 3 days after reperfusion, which was prevented by the treatment of aminoguanidine (100 mg/kg and 200 mg/kg). Our findings showed that (1) the generation of peroxynitrite in the hippocampal CA1 area 3 days after ischemia-reperfusion was dependent on the iNOS activity; (2) the postischemic delayed neuronal death was attenuated in the early phase through the prevention of peroxynitrite generation by an iNOS inhibitor.