[39] Immunohistochemical methods to detect nitrotyrosine

Viera, Liliana; Ye, Yao Zu; Estévez, Álvaro G.; Beckman, Joseph S.

doi:10.1016/s0076-6879(99)01101-5

Cited by 128 publications

(81 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The Histomouse™-MAX kit (Zymed, South San Francisco, CA) was used for immunostaining of liver sections, and 12% gel was run to separate liver homogenates (50 μg protein/lane) for Western analysis. Specificity of the antibody against nitrotyrosine was verified by using 10 mM sodium dithionite to reduce nitrotyrosine on the blotting membrane and incubating the antibody with 20 μM 3-nitrotryosine overnight for a prior block [37]. Protein concentration was measured by the Lowry method [38].…”

Section: Hepatic Protein Nitration Antibody Verification and Plasmamentioning

confidence: 99%

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

Zhu

Zhang

Roneker

et al. 2008

Free Radical Biology and Medicine

View full text Add to dashboard Cite

The solely 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 catalyzed 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, but 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 4 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 was due to the lack of SOD1 activity per se. Keywords SOD1; Protein nitration; Acetaminophen; Peroxynitrite; Lipopolysaccharide; Mouse Although copper,zinc-superoxide dismutase (SOD1) has been widely considered a major intracellular antioxidant enzyme in mammals, its solely known function is to catalyze the conversion of superoxide anion into less active hydrogen peroxide. Indeed, SOD1 is protective against oxidative stress associated with acute paraquat toxicity, myocardial ischemia/reperfusion injury, and N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurodegeneration [1][2][3]. Mice deficient in SOD1 develop Address correspondence to: Xin Gen Lei, Department of Animal Science, Cornell University, Ithaca, NY 14853, Tel. 607-254-4703; Fax. 607-255-9829; E-Mail: XL20@cornell.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. [7,8]. These paradoxical findings strongly suggest that SOD1 may exert functions more than just superoxide dismutation. In fact, SOD1 was shown to promote peroxynitrite-mediated nitrotyrosine formation in vitro [9,10]. NIH Public AccessPeroxynitrite is formed by a rapid diffusion-limited, radical-radical coupling reaction between nitric oxide (NO) and superoxide anion [11,12] [24,25,33,34] provide us with an unprecedented opportunity to study the in vivo role of SOD1 in nitrotyrosine formation. Furtherm...

show abstract

Section: Hepatic Protein Nitration Antibody Verification and Plasmamentioning

confidence: 99%

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

Zhu

Zhang

Roneker

et al. 2008

Free Radical Biology and Medicine

View full text Add to dashboard Cite

show abstract

“…To verify the specificity of the assay system, samples were pre-treated with sodium hydrosulfite as described by Ischiropoulos et al [19] to convert 3-nitrotyrosine to aminotyrosine [20][21][22]. In brief, ischemic/reperfused heart tissue (n=3 rats) was homogenized and the supernatant pH was adjusted to 9.0 by addition of sodium hydroxide.…”

Section: Quantitation Of Tissue Nitrotyrosine Contentmentioning

confidence: 99%

Nitrative thioredoxin inactivation as a cause of enhanced myocardial ischemia/reperfusion injury in the aging heart

Zhang

Tao

Jiao

et al. 2007

Free Radical Biology and Medicine

View full text Add to dashboard Cite

Objective-Several recent studies have demonstrated that thioredoxin (Trx) is an important antiapoptotic/cytoprotective molecule. The present study was designed to determine whether Trx activity is altered in the aging heart in a way that may contribute to increased susceptibility to myocardial ischemia/reperfusion (MI/R).Methods and Results-Compared to young animals, MI/R-induced cardiomyocyte apoptosis and infarct size were increased in aging animals (P<0.01). Trx activity was decreased in the aging heart before MI/R, and this difference was further amplified after MI/R. Trx expression was moderately increased and Trx nitration, a post-translational modification that inhibits Trx activity, was increased in the aging heart. Moreover, Trx-ASK1 complex formation was reduced and activity of p38 MAPK was increased. Treatment with FP15 (a peroxynitrite decomposition catalyst) reduced Trx nitration, increased Trx activity, restored Trx-ASK1 interaction, reduced P38 MAPK activity, attenuated caspase 3 activation and reduced infarct size in aging animals (p<0.01). Conclusions-Our results demonstrated that Trx activity is decreased in the aging heart by posttranslational nitrative modification. Interventions that restore Trx activity in the aging heart may be novel therapies to attenuate MI/R injury in aging patients.

show abstract

“…Tg SOD1 WT and G93A mice express transgenic SOD1 at the same level, as measured by densitometric analysis (data not shown), allowing accurate proteome map comparisons. Technical problems exist in the detection of nitrated proteins because (i) the modification is rare in tissues and in biological fluids, even in pathological conditions (10); (ii) the modification is lost in strongly reducing conditions and possibly also under the action of not yet identified denitrase enzymes (17,18); and (iii) common chemical and immunological methods may detect false-positive nitrated proteins (35,36). Therefore, special care was needed in each experiment to avoid under-or overestimation, as described under "Materials and Methods."…”

Section: Fig 3 Nt Immunoreactivity In Ventral Horn Of Transverse Sementioning

confidence: 99%

Protein Nitration in a Mouse Model of Familial Amyotrophic Lateral Sclerosis

Casoni

Basso

Massignan

et al. 2005

Journal of Biological Chemistry

172

119

View full text Add to dashboard Cite

Multiple mechanisms have been proposed to contribute to amyotrophic lateral sclerosis (ALS) pathogenesis, including oxidative stress. Early evidence of a role for oxidative damage was based on the finding, in patients and murine models, of high levels of markers, such as free nitrotyrosine (NT). However, no comprehensive study on the protein targets of nitration in ALS has been reported. We found an increased level of NT immunoreactivity in spinal cord protein extracts of a transgenic mouse model of familial ALS (FALS) at a presymptomatic stage of the disease compared with age-matched controls. NT immunoreactivity is increased in the soluble fraction of spinal cord homogenates and is found as a punctate staining in motor neuron perikarya of presymptomatic FALS mice. Using a proteome-based strategy, we identified proteins nitrated in vivo, under physiological or pathological conditions, and compared their level of specific nitration. ␣-and ␥-enolase, ATP synthase ␤ chain, and heat shock cognate 71-kDa protein and actin were overnitrated in presymptomatic FALS mice. We identified by matrix-assisted laser desorption/ionization mass spectrometry 16 sites of nitration in proteins oxidized in vivo. In particular, ␣-enolase nitration at Tyr 43 , target also of phosphorylation, brings additional evidence on the possible interference of nitration with phosphorylation. In conclusion, we propose that protein nitration may have a role in ALS pathogenesis, acting directly by inhibiting the function of specific proteins and indirectly interfering with protein degradation pathways and phosphorylation cascades.

show abstract

[39] Immunohistochemical methods to detect nitrotyrosine

Cited by 128 publications

References 29 publications

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

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

Nitrative thioredoxin inactivation as a cause of enhanced myocardial ischemia/reperfusion injury in the aging heart

Protein Nitration in a Mouse Model of Familial Amyotrophic Lateral Sclerosis

Contact Info

Product

Resources

About