The Determination of Nitrotyrosine Residues in Proteins

Gow, Andrew J.; McClelland, Molly; Garner, Sarah; Malcolm, Stuart; Ischiropoulos, Harry

doi:10.1385/1-59259-749-1:291

Cited by 10 publications

(12 citation statements)

References 17 publications

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“…For immunochemical detection of protein nitrotyrosine residues (3NT) in liver homogenate, a slot-blot technique was used. 30 A total of 0.5 g of homogenate protein were loaded onto nitrocellulose and placed in a Slot-Blot microfiltration unit (Bio-Rad Laboratories, Hercules, CA). For nitrotyrosine determination, the polyclonal antibody previously described for immunohistochemical detection was used.…”

Section: Methodsmentioning

confidence: 99%

Alcohol-induced liver injury in mice lacking Cu, Zn-superoxide dismutase

et al. 2003

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Because alcoholic liver disease has been linked to oxidative stress, we investigated the effect of a compromised antioxidant defense system, Cu, Zn-superoxide dismutase (Sod1) deficiency, on alcohol-induced liver injury. C57BL/129SV wild-type (Sod1 ؉/؉ ) and Sod1 knockout (Sod1 ؊/؊ ) mice were fed dextrose or ethanol (10% of total calories) liquid diets for 3 weeks. Histologic evaluation of liver specimens of Sod1 ؊/؊ mice fed ethanol showed the development of liver injury ranging from mild to extensive centrilobular necrosis and inflammation. Sod1 ؉/؉ mice fed ethanol showed mild steatosis; both Sod1 ؉/؉ and Sod1 ؊/؊ mice fed the dextrose diet had normal histology. Alanine transaminase levels were significantly elevated only in Sod1 ؊/؊ mice fed ethanol. Cytochrome P450 2E1 (CYP2e1) activity was elevated about 2-fold by ethanol in Sod1 ؉/؉ and Sod1 ؊/؊ mice. Ethanol consumption increased levels of protein carbonyls and lipid peroxidation aldehydic products in the liver of Sod1 ؊/؊ mice. Hepatic adenosine triphosphate (ATP) content was reduced dramatically in Sod1 ؊/؊ mice fed ethanol in association with a decrease in the mitochondrial reduced glutathione (GSH) level and activity of MnSOD. Immunohistochemical determination of 3-nitrotyrosine (3NT) residues in liver sections of the Sod1 knockout mice treated with ethanol showed a significant increase of 3NT staining in the centrilobular areas. In conclusion, a rather moderate ethanol consumption promoted oxidative stress in Sod1 ؊/؊ mice, with increased formation of peroxynitrite, protein carbonyls, and lipid peroxidation and decreased mitochondrial GSH and MnSOD. We speculate that the increased oxidative stress causes mitochondrial damage and reduction of ATP content, leading to alcoholic liver injury. This model may be useful in further mechanistic studies on alcohol-induced liver injury. (HEPATOLOGY 2003;38:1136-1145

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Section: Methodsmentioning

confidence: 99%

Alcohol-induced liver injury in mice lacking Cu, Zn-superoxide dismutase

et al. 2003

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“…Paraffin sections prepared from the lungs of SP-D (Ϫ/Ϫ) and (ϩ/ϩ) mice were stained with H&E for evaluation of airway inflammation. Sections were also used for immunohistochemistry for the detection of SNO and nitrotyrosine as previously described (24,30). For the purposes of inflammation scoring, blinded slides were assessed as follows; 0, no lesions; 1, minimal lymphocytic restricted to perivascular and peribronchiolar regions; 2, moderate perivascular and peribronchiolar inflammation with mildly increased numbers of alveolar macrophages, lymphocytes, and eosinophils; 3, marked perivascular and peribronchiolar inflammation with moderately increased numbers of alveolar macrophages, lymphocytes, eosinophils, and multinucleated giant cells; 4, severe perivascular and peribronchiolar inflammation with markedly increased numbers of alveolar macrophages, eosinophils, lymphocytes, and multinucleated giant cells; 5, severe, perivascular, and peribronchiolar inflammation with effacement of alveolar parenchyma and small airways by sheets of inflammatory cells.…”

Section: Lung Tissue Histologymentioning

confidence: 99%

Surfactant Protein-D, a Mediator of Innate Lung Immunity, Alters the Products of Nitric Oxide Metabolism

Atochina

Beers

Hawgood

et al. 2004

Am J Respir Cell Mol Biol

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Surfactant protein (SP)-D, a 43-kD multifunctional collagen-like lectin, is synthesized and secreted by the airway epithelium. SP-D knockout (SP-D [-/-]) mice exhibit an increase in the number and size of airway macrophages, peribronchiolar inflammation, increases in metalloproteinase activity, and development of emphysema. Nitric oxide (NO) is involved in a variety of signaling processes, and because altered NO metabolism has been observed in inflammation, we hypothesized that alterations in its metabolism would underlie the proinflammatory state observed in SP-D deficiency. Examination of the bronchial alveolar lavage (BAL) from SP-D (-/-) mice reveals a significant increase in protein and phospholipid content and total cell count. NO production and inducible NO synthase expression were increased in the BAL; however, there was a decline in S-nitrosothiol (SNO) content in the BAL and a loss of SNO immunoreactivity within the tissue. This decline in SNO was accompanied by an increase in nitrotyrosine staining. We conclude that inflammation that occurs in SP-D deficiency results in an increase in NO production and a shift in the chemistry and targets of NO. We speculate that the proinflammatory response due to SP-D deficiency results, in part, from a disruption of NO-mediated signaling within the innate immune system.

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“…The 3-NT content of 20 mg of each lung homogenate was analyzed by use of solid-phase ELISA using an anti-3-NT monoclonal antibody (1:1000 dilution) and a sheep antimouse secondary antibody linked to horseradish peroxidase, as described elsewhere [45,46]. Relative units were calculated by comparison to a standard of nitrated bovine serum albumin.…”

Section: Solid-phase Elisa For 3-ntmentioning

confidence: 99%

“…Data are percentage of uninfected wt levels. Total mean ‫ע‬ SEM lung homogenate 3-NT content was determined by solid-phase ELISA dotblotting, as described elsewhere[45,46].a , vs. corresponding uninfected wt level. P !…”

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confidence: 99%

Delayed Clearance ofPneumocystis cariniiInfection, Increased Inflammation, and Altered Nitric Oxide Metabolism in Lungs of Surfactant Protein–D Knockout Mice

et al. 2004

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Surfactant protein-D (SP-D), a member of the "collectin" family, has been shown to play a role in innate immunity through modulation of inflammation and clearance of organisms. The role of SP-D in host defense against Pneumocystis carinii pneumonia was assessed using SP-D knockout (KO) mice. When inoculated with P. carinii, both wild-type (wt) and SP-D KO mice required CD4 cell depletion to develop infection. In CD4 cell-depleted models, 2 weeks after infection with P. carinii, SP-D KO mice developed increased intensity of infection, compared with wt mice, despite higher lung-inflammation scores and increased amounts of alveolar inflammatory cells. The increased inflammation seen in SP-D KO mice was accompanied by increases in lung weight, expression of inducible nitric oxide (NO) synthase, total NO levels, and 3-nitrotyrosine levels in lung tissue. These results indicate that SP-D plays a role in host defense against P. carinii in vivo by modulating clearance of organisms, lung inflammation, and metabolism of NO.

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The Determination of Nitrotyrosine Residues in Proteins

Cited by 10 publications

References 17 publications

Alcohol-induced liver injury in mice lacking Cu, Zn-superoxide dismutase

Alcohol-induced liver injury in mice lacking Cu, Zn-superoxide dismutase

Surfactant Protein-D, a Mediator of Innate Lung Immunity, Alters the Products of Nitric Oxide Metabolism

Delayed Clearance ofPneumocystis cariniiInfection, Increased Inflammation, and Altered Nitric Oxide Metabolism in Lungs of Surfactant Protein–D Knockout Mice

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