title: Immunohistochemical detection and Western blot analysis of nitrated protein in stored human corneal epithelium.
abstract: While the production of nitric oxide by human corneas in storage has recently been demonstrated, protein nitration as a result of this production has not been demonstrated. In this study, nitrated protein accumulation in the epithelium of stored human corneas was assessed. One half of five donor corneas maintained in storage media for 3 days were prepared for immunohistochemical studies. The other halves remained in storage media for 7 additional days and were also processed for immunohistochemistry. Mouse monoclonal antibody to nitrotyrosine adducts was used to define the localisation of these epitopes. The density of antibody staining was observed and quantified on a digital camera system and statistically analysed. Immunostaining in the epithelium was greater in tissues recovered after 10 days in storage compared to the intensity of staining after 3 days of storage (p<0.0001). No staining was evident in the epithelium in sections exposed to non-immune mouse IgG. Western blot analysis was performed on epithelial cells scraped from corneal surfaces of one-half of four donor corneas in storage for 3 days and from the other half at 10 days of storage. Nitrated BSA was used as a positive control. After extraction and homogenisation, identical protein concentrations of each sample were loaded per lane on 10% gels and subjected to SDS-PAGE. Proteins were blotted and probed with the anti-nitrotyrosine antibody. Western blot immunoreactivity was detected in epithelial samples at the 3 and 10 day recovery times with the latter samples showing greater staining intensity. Nitrated protein, thought to indicate toxic peroxynitrite formation, accumulates in the human corneal epithelium with time of storage. Our study shows that there is an association between increased nitrated protein and storage time.
authors.affiliation: Cole Eye Institute, i-32, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA.
authors.forename: Bennie H
authors.initials: BH
authors.lastname: Jeng
authors.forename: Karen G
authors.initials: KG
authors.lastname: Shadrach
authors.forename: David M
authors.initials: DM
authors.lastname: Meisler
authors.forename: Joe G
authors.initials: JG
authors.lastname: Hollyfield
authors.forename: Jason T
authors.initials: JT
authors.lastname: Connor
authors.forename: Thomas
authors.initials: T
authors.lastname: Koeck
authors.forename: Kulwant S
authors.initials: KS
authors.lastname: Aulak
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Stuehr
mesh_terms: D000328:Adult; D015153:Blotting, Western / Q000379:methods; D004847:Epithelial Cells / Q000737:chemistry; D019573:Epithelium, Corneal / Q000737:chemistry*; D005136:Eye Proteins / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008875:Middle Aged; D009569:Nitric Oxide / Q000378:metabolism; D016877:Oxidants / Q000032:analysis / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000032:analysis / Q000378:metabolism*; D046509:Tissue Culture Techniques / Q000379:methods; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000276:immunology
chemical_list: D005136:Eye Proteins; D016877:Oxidants; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine