title: Peroxynitrite modified DNA presents better epitopes for anti-DNA autoantibodies in diabetes type 1 patients.
abstract: Peroxynitrite (ONOO(-)), formed by the reaction between nitric oxide (NO) and superoxide (O2(-)), has been implicated in the etiology of numerous disease processes. Peroxynitrite interacts with DNA via direct oxidative reactions or via indirect radical-mediated mechanism. It can inflict both oxidative and nitrosative damages on DNA bases, generating abasic sites, resulting in the single strand breaks. Plasmid pUC 18 isolated from Escherichiacoli was modified with peroxynitrite, generated by quenched flow process. Modifications incurred in plasmid DNA were characterized by ultraviolet and fluorescence spectroscopy, circular dichroism, HPLC and melting temperature studies. Binding characteristics and specificity of antibodies from diabetes patients were analyzed by direct binding and inhibition ELISA. Peroxynitrite modification of pUC 18 plasmid resulted in the formation of strand breaks and base modification. The major compound formed when peroxynitrite reacted with DNA was 8-nitroguanine, a specific marker for peroxynitrite induced DNA damage in inflamed tissues. The concentration of 8-nitroguanine was found to be 3.8 μM. Sera from diabetes type 1 patients from different age groups were studied for their binding to native and peroxynitrite modified plasmid. Direct binding and competitive-inhibition ELISA results showed higher recognition of peroxynitrite modified plasmid, as compared to the native form, by auto-antibodies present in diabetes patients. The preferential recognition of modified plasmid by diabetes autoantibodies was further reiterated by gel shift assay. Experimentally induced anti-peroxynitrite-modified plasmid IgG was used as a probe to detect nitrosative lesions in the DNA isolated from diabetes patients.
authors.affiliation: Department of Biochemistry, Jawaharlal Nehru Medical College, AMU, Aligarh, India.
authors.forename: Prashant
authors.initials: P
authors.lastname: Tripathi
authors.affiliation: Department of Biochemistry, Jawaharlal Nehru Medical College, AMU, Aligarh, India. Electronic address: moin_u@rediffmail.com.
authors.lastname: Moinuddin
authors.affiliation: Department of Biochemistry, Jawaharlal Nehru Medical College, AMU, Aligarh, India.
authors.forename: Kiran
authors.initials: K
authors.lastname: Dixit
authors.affiliation: Department of Biochemistry, Jawaharlal Nehru Medical College, AMU, Aligarh, India.
authors.forename: Abdul Rouf
authors.initials: AR
authors.lastname: Mir
authors.affiliation: Department of Biochemistry, Jawaharlal Nehru Medical College, AMU, Aligarh, India.
authors.forename: Safia
authors.initials: S
authors.lastname: Habib
authors.affiliation: Department of Biochemistry, Jawaharlal Nehru Medical College, AMU, Aligarh, India.
authors.forename: Khursheed
authors.initials: K
authors.lastname: Alam
authors.affiliation: Department of Biochemistry, Jawaharlal Nehru Medical College, AMU, Aligarh, India.
authors.forename: Asif
authors.initials: A
authors.lastname: Ali
mesh_terms: D000974:Antibodies, Antinuclear / Q000276:immunology*; D004247:DNA / Q000737:chemistry*; D018736:DNA Adducts / Q000235:genetics; D053960:DNA Breaks; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology*; D000939:Epitopes / Q000276:immunology; D006147:Guanine / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000097:blood / Q000737:chemistry; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D009573:Nitrites / Q000097:blood / Q000737:chemistry; D030421:Peroxynitrous Acid / Q000097:blood / Q000737:chemistry*; D010957:Plasmids / Q000737:chemistry / Q000235:genetics* / Q000276:immunology
chemical_list: C095838:8-nitroguanine; D000974:Antibodies, Antinuclear; D018736:DNA Adducts; D000939:Epitopes; D007074:Immunoglobulin G; D009573:Nitrites; D030421:Peroxynitrous Acid; D006147:Guanine; D004247:DNA
keywords: Antibodies; DNA damage; Diabetes; Peroxynitrite