ABSTRACT
Immunofluorescence assay was applied for determination of 8-oxoguanine (8-oxoG) in DNA. The 8-oxoG content in liver and lung DNA of 2- and 18-month-old Wistar rats was compared with that of prematurely aging OXYS rats. It was shown that for rats of both strains, 8-oxoG content in lung DNA compared with liver DNA was 1.7-2.0-fold and 1.3-1.7-fold higher for 2- and 18-month-old rats, respectively. However, the degree of oxidative damage in liver DNA of OXYS rats was 2.4- (p < 0.01) and 1.5-fold (p < 0.05) higher for 2- and 18-month-old animals, respectively, than that in liver DNA of Wistar rats. Oxidation of guanine in lung DNA of OXYS rats was 2- (p < 0.01) and 1.7-fold (p < 0.05) higher for 2- and 18-month-old animals, respectively, than that in lung DNA of Wistar rats. The data indicate that elevated DNA oxidative damage in various organs of OXYS rats may be an important factor of accelerated aging and progression of age-related diseases--cataract, macular dystrophy, hypertension, osteoporosis, cognitive and behavioral dysfunctions, and also lung and liver pathologies.
Subject(s)
Aging, Premature/metabolism , DNA/metabolism , Guanine/metabolism , Liver/metabolism , Lung/metabolism , Oxidative Stress , Animals , DNA/chemistry , Guanine/analogs & derivatives , Male , Rats , Rats, WistarABSTRACT
The specific light-induced, non-enzymatic digestion of chicken skeletal muscle myosin heavy chain by xanthene dye-conjugated antibodies is reported. The xanthene dye Rose Bengal was conjugated to either a mouse monoclonal anti-myosin primary specific antibody or to goat anti-mouse IgG secondary antibodies. Under our experimental conditions, visible light induced the non-enzymatic breakdown of myosin heavy chains when chicken skeletal muscle myosin either directly formed a complex with Rose Bengal-conjugated anti-myosin antibodies or indirectly formed a complex with anti-myosin antibody-Rose Bengal-conjugated secondary antibodies. The rate of the photochemical reaction depended on irradiation time and temperature. Although SDS-PAGE and immunoblot analyses showed that fragments migrating below the myosin heavy chain polypeptide predominated, these analyses also showed higher molecular mass polypeptides were generated.
Subject(s)
Antibodies, Monoclonal/metabolism , Fluorescent Dyes , Myosin Heavy Chains/metabolism , Rose Bengal , Animals , Antibody Specificity , Chickens , Endopeptidases/metabolism , Fluorescent Antibody Technique, Direct , Fluorescent Antibody Technique, Indirect , Goats , Immunoglobulin G , Light , Mice , Muscle, Skeletal/metabolism , Myosin Heavy Chains/immunology , Photochemistry , Temperature , Time FactorsSubject(s)
Fluorescent Dyes , Microscopy, Confocal/methods , Microscopy, Fluorescence/methods , Animals , Drosophila/chemistry , Fixatives/chemistry , Fluorescein-5-isothiocyanate , Fluorescent Antibody Technique , Formaldehyde/chemistry , Glutaral/chemistry , Nuclear Proteins/analysis , Polymers/chemistry , Rhodamines , Tissue Fixation/methodsABSTRACT
A one step method to cross-link DNA bases containing aromatic amino groups directly to proteins was developed. No chemical modification of the base is required prior to conjugation, which is performed at neutral pH. Work focused on 8-oxoguanine and the carrier protein, bovine serum albumin. Conjugates were stable after sodium dodecyl sulfate (SDS)-induced protein denaturation and were characterized by UV spectroscopy, enzyme linked immunosorbent assay (ELISA), SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses. This method is a viable alternative to existing procedures for generating DNA base-protein conjugates for antibody characterization and affinity purification.
