The long-term goal of the proposed study is to understand the nature of the mechanisms by which caloric restriction prolongs the life span of animals. The specific hypothesis to be tested is that """"""""extension of life span of mice by caloric restriction is dependent upon attenuations of oxidative stress and the rate of accrual of oxidative damage to specific proteins."""""""" The experimental approach to test the hypothesis will involve two different strains of mice. One strain, C57BL/6, exhibits life span extension as a result of caloric restriction, while the other, DBA/2, shows no such effect. Thus, both a positive and negative control for the caloric restriction (CR) regimen will be used. The mean and maximum life span of ad libitum-fed (AL) DBA/2 mice are similar to those of AL C57BL/6 mice. Comparisons will be made between AL and CR mice, within each strain and between the two strains of mice, in order to determine whether CR attenuates: (a) the age related elevations in the level of oxidative stress and (b) the accrual of oxidative damage and loss of catalytic activity of specific proteins. The key question will be whether these effects occur in both strains or only in C57BL/6, which shows life span prolongation by CR. The hypothesis will be supported if CR lowers the level of oxidative stress and attenuates oxidative damage to selective proteins in C57BL/6 mice, which show life span prolongation by CR, but not in DBA/2 mice, in which CR has no effect. The level of oxidative stress will be determined using a battery of tests involving measurements of generation of reactive oxygen species, antioxidative enzymes, glutathione disulfide/glutathione redox couple, and amounts of glutathionylated proteins. Proteins exhibiting oxidative modifications such as carbonylation, 4-hydroxy 2-nonenal adducts, malondialdehyde adducts and 3-nitro modification of tyrosine residues will be detected immunochemically. Proteins will be identified by microsequencing and/or mass spectrometry. The significance of this study is that the results should provide important new knowledge about: (1) mechanisms linking oxidative stress to the losses in physiological functions during aging, and (2) the identity of specific targets of protein oxidative damage during aging and the consequent metabolic failures. Thus, it will also provide a further critical test of the validity of oxidative stress hypothesis of aging.
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