Life in air is possible because uncatalyzed reactions of dioxygen in living organisms are usually slow. In addition, oxidative damage to components of healthy cells is frequently prevented or repaired by antioxidant, replacement, or repair systems that exist in the cells for the purpose of maintaining and restoring redox balance. Our approach is to study the roles of superoxide, hydrogen peroxide, metal ions, and small molecule antioxidants in the yeast S. cerevisiae. The yeast S. cerevisiae is a simple eucaryote for which extensive genetics and molecular biology exist. Many genes from higher organism have been shown to substitute functionally for their yeast analogs, and more are expected now the entire yeast genome has been sequenced and is available. When grown on a non-fermentable carbon source, yeast cells metabolize dioxygen in a fashion similar to human cells, and the cellular systems for prevention, repair, and replacement of oxidatively damaged cell components are also similar. Yeast is thus an excellent system in which to study how redox balance is maintained in healthy eucaryotic cells. These studies are expected to lead to a better understanding of redox balance in eucaryotic organisms and the role of """"""""oxidative stress"""""""" in processes leading to human aging, cell death, and disease. We will determine the major natural sources of superoxide and hydrogen peroxide in yeast and measure their concentrations and those of small molecule antioxidants (reduced and oxidized) within the cells. These methods will be applied to the wild type strains and to a variety of mutant strains in which antioxidant systems and/or dioxygen metabolism have been modified; the results will be used to interpret the phenotypes observed for these mutant strains. Naturally occurring sensors of hydrogen peroxide levels that are involved in regulation of gene expression will be studied. Levels of copper, zinc, manganese, and calcium metal ions will be determined in wild type and mutant strains in which levels of various antioxidant systems have been modified. Effects of addition or depletion of metal ions on the nature and rates of antioxidant and pro-oxidant processes and the maintenance of redox balance will be examined in the wild type and mutant strains as well. The roles played by the different metal ions will also be examined in yeast model systems designed to investigate processes related to aging, cell death and disease.
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