Among the most sensitive neuropatholgic effects of gestational methylmercury exposure in both animals and humans is impaired cell proliferation. Previous investigations in this laboratory have revealed a concentration-dependent effect of MeHg on the in vitro proliferation of rat midbrain neuroepithelial cells, and these effects were associated with an increase in the mRNA and the protein content of the cell cycle regulatory molecules p21, GADD 45 and GADD153. Because MeHg is known to inhibit both mRNA and protein synthesis, it is suspected that the observed increase in mRNA and protein content arise from impaired degradation rather than induced synthesis, and propose experiments to examine this hypothesis. The principal investigator proposes to examine whether methylmercury can inhibit calpain- and ubiquitin/proteasome-mediated proteolysis, whether this inhibition increases the mRNA and protein content of cell cycle regulatory molecules, and whether these increases are associated with altered neuroepithelial cell proliferation. The PI proposes to use positive control agents known to inhibit calpain- and ubiquitin-mediated proteolysis, and to examine the relative effect of proteolytic inhibition on the intracellular content of specific cell cycle regulatory mRNA and proteins. Finally, the PI proposes to examine the observed elevation in cell cycle phase dependencies in mRNA and protein content following exposure to MeHg or thiol protease inhibitory agents by using flow cytometrically sorted neuroepithelial cell populations.
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