This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. To better understand the mechanism for human health consequences of exposure to high concentrations of sugars, as in diabetes, changes in the cell cycle progression will be studied. It has been previously shown that one of the most significant changes in diabetic and galactosemic LECs are changes in the rate of mitosis when compared to controls as demonstrated using several techniques. When LECs are exposed to high galactose (40 mM) in the culture medium for 4 days an increase in the rate of mitosis is observed as evidenced by an increase in the number of mitotic figures and in H3 incorporation experiments. By 7 days, the rate of mitosis decreases dramatically when compared to control using the techniques listed above. The PI analyzed the cell cycle progression of bovine LECs using flow cytometry and acridine orange and determined that galactosemic LECs exhibited a high mitotic rate at 7 days of exposure to 40 mM galactose because of the amount and conformation of the DNA of these cells. This finding contradicts the results obtained by counting mitotic figures and H3-incorporation experiments. The hypothesis is that changes in the cell cycle progression of galactosemic LECs are a consequence of the activation of a checkpoint at the G2 to mitosis transition regulated by the cdc2/p34 complex. The purpose of this study is to determine the status of the cdc2/p34 complex in normal and galactosemic (40 mM galactose treated) bovine LECs in culture. This study will establish the parameters to be used in the future to expand research using real-time RT-PCR and human LECs obtain from local sources. Results could be used to develop strategies to control the onset of sugar cataracts in susceptible populations.
The specific aims of this proposal are two-fold: Scientific: - To establish primary cultures of bovine lens epithelial cells. - To synchronize primary cultures of bovine LECs using - To determine the levels and activation status ofcdc2/p34 complex in normal and galactosemic LECs. The complexes will be isolated using a peptide-agarose bead complex available commercially specific for the cdc2/p34 complex and Western blot analysis with antibodies specific for the complex and anti-phosphotyrosine antibodies. The activation status of the cdc2/p34 complex will be studied using a kinase activity assay. - To determine the levels of the regulators, wee-1 protein and cdc25, in normal and galactosemic LECs. This will be accomplished through Western blot analysis and commercially available antibodies to wee-t protein and cdc25. Academic: - To increase the number of minority science students doing research at UMET through exposure to the latest techniques in cellular biology and travel opportunities to scientific meetings to present their findings. - To strengthen the research environment at UMET through enhancing student's awareness to continue graduate studies' in biomedical sciences. - To integrate research achievements and environment into the academic experience.
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