CELL CYCLE CHANGES IN THE GALACTOSEMIC LENS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。为了更好地了解暴露于高浓度糖（如糖尿病）对人类健康的影响机制，将研究细胞周期进程的变化。以前的研究表明，与对照相比，糖尿病和半乳糖凝血症LECs最显著的变化之一是有丝分裂率的变化，这是通过几种技术证明的。当lec在培养基中暴露于高半乳糖（40 mM） 4天时，观察到有丝分裂率的增加，有丝分裂图的数量和H3掺入实验的增加证明了这一点。到第7天，与使用上述技术的对照相比，有丝分裂率急剧下降。PI使用流式细胞术和吖啶橙分析了牛LECs的细胞周期进程，并确定半乳糖LECs在暴露于40 mM半乳糖7天后表现出较高的有丝分裂率，这是由于这些细胞DNA的数量和构象。这一发现与计算有丝分裂图和h3融合实验所得的结果相矛盾。该假说认为，半乳糖LECs细胞周期进程的变化是cdc2/p34复合体调控的G2到有丝分裂过渡检查点激活的结果。本研究的目的是确定cdc2/p34复合物在正常和半乳糖血症（40 mM半乳糖处理）培养的牛LECs中的状态。本研究将为将来使用实时RT-PCR和从当地获得的人类LECs扩大研究建立参数。结果可用于制定控制易感人群糖性白内障发病的策略。本建议的具体目的有两个方面：科学：-建立牛晶状体上皮细胞的原代培养。测定正常和半乳糖凝集细胞中cdc2/p34复合物的水平和激活状态。这些复合物将使用可用于cdc2/p34复合物的肽-琼脂糖头复合物进行分离，并使用该复合物的特异性抗体和抗磷酸酪氨酸抗体进行Western blot分析。cdc2/p34复合物的激活状态将使用激酶活性测定法进行研究。-测定正常和半乳糖凝集细胞中调节因子wee-1蛋白和cdc25的水平。这将通过Western blot分析和市售的wee-t蛋白和cdc25抗体来完成。学术：通过接触细胞生物学的最新技术，增加在UMET做研究的少数民族理科生的数量，并有机会参加科学会议，展示他们的发现。-通过提高学生继续生物医学研究生学习的意识，加强UMET的研究环境。-将研究成果和环境融入学术体验。

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.