CELL CYCLE CHANGES IN THE GALACTOSEMIC LENS

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。为了更好地了解暴露于高浓度糖的人类健康后果的机制，如糖尿病，将研究细胞周期进程的变化。先前已经表明，与对照组相比，糖尿病和半乳糖血症LEC中最显著的变化之一是有丝分裂速率的变化，如使用几种技术所证明的。当LEC暴露于培养基中的高半乳糖（40 mM）4天时，观察到有丝分裂速率的增加，如有丝分裂图数目的增加和H3掺入实验所证明的。到7天时，与使用上述技术的对照相比，有丝分裂速率显著降低。PI使用流式细胞术和吖啶橙子分析了牛LEC的细胞周期进程，并确定半乳糖LEC在暴露于40 mM半乳糖7天时表现出高有丝分裂速率，因为这些细胞的DNA的量和构象。这一发现与有丝分裂计数和H3掺入实验的结果相矛盾。 假设半乳糖血症LEC的细胞周期进程的变化是由cdc2/p34复合物调节的G2到有丝分裂转变的检查点激活的结果。本研究的目的是确定的cdc2/p34复合物在正常和半乳糖（40 mM半乳糖处理）的牛晶状体上皮细胞培养的状态。本研究将建立未来使用的参数，以扩大使用实时RT-PCR和从当地来源获得的人类LEC的研究。研究结果可用于制定控制易感人群糖性白内障发病的策略。 该提案的具体目标是双重的：科学：-建立牛透镜上皮细胞的原代培养。- 同步化牛晶状体上皮细胞的原代培养，测定正常和半乳糖血症晶状体上皮细胞中cdc2/p34复合物的水平和激活状态。使用市售的cdc2/p34复合物特异性肽-琼脂糖珠复合物分离复合物，并使用复合物特异性抗体和抗磷酸酪氨酸抗体进行Western印迹分析。cdc2/p34复合物的活化状态将使用激酶活性测定法进行研究。- 测定正常和半乳糖血症LEC中调节因子wee-1蛋白和cdc25的水平。这将通过Western印迹分析和市售wee-t蛋白和cdc25抗体来实现。 学术：- 通过接触细胞生物学的最新技术和有机会参加科学会议以介绍其发现，增加少数民族理科学生在UMET进行研究的人数。- 通过提高学生继续生物医学科学研究生学习的意识来加强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.