LENS METABOLIC COOPERATION, GAP JUNCTIONS AND CATARACT

这一应用程序提出了识别晶状体的蛋白质成分 纤维细胞间连接。这一识别的策略将 用分离的BE制备单抗和多克隆抗体 晶状体纤维膜作为抗原。这些抗体将通过以下方式进行筛查 选择产生黄斑染色图案的试剂 免疫荧光标记的全晶状体冰冻切片，识别 免疫复制物上的特异性多肽，干扰晶状体 用细胞内法测定纤维细胞间通讯 微量注射荧光染料，荧光黄CH。抗原将会是 用免疫电子显微镜观察以验证它们在晶状体上的位置 纤维连接。成功筛选的抗血清将用于 显示晶状体上皮到晶状体纤维的分子组成 连接，以及上皮细胞对上皮细胞的连接。这些 抗血清将被用来研究连接素的体外合成 无细胞系统中的蛋白质，并定义连接相互作用 在培养的分化和未分化细胞之间。抗血清 还将被用来尝试克隆晶状体纤维连接基因，使用 将Lens cDNA文库克隆到Gamma gtll表达载体中。如果 如果成功，从该基因衍生的氨基酸序列将允许 将额外的抗血清提升为确定的氨基酸序列 连接分子和蛋白质结构的拓扑图 在连接膜内。如果抗血清显示为天然细胞 培养细胞之间的连接，直接影响白内障的发生 将尝试细胞间通信的条件。

This application proposes to identify the protein components of the lens fiber intercellular junctions. The strategy for this identification will be to prepare monoclonal antibodies and polyclonal antisers using isolated lens fiber membranes as antigen. These antibodies will be screened by selecting for reagents which produce a macular staining pattern on immunofluorescently-labeled frozen sections of whole lens, which recognize specific polypeptides on immune replicas, and which interfere with lens fiber intercellular communication as assayed by intracellular microinjection of the fluorescent dye, Lucifer Yellow CH. Antigens will be visualized by immunoelectron microscopy to verify their location on lens fiber junctions. Successfully screened antisera will be used to demonstrate the molecular components of lens epithelium to lens fiber junctions, and epithelial cell-to-epithelial cell junctions. These antisera will be used to study the in vitro synthesis of junctional proteins in cell-free systems, and to define the junctional interactions between differentiated and undifferentiated cells in culture. The antisera will also be used to attempt cloning the lens fiber junction gene, using a lens cDNA library cloned into the Gamma gtll expression vector. If successful, the derived amino acid sequence from this gene will permit raising additional antisera to defined amino acid sequences within the junctional molecule, and topological mapping of the protein structure within the junctional membranes. If the antisera demonstrate native cell junctions between the cultured cells, direct effects of cataractogenic conditions on intercellular communication will be attempted.