LENS METABOLIC COOPERATION, GAP JUNCTIONS AND CATARACT

晶状体代谢协作、间隙连接和白内障

• 批准号: 2158430
• 负责人: DANIEL A. GOODENOUGH
• 金额: $ 23.57万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-03-01 至 1996-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2158430
• 关键词: Xenopus; alternatives to animals in research; aminoacid metabolism; biological information processing; cataract; cell differentiation; cell free system; chick embryo; congenital eye disorder; electrophysiology; embryo /fetus tissue /cell culture; gap junctions; gene expression; genetic library; glucose metabolism; histochemistry /cytochemistry; immunochemistry; immunoelectron microscopy; immunofluorescence technique; lens proteins; membrane proteins; monoclonal antibody; nonmammalian vertebrate embryology; nucleic acid probes; nucleic acid sequence; protein sequence; retinal pigment epithelium

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.

该应用旨在鉴定晶状体的蛋白质成分 纤维细胞间连接。 该识别策略将 使用分离的抗体制备单克隆抗体和多克隆抗血清 晶状体纤维膜作为抗原。 这些抗体将被筛选 选择能够产生黄斑染色图案的试剂 免疫荧光标记的整个晶状体冰冻切片，可识别 免疫复制品上的特定多肽，会干扰晶状体 细胞内纤维通讯测定 荧光染料 Lucifer Yellow CH 的显微注射。 抗原将是 通过免疫电子显微镜观察以验证它们在镜片上的位置 纤维接头。 成功筛选的抗血清将用于 展示晶状体上皮和晶状体纤维的分子成分 连接和上皮细胞与上皮细胞的连接。 这些 抗血清将用于研究连接的体外合成 无细胞系统中的蛋白质，并定义连接相互作用 培养物中分化细胞和未分化细胞之间的差异。 抗血清 还将用于尝试克隆晶状体纤维连接基因，使用 将透镜 cDNA 文库克隆到 Gamma gtll 表达载体中。 如果 成功的话，从该基因衍生的氨基酸序列将允许 提高额外的抗血清以限定氨基酸序列 连接分子和蛋白质结构的拓扑图 交界膜内。 如果抗血清显示天然细胞 培养细胞之间的连接，导致白内障的直接影响 将尝试细胞间通讯的条件。