MOLECULAR BIOLOGY OF ZONULAE OCCLUDENTES

闭锁小带的分子生物学

• 批准号: 3276314
• 负责人: DANIEL A. GOODENOUGH
• 金额: $ 13.44万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-04-01 至 1989-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3276314
• 关键词: antibody formation; antibody specificity; bile ducts; brush border membrane; density gradient ultracentrifugation; electron microscopy; electrophysiology; freeze etching; gastrointestinal epithelium; gel electrophoresis; immunochemistry; immunoelectron microscopy; immunofluorescence technique; immunoprecipitation; intercellular connection; intestines; liver; membrane permeability; molecular biology; surface antigens; tissue /cell culture

Biological epithelia which separate physiological compartments act as permeability barriers to the transepithelial passage of ions and molecules. The spaces between the cells, called the paracellular pathway, are sealed by a gasket-like intercellular junction, the zonula occludens (ZO). The ZO is the apical-most member of a series of intercellular junctions, collectively termed the junctional complex. A novel preparation of isolated junctional complexes from rodent liver has prepared, which is demonstrated by morphological techniques to contain structural components of the ZO. Polyacrylamide gel analysis of these complexes reveals a greatly simplified profile of polypeptides, compared to the whole hepatocyte plasma membrane. Using monoclonal and polyclonal antibody approaches, it is proposed in this application to generate specific antisera to these junctional complex preparations and to identify the specificity of these antisera by immunoreplica techniques. Using immunoelectron microscopy, the anatomical locations of the antigens recognized by these antisera will be mapped within the junctional complex, with the goal of specifically identifying the molecular components of the ZO. Once identified, the biogenesis and assembly of the ZO will be studied using immune precipitation of antigens following synthesis in either whole liver or rough microsome enriched fractions of polyA mRNA. Hopefully, these antisera will also prove valuable in studies of biological transport in cultures of transporting epithelia.

分离生理隔室的生物上皮起着 跨上皮离子通道的通透性障碍和 分子。细胞之间的间隙，称为细胞旁通路， 由垫片状的细胞间连接密封，小带闭塞 (ZO)。ZO是一系列细胞间质中最顶端的成员 联结，统称为联结复合体。一种新型制剂 已从啮齿动物肝脏中分离出连接复合体，这是 通过形态技术证明含有结构成分 是ZO的。对这些络合物进行的聚丙烯酰胺凝胶分析显示 与整体相比，大大简化了多肽的轮廓 肝细胞质膜。使用单抗和多克隆抗体 方法，建议在此应用程序中生成特定的 这些连接复合体制剂的抗血清，并鉴定 用免疫复制技术鉴定这些抗血清的特异性。vbl.使用 免疫电子显微镜，抗原的解剖位置 由这些抗血清识别的抗体将被映射到连接复合体中， 其目标是专门识别该分子的组成 佐伊。一旦确定，将研究ZO的生物发生和组装 使用合成后的抗原的免疫沉淀 肝脏或粗微体富含部分Polya mRNA。但愿能去, 这些抗血清也将被证明在生物运输研究中很有价值。 在运输上皮细胞的培养中。