MOLECULAR BIOLOGY OF ZONULAE OCCLUDENTES

闭锁小带的分子生物学

• 批准号: 3276315
• 负责人: DANIEL A. GOODENOUGH
• 金额: $ 14.52万
• 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/3276315
• 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.

分隔生理区室的生物上皮细胞充当 离子跨上皮通道的渗透性屏障， 分子。 细胞之间的空间，称为细胞旁通路， 被一个垫圈状的细胞间连接封闭起来， （ZO）。 ZO是一系列细胞间连接的最顶端成员， 连接，统称为连接复合体。 新的制备 从啮齿动物肝脏分离的连接复合物制备， 通过形态学技术证明含有结构成分 的ZO。 这些复合物的聚丙烯酰胺凝胶分析显示， 与整个多肽相比， 肝细胞质膜 使用单克隆和多克隆抗体 方法，在本申请中提出生成特定的 这些连接复合物制剂的抗血清，并鉴定 这些抗血清的特异性。 使用 免疫电子显微镜，抗原的解剖位置 被这些抗血清识别的抗体将定位在连接复合物内， 其目的是特异性地鉴定 ZO 一旦确定，将研究ZO的生物起源和组装 在整个合成后使用抗原的免疫沉淀 肝脏或粗微粒体富集的polyA mRNA组分。 但愿， 这些抗血清在生物运输的研究中也将证明是有价值的 在运输上皮细胞的培养中。