BIOLOGY OF INTESTINAL EPITHELIAL PERMEABILITY

在过去的12年里，申请者实验室已经制作了许多 帮助定义了大脑功能解剖学的观察 肠上皮细胞间连接。它显示在这些 研究表明，无论是在健康还是在疾病中，肠道上皮细胞都是紧密的 在高度动态的情况下，连接可以调节上皮的通透性 时尚。鉴于这种屏障对肠道功能的重要性， 现在把重点放在紧致的分子结构上是合适的 紧密连接及其调控的分子基础 装配性和渗透性。这种新的方法应该包括分子 结构/功能方面的工作集中在有助于紧密结合的分子上 结合部结构。主办实验室(丹尼尔·卢瓦德教授，主任 巴黎巴斯德研究所细胞和分子生物学)不仅在 这种基于分子的结构/功能在肠道上皮细胞中发挥作用， 但这个实验室最近意外地发现了一种紧密的- Ra家族的连接特异性小间隙结合蛋白。这部小说 东道主实验室已经克隆了蛋白质并进行了测序。特定的 这个大的蛋白质家族的成员通常位于不同的 以及细胞内高度特定的位置。此外，这个家庭的成员 通常充当控制薄膜的功能开关 在这种特定情况下的相互作用/靶向和跨膜信号 网站。因此，该奖学金申请建议启动 这种紧密连接特异性Ra的分子结构/功能研究 在紧密连接调控中发挥作用的(J-ra)蛋白 和/或组件。在第一次访问期间(1993年8月1日至1月30日， 19994)，在卢瓦德博士的监督下，并与 他实验室里的分子生物学家(包括Arnold Zahraoui博士，他 是最早克隆人类ra蛋白的人之一)，申请者将 制备表达标签形式和突变形式的J-ra的质粒 转染人肠上皮细胞(Caco-2)。瞬变 将进行转染性实验，以确定是否转染性 蛋白质得到适当的表达。随后，稳定的扩张 转基因细胞系的建立及其功能的详细评价 交叉口组装-渗透性/监管将在波士顿进行 在申请者波士顿实验室，该实验室非常适合这些功能 学习。第二次访问(1994年6月1日-1994年10月30日)结果 为从形式化功能上筛选突变体提供了依据 获取更详细的突变/嵌合体构建，目的是识别 给予特定于结点的靶向和/或 控制接头组装/渗透性/调节。随后，一个 转导STATEL基因细胞的第二轮功能筛选 将在申请者波士顿实验室开始。这些研究将 向申请人介绍分子结构/功能分析，可 提供对紧密连接组装和/或监管的关键见解，以及 将为长期协作互动提供跳板 在东道主和申请者实验室之间。预计这将是 该项目将是NIH资助的正在进行的研究的重要组成部分 未来5-7年在胃肠道病理科工作 布里格姆妇女医院。

During the past 12 years the applicants laboratory has made many observations which helped to define the functional anatomy of the intercellular junctions of intestinal epithelial. It was shown in these studies that, both in health and disease, intestinal epithelial tight junctions could regulate epithelial permeability in a highly dynamic fashion. Given the importance of this barrier to intestinal function, it is now appropriate to focus on the molecular structure of the tight junction and on the molecular basis for the regulation of tight junction assembly and permeability. Such new approaches should include molecular structure/function work focused on molecules which contribute to tight junction structure. The host lab (Professor Daniel Louvard, Director of Cell and Molecular Biology, Pasteur Institute, Paris) not only excels in such molecularly based structure/function work in intestinal epithelia, but this lab has recently made the serendipitous discovery of a tight- junction specific small GAP binding protein of the ra family. This novel protein has been cloned and sequenced by the host laboratory. Specific members of this large protein family are generally located at different and highly specific sites within cells. Moreover, members of this family typically behave as functional switches to control membrane interactions/targeting and transmembrane signaling at such specific sites. Accordingly, this fellowship application proposes to initiate molecular structure/function studies of this tight junction-specific ra (J-ra) protein which could play a role in tight junction regulation and/or assembly. During the first visit (August 1, 1993 - January 30, 19994), under the supervision of Dr. Louvard and in association with molecular biologists in his laboratory (including Dr. Arnold Zahraoui who was one of the first to clone human ra proteins), the applicant will prepare plasmids which will express tagged and mutant forms of J-ra when transfected into human intestinal epithelial cells (Caco-2). Transient transfection experiments will be carried out to determine if transfected proteins are appropriately expressed. Subsequently, expansion of stabely transfected cell lines and detailed functional assessment of tight junction assembly-permeability/regulation will be carried out in Boston in the applicants Boston lab which is well suited for these functional studies. On the second trip (June 1, 1994 - October 30, 1994) results from the formalized functional screening of mutants will provide a basis for more detailed mutagenesis/chimaera construction aimed at identifying sequence information which imparts junction-specific targeting and/or controls junction assemble/permeability/regulation. Subsequently, a second cycle of functional screening of stabel transfected cell lines will begin in the applicants Boston laboratory. These studies will introduce the applicant to molecular structure/function analyses, may provide key insights into tight junction assemble and/or regulation, and will provide a springboard for long-term collaborative interactions between the host and applicant laboratories. It is anticipated that this project will be an important component of the ongoing NIH-funded research for the next 5-7 years in the Division of Gastrointestinal Pathology at the Brigham and Women' Hospital.