NA, K-ATPASE GENE EXPRESSION IN THE HUMAN CILIARY BODY

人类睫状体中的 NA、K-ATP 酶基因表达

• 批准号: 3265997
• 负责人: MIGUEL COCA-PRADOS
• 金额: $ 16.88万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-08-06 至 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3265997
• 关键词: antibody formation; cell differentiation; conformation; epithelium; gene expression; genetic library; genetic transcription; genetic translation; glaucoma; histogenesis; human genetic material tag; human tissue; in situ hybridization; isozymes; messenger RNA; northern blottings; nucleic acid probes; nucleic acid sequence; posttranslational modifications; potassium; proteins; radioassay; sodium; sodium potassium exchanging ATPase; tissue /cell culture; uvea ciliary body; western blottings

The human ciliary epithelium is an excellent cellular model to understand the function and structure of the Na,K-ATPase. The Na-pump is a pivotal ion pump in the formation and transport of aqueous humor fluid by the ciliary epithelium which in Open Angle Glaucoma, leads to an elevation of intraocular fluid pressure in the eye. The objective of the proposed project is to investigate the differential regulation of gene expression of the alpha and beta subunit isoforms of the NaK-ATPase in the human ciliary body. To achieve this goal we will construct a human cDNA library from the ciliary body of a young eye donor. This cDNA library will serve as an instrumental tool to address basic questions with respect to the differential regulation of activity of the alpha isoforms of the Na,K-ATPase in normal and in Glaucoma eyes. The experimental design and methods for achieving these goals are the following: A) Isolation and characterization of full length human cDNA clones specific for the alpha and beta subunit isoforms of the ciliary epithelium Na,K-ATPase. B) The localization of mRNAs for alpha and beta subunit isoforms of the Na,K-ATPase by in situ hybridization in the human ciliary epithelium in various stages of development and differentiation. C) Regulation of ATPase gene expression in eyes with Glaucoma. By Northern blot analysis, in situ hybridization and immunoblotting we will determine whether the expression of Na,K-ATPase alpha and beta isoforms is altered at the transcription or translational level when compared to normal eyes. D) Development of antibodies against specific domains of the human alpha and beta isoforms of the Na,K-ATPase. These antibodies will provide valuable information on the cellular distribution, regional differences, structure and conformational changes during development of the ciliary epithelium. By immunoblotting analysis it will be possible to determine whether the expression of the alpha and beta polypeptides is regulated at the translational level and/or to characterize any post-translational modifications. E) Expression of functional Na,K-ATPase from cloned cDNAs. The introduction of cloned cDNA coding for the alpha isoforms into ciliary epithelial cells lacking one of the three alpha isoforms will allow us to study aspects of the differential activity of the alpha isoforms.

人类睫状上皮是一个很好的理解细胞模型 Na,K-ATP酶的功能和结构。 Na泵是关键离子 通过睫状体泵入房水的形成和运输 在开角型青光眼中，上皮细胞会导致 眼睛内的眼内液压力。 拟议项目的目的是调查差异 α和β亚基亚型基因表达的调控 人体睫状体中的 NaK-ATP 酶。为了实现这一目标，我们将 从年轻眼睛捐赠者的睫状体构建人类 cDNA 文库。 该 cDNA 文库将作为解决基本问题的工具 关于对活动的区别监管的问题 正常眼和青光眼眼中 Na,K-ATP 酶的 α 亚型。这 实验设计和实现这些目标的方法是 下列的： A) 全长人类 cDNA 克隆的分离和表征 对于睫状上皮的 α 和 β 亚基亚型 Na,K-ATP 酶。 B) α 和 β 亚基亚型的 mRNA 定位 Na,K-ATP酶在人睫状上皮中的原位杂交 不同的发展阶段和分化。 C) 青光眼眼中 ATP 酶基因表达的调节。由北方 我们将确定印迹分析、原位杂交和免疫印迹 Na,K-ATPase α 和 β 亚型的表达是否在 与正常眼睛相比的转录或翻译水平。 D) 开发针对人类α特定结构域的抗体 和 Na,K-ATP 酶的 β 亚型。这些抗体将提供 有关细胞分布、区域差异的宝贵信息， 睫状体发育过程中结构和构象的变化 上皮。通过免疫印迹分析可以确定 α和β多肽的表达是否受到调节 翻译水平和/或表征任何翻译后 修改。 E) 从克隆的 cDNA 表达功能性 Na,K-ATP 酶。简介 将编码α亚型的克隆cDNA导入睫状上皮细胞 缺乏三种α亚型之一将使我们能够研究 α亚型的差异活性。