FOLATE BINDERS IN HEMATOPOIESIS AND CELL REPLICATION

叶酸结合剂在造血和细胞复制中的作用

• 批准号: 3170323
• 负责人: SHELDON P ROTHENBERG
• 金额: $ 18.66万
• 依托单位: SUNY DOWNSTATE MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-05-01 至 1992-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3170323
• 关键词: aplastic anemia; autoradiography; binding proteins; biological polymorphism; cell biology; cell growth regulation; cell transformation; chemical structure function; cytogenetics; deficient growth media; dementia; dyserythropoietic anemia; electron microscopy; folate; folate deficiency; fragile X syndromes; gel electrophoresis; gene expression; genetic library; genetic manipulation; genetic mapping; hematopoiesis; human population genetics; human subject; immunochemistry; immunoprecipitation; laboratory mouse; laboratory rabbit; ligands; membrane activity; membrane proteins; molecular cloning; monoclonal antibody; neoplastic cell culture for noncancer research; nucleic acid probes; nucleic acid sequence; nutrition related tag; protein biosynthesis; protein metabolism; protein sequence; radiotracer; schizophrenia; tissue /cell culture; vitamin metabolism

A number of human diseases have been described which are not due to a quantitative deficiency of folic acid but which may respond to large doses of the vitamin. These include hematopoietic disorders, such as some forms of aplastic anemia, a hereditary form of dyserythropoiesis, neurologic disorders such as dementia, some forms of schizophrenia, the recently recognized fragile X chromosome syndrome, and drug induced disorders of folate metabosism. The response to large doses of folate suggests that a common defect may underlie these disorders. The folate binding proteins (FBPs) which have now been identified in the membrane, in the soluble, and most recently in the nuclear fraction of mammalian cells could be the common link. Most of the work on these proteins from mammalian cells have focused on their structural properties rather than on their function and turnover in the cell. In addition, there have been no studies on the molecular genetics of the FBP to characterize the structure, organization and expression of the gene. The primary aims of this project, therefore, are to establish the precise function(s) of the folate binding proteins (FBPs) in the cellular uptake and intracellular metabolism of folate and to characterize fully the molecular genetics of this protein(s). The experiments will be designed to achieve the following objectives: 1) define the kinetics for the synthesis and degradation of the FBP in human KB cells and study the factors that affect these parameters using intrinsic labeling with (35S)methionine 2) map the cellular and topographic distribution of the FBP in the KB cell using electron microscopy immunocytology and study the factors that modify this distribution using monoclonal antibodies 3) clone the FBP gene from KB cells and then characterize its organization and nucleotide sequence, determine its chromosomal location, and examine different human cell lines and tissues for expression and restriction fragment length polymorphism of the gene 4) determine if transfection of the functional FBP gene into cells which express little or no FBP improves the viability of these cells in folate deficient medium. The study could provide definitive evidence that the FBP provides a critical function in cellular metabolism.

已经描述了许多人类疾病，这些疾病并没有 由于叶酸的数量不足，但可能 对大剂量的维生素有反应。这些措施包括 造血障碍，如某些形式的再生障碍性贫血， 遗传性红细胞生成障碍，神经系统疾病，如 痴呆症，精神分裂症的一些形式，最近认识到 脆性X染色体综合征和药物所致的疾病 叶酸代谢症。对大剂量叶酸的反应表明 一种常见的缺陷可能是这些疾病的根源。叶酸 结合蛋白(FBPs)，现已在 膜，在可溶的，最近在核中 哺乳动物细胞的一部分可能是共同的联系。大部分 对哺乳动物细胞中这些蛋白质的研究主要集中在 它们的结构属性，而不是它们的功能和 牢房里的周转率。此外，目前还没有关于 FBP的分子遗传学来表征其结构， 基因的组织和表达。 因此，这个项目的主要目标是建立 叶酸结合蛋白的精确功能(S) 叶酸和TO的细胞摄取和细胞内代谢 充分描述这种蛋白质的分子遗传学(S)。这个 将设计实验，以实现以下目标： 1)确定FBP的合成和降解动力学 并研究影响这些因素的因素 用(~(35)S)蛋氨酸2)图进行本征标记的参数 纤毛膜结合蛋白在KB细胞中的细胞和形态分布 用电子显微镜免疫细胞学方法研究影响因素 用克隆的单抗来改变这种分布 从KB细胞中克隆FBP基因并鉴定其特性 组织和核苷酸序列，确定其染色体 定位，并检查不同的人类细胞系和组织 基因的表达及限制性片段长度多态 基因4)确定是否将功能性FBP基因导入 表达很少或不表达FBP的细胞可提高细胞的存活率 这些细胞在叶酸缺乏的培养液中。这项研究可能会提供 确凿的证据表明，FBP在 细胞新陈代谢。