SOMATIC CELL MUTANT IN CELLULAR CHOLESTEROL HOMEOSTASIS

细胞胆固醇稳态中的体细胞突变

• 批准号: 2735005
• 负责人: JUDETH J KLANSEK
• 金额: $ 3.15万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2735005
• 关键词: cholesterol; gene mutation; intracellular transport; lipid metabolism; lipid transport

The primary goal of this research is to identify gene products involved in intracellular cholesterol transport and regulation of cholesterol levels. Using isolated Chinese hamster ovary (CHO) cell mutants which are defective in LDL-trafficking we will identify factors involved in cholesterol movement and regulation. One mutant, 3-6, by complementation analysis was found to be in a separate class; and thus, designated ced2. Mutant 3-6 shows impaired LDL-stimulation of cholesterol esterification and LDL-suppression of HMG CoA reductase, but normal movement of LDL- cholesterol from the lysosomes to the plasma membrane. Specific aim 1 involves the identification of gene(s) that are defective in the mutant ced2/3-6 cell line. This aim will be achieved by isolating cDNAs that correct the defective phenotype in the mutant. Initially, mutant 3-6 cells will be transfected with a normal cDNA library in a mammalian expression vector. Cells with normal phenotype will be identified using a two-step screening process, and the cDNAs expressed in the cloned cell lines will be isolated using plasmid rescue techniques. Finally, sequencing the corresponding genes in normal and mutant CHO cells will allow identification of the gene defects causing mutant phenotypes. This work should reveal important information on gene products that play a direct role in cholesterol homeostasis. Specific aim 2 involves analysis of the biochemical phenotype of the ced2 complementation group. Analysis of cholesterol movement, esterification by ACAT, sensitivity to Amphotericin B, and cell growth will be done to determine how this gene defect alters key aspects of intracellular cholesterol transport and metabolism.

这项研究的主要目标是确定所涉及的基因产物 细胞内胆固醇转运和胆固醇调节 水平。使用分离的中国仓鼠卵巢（CHO）细胞突变体 如果 LDL 贩运有缺陷，我们将确定涉及的因素 胆固醇的运动和调节。一种突变体，3-6，通过互补 分析发现是在一个单独的类中；因此，指定为 ced2。 突变体 3-6 显示胆固醇酯化的 LDL 刺激受损 HMG CoA 还原酶的 LDL 抑制，但 LDL- 的正常运动 胆固醇从溶酶体转移到质膜。具体目标1 涉及识别突变体中有缺陷的基因 ced2/3-6 细胞系。这一目标将通过分离 cDNA 来实现， 纠正突变体中的缺陷表型。最初，突变体3-6 细胞将被哺乳动物的正常 cDNA 文库转染 表达载体。将使用以下方法鉴定具有正常表型的细胞 两步筛选过程，以及克隆细胞中表达的 cDNA 将使用质粒救援技术分离品系。最后， 对正常和突变 CHO 细胞中的相应基因进行测序将 允许识别导致突变表型的基因缺陷。这 工作应该揭示有关基因产物的重要信息，这些基因产物发挥着 对胆固醇稳态有直接作用。具体目标2涉及分析 ced2互补组的生化表型。分析 胆固醇运动、ACAT 酯化、对 两性霉素 B，并且将进行细胞生长以确定该基因如何 缺陷改变了细胞内胆固醇运输的关键方面 代谢。

项目成果

Effects of a combination hemoglobin based oxygen carrier-hypertonic saline solution on oxygen transport in the treatment of traumatic shock.

基于血红蛋白的氧载体-高渗盐溶液组合对创伤性休克治疗中氧输送的影响。

• DOI: 10.1016/j.resuscitation.2011.03.018
• 发表时间: 2011
• 期刊: Resuscitation
• 影响因子: 6.5
• 作者: Leong,Benjamin;Reynolds,PennyS;Tiba,MohamadH;Holbert,WilliamH;Draucker,GerardT;Medina,JulianaA;Barbee,RobertW;White,NathanJ;Ward,KevinR
• 通讯作者: Ward,KevinR