ETHANOL RESPONSIVE GENE EXPRESSION IN NEURAL CULTURES

神经培养中的乙醇响应基因表达

• 批准号: 2042718
• 负责人: MICHAEL F MILES
• 金额: $ 10.37万
• 依托单位: ERNEST GALLO CLINIC AND RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2042718
• 关键词: G protein; adenylate cyclase; antisense nucleic acid; biological signal transduction; ethanol; gene expression; genetic strain; guanine nucleotide binding protein; in situ hybridization; laboratory mouse; molecular cloning; molecular genetics; neurons; northern blottings; nucleic acid probes; phospholipase C; phosphoproteins; protein isoforms; protein structure function; tissue /cell culture; transfection; western blottings

This is a detailed study of an ethanol-responsive potential modulator of GTP-binding protein function which our laboratory has recently isolated by subtractive cloning. Changes in signal transduction, mediated by heterotrimeric GTP-binding proteins (G-proteins), may have an important role in adaption of the central nervous system to chronic ethanol. This phosducin-like protein (PhLP) is prominently induced in NG108-15 neuroblastoma x glioma cells upon chronic exposure to ethanol. Phosducin is a retinal phosphoprotein known to regulate diverse classes of G- proteins. PhLP is widely expressed, highly conserved across species and exists as multiple isoforms, possibly generated through alternative splicing. We hypothesize that ethanol-induced changes in PhLP isoform abundance may mediate at least some of the effects of chronic ethanol on G-protein function. The goal of this project is to identify ethanol- responsive PhLP isoforms and determine their function is possibly mediating ethanol effects on two G-protein coupled signal transduction system: adenylyl cyclase (AC) and phospholipase C (PLC). PhLP isoforms will be characterized by isolating a PhLP genomic clone followed by determining which of these respond to chronic ethanol exposure. In vitro membranae assays will be combined with in vivo overexpression (stable transfection) and underexpression (antisense oligonucleotides) of ethanol-responsive PhLP isoforms to determine whether ethanol induction of these isoforms alters AC and PLC functioning. Finally, mice strains that have been selected for sensitivity or resistance to seizures following chronic ethanol exposure will be used to determine whether PhLP isoforms are regulated by ethanol in selective brain regions and whether there are difference in PhLP expression or regulation between these two lines of mice. These studies could lead to novel therapeutic approaches to alcoholism or new molecular probes for the study of alcoholism.

这是对乙醇响应潜在调节剂的详细研究 我们实验室最近分离的GTP结合蛋白功能 通过减法克隆。 信号转导的变化，介导 异三聚体GTP结合蛋白（G蛋白）可能具有重要的 中枢神经系统对慢性乙醇的适应性作用。这 NG108-15在NG108-15中显着诱导phosducin样蛋白（PHLP） 长期暴露于乙醇后，神经母细胞瘤X神经瘤细胞。 磷酸素 是一种已知的视网膜磷蛋白，可调节各种类别的G- 蛋白质。 PHLP广泛表达，在物种之间高度保守， 作为多种同工型存在，可能通过替代生成 剪接。 我们假设乙醇诱导的PHLP同工型变化 丰度至少可以介导慢性乙醇对 G蛋白功能。 该项目的目的是确定乙醇 - 响应式PHLP同工型并确定其功能可能是 介导乙醇对两个G蛋白偶联信号转导的影响 系统：腺苷酸环化酶（AC）和磷脂酶C（PLC）。 PHLP同工型 将以分离PHLP基因组克隆，然后是 确定这些对慢性乙醇暴露的反应。 体外 膜测定将与体内过表达结合（稳定 转染）和不渗透的（反义寡核苷酸） 乙醇反应性PHLP同工型，以确定乙醇是否诱导 这些同工型改变了AC和PLC功能。最后，小鼠菌株 已选择以敏感或抗癫痫发作的抗性 慢性乙醇暴露后将使用用于确定PHLP是否 同工型在选择性大脑区域受到乙醇的调节，以及是否是否 这两者之间的PHLP表达或调节有差异 小鼠线。 这些研究可能导致新的治疗方法 酗酒或新分子探针研究酒精中毒。