TRANSMITTER-SPECIFIC GENES AS TARGETS IN LEAD TOXICITY

递质特异性基因作为铅毒性的靶标

• 批准号: 6055969
• 负责人: Janusz Bogdan Suszkiw
• 金额: $ 21.3万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6055969
• 关键词: DNA binding protein; PC12 cells; brain mapping; chemical stability; choline acetyltransferase; embryo /fetus tissue /cell culture; embryo /fetus toxicology; enzyme induction /repression; gene expression; genetic regulatory element; genetic transcription; laboratory rat; lead poisoning; messenger RNA; neurogenetics; neurotransmitter metabolism; nuclear runoff assay; protein kinase C; toxin metabolism; transfection; tyrosine 3 monooxygenase

DESCRIPTION: (Adapted from the Investigator's Abstract) Alterations in the expression of neurotransmitter phenotype-specific genes are of key interest as a potential mechanism underlying long-term neurobehavioral disturbances associated with low-level lead (Pb) toxicity. Previous results from our laboratory indicate that Pb alters the expression of key cholinergic (cholineacetyltransferase, ChAT) and catecholaminergic (tyrosine hydroxylase, TH) neurotransmitter biosynthetic enzymes. The objective for the current project period is to further characterize the effects of toxicological concentrations of inorganic lead (Pb) on the tyrosine hydroxylase gene expression. (1) Experiments will be conducted to verify the preliminary observations that perinatal Pb-exposure upregulates TH gene expression in noradrenergic neurons in locus coeruleus (LC) and superior cervical ganglia (SCG) in the rat in vivo: the dose-dependence and time course of Pb upregulation of TH will be determined using LC and SCG explants in culture; (2) Effects of Pb on TH-gene transcription rates and TH-mRNA stability will be analyzed in PC12 cells, using the nuclear run on transcription assay and determination of TH mRNA half-lives, respectively; (3) The role of protein kinase C (PKC) in Pb-induced upregulation of TH will be assessed by determining the effect of PKC inhibitors on Pb-Dependent upregulation of TH activity and mRNA levels, and on Pb-enhancement of TH transcription rate; (4) A hypothesis will be tested that Pb induces DNA binding protein(s) which interact with specific lead-responsive elements (LRE) in the TH gene: (a) reporter plasmids containing specific TH 5'-flanking sequences fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and transfected into PC12 cells will be used to identify the putative LERs in the TH gene. (b) gel mobility shift assays will be performed with a series of synthetic oligonucleotides containing specific cis sequences to search for and identify the putative Pb-inducible TH-DNA binding protein(s). This research should advance our understanding of Pb actions at the molecular genetic level and provide a model for the future investigations of Pb effects on ChAT gene expression as well as on other neuronal phenotype-specific genes that may be targeted by Pb.

描述：（根据研究者的摘要进行了改编） 神经递质表型特异性基因的表达是关键的兴趣 作为长期神经行为障碍的潜在机制 与低级铅（PB）毒性有关。 我们先前的结果 实验室表明PB会改变钥匙胆碱能的表达 （cholineacetyltransferase，Chat）和儿茶酚胺能（酪氨酸） 羟化酶，神经递质生物合成酶。 目标 当前的项目期间是进一步表征 酪氨酸上无机铅（PB）的毒理学浓度 羟化酶基因表达。 （1）将进行实验以验证 围产期PB暴露于TH基因的初步观察结果 在去甲肾上腺素能神经元（LC）中的甲肾上腺素能神经元中的表达 大鼠体内大鼠的宫颈神经节（SCG）：剂量依赖性和时间 TH的PB课程将使用LC和SCG Epplants确定 在文化中； （2）PB对TH-GENE转录率和Th-MRNA的影响 稳定性将在PC12细胞中分析，使用核运行 转录测定和测定TH mRNA的半衰期； （3）蛋白激酶C（PKC）在PB诱导的TH上调中的作用 通过确定PKC抑制剂对PB依赖性的影响来评估 TH活性和mRNA水平的上调，以及TH的PB增强 转录率； （4）将检验一个假设Pb诱导DNA 与特定铅响应元件相互作用的结合蛋白 （LRE）在第基因中：（a）含有特定th的报告质粒 融合到细菌氯霉素的5'频序列 乙酰基转移酶（CAT）基因并转染到PC12细胞中 识别TH基因中的假定LER。 （b）凝胶机动性转移测定法 将使用一系列包含的合成寡核苷酸进行 特定的顺式序列以搜索和识别推定的PB诱导 Th-DNA结合蛋白（S）。 这项研究应该提高我们的理解 在分子遗传水平上的PB作用，为该模型提供了模型 对PB对聊天基因表达的影响以及未来的研究 Pb可能针对的其他神经元表型特异性基因。