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The Roles of PCB-regulated TR-binding Proteins during brain development.

PCB 调节的 TR 结合蛋白在大脑发育过程中的作用。

基本信息

批准号：
17510039
负责人：
IWASAKI Toshiharu
金额：
$ 2.3万
依托单位：
Gunma University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17510039/
关键词：
PCB transcriptional regulation brain development corepressor thyroid hormone receptor Sos-Ras Yeast Two-hybrid

项目摘要

Environmental chemicals (ECs) such as Polychlorinated biphenyls (PCBs), dibenzo-p-dioxin (PCDDs) and dibenzofurans (PCDFs) were exerted abnormalities on brain development. We recently reported that low-dose bydroxylated-PCB whose toxicity equivalent factor (TEF) is almost negligible, suppressed TH receptor (TR)-mediated transcription by dissociation of TR from TH response element (TRE).#1. In the present study, we investigated effects of several ECs on TR-mediated transcription.TCDD and PCDFs did not affect on TR-mediated transcription and various effects of PCBs were observed. The degree of TR dissociation from TRE is correlated with that of suppression by ECs. These results suggest that PCBs effects are caused through TR-DNA binding domain (DBD), thus we performed transient transfection assay with chimeric receptors. The suppressions by PCBs were observed on chimeric receptors containing TR-DBD mediated transcription. Furthermore, the proteins contained TR-DBD dissociated from TRE. We also have reported that the magnitude of dissociation of TR from TRE by each congener was correlated with that of suppression. These findings led us to propose a hypothesis that ECs may act on DNA binding domain on TR.Thus, the toxic effects of these compounds on the development of central nerve system might be exerted through several different mechanisms. Especially PCBs affects TR-mediated transcription through DBD.#2. To screen above mentioned findings, we develop a novel in vitro DNA-protein binding assay. This assay is based on a fluorescence-labeled DNAs and GST-fused proteins. After washing steps, immunological reaction was performed seconds.#3. To investigate the roles of PCB on TR, we cloned a novel nuclear protein, using Sos-Ras Yeast Two-hybrid screening, and the functional examinations are under investigation.In conclusion, we propose that the toxicity index should be rearranged based on several critical factors.

多氯联苯（PCBs）、二苯并二恶英（PCDDs）和二苯并呋喃（PCDFs）等环境化学物对脑发育有不良影响。我们最近报道，低剂量的bydroxylated-PCB的毒性等效因子（TEF）几乎可以忽略不计，抑制TH受体（TR）介导的转录从TH反应元件（TRE）的TR解离。1.在本研究中，我们研究了几种EC对TR介导的转录的影响，TCDD和PCDFs不影响TR介导的转录，而PCBs则有不同的影响。TR从TRE解离的程度与EC的抑制程度相关。这些结果表明，PCBs的影响是通过TR-DNA结合域（DBD），因此，我们进行了瞬时转染实验与嵌合受体。在含有TR-DBD介导的转录的嵌合受体上观察到PCBs的支持。此外，蛋白质含有从TRE解离的TR-DBD。我们还报道了TR从TRE的每个同源物的解离的大小与抑制的大小相关。这些发现使我们提出一个假设，即ECs可能作用于TR上的DNA结合结构域。因此，这些化合物对中枢神经系统发育的毒性作用可能是通过几种不同的机制来实现的。特别是PCB通过DBD影响TR介导的转录。# 2.为了筛选上述结果，我们开发了一种新的体外DNA-蛋白结合试验。该测定基于荧光标记的DNA和GST融合蛋白。洗涤步骤后，进行免疫反应。3.为了研究PCB对TR的影响，我们利用Sos-Ras酵母双杂交技术克隆了一个新的核蛋白，并对其功能进行了初步研究。