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

多氯联苯(PCBS)、二苯并对二恶英(PCDDs)、二苯并呋喃(PCDFs)等环境化学物质对脑发育产生异常影响。我们最近报道了毒性当量因子(TEF)几乎可以忽略不计的低剂量羟基化多氯联苯，通过将TR从TH反应元件(TRE)中解离来抑制TH受体(TR)介导的转录。#1.在本研究中，我们研究了几种ECS对TRR介导转录的影响。TCDD和PCDFs对TRR介导的转录没有影响，并且观察到多氯联苯的不同作用。TRE对TRR的解离程度与ECs的抑制程度相关。这些结果表明，多氯联苯的效应是通过TR-DNA结合域(DBD)引起的，因此我们进行了嵌合受体的瞬时转染实验。在含有tr-DBD介导的转录的嵌合受体上观察到多氯联苯的抑制作用。此外，含有tr-DBD的蛋白质从tre中解离出来。我们还报道了每个同系物从TRE解离的幅度与抑制的程度相关。这些发现导致我们提出一种假设，即内皮细胞可能作用于TRDNA结合区，因此，这些化合物对中枢神经系统发育的毒性作用可能通过几种不同的机制发挥。尤其是多氯联苯通过DBD影响tr介导的转录。2.为了筛选上述发现，我们发展了一种新的体外DNA-蛋白质结合试验。这种分析是基于荧光标记的DNA和GST融合蛋白。洗涤步骤后，进行免疫反应。#3.为了研究多氯联苯对tr的作用，我们利用SOS-RAS酵母双杂交技术克隆了一个新的核蛋白，并进行了功能检测。

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.