ENVIRONMENTAL NEUROTOXICANTS & THE AXONAL CYTOSKELETON

环境神经毒剂

• 批准号: 3253406
• 负责人: ANTHONY P DECAPRIO
• 金额: $ 10.12万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3253406
• 关键词: acrylamides; adduct; aminoacid analyzer; axon reaction; binding proteins; chemical binding; chemical fingerprinting; chemical structure function; covalent bond; cyanides; cytoskeleton; electron microscopy; environmental toxicology; fluorescence microscopy; gel electrophoresis; high performance liquid chromatography; laboratory rat; lysine; mass spectrometry; microtubules; morphology; neurofilament; neurotoxins; occupational hazard; protein purification; protein structure; scintillation counter; tissue /cell culture; toxicant interaction; video recording system

The broad, long-term objective of this project is to elucidate the molecular mechanisms of action of environmental and occupational neurotoxins which induce axonal degeneration in experimental animals and man. This proposal examines an important subclass of this group, the neurofilament (NF) neurotoxins, which includes n-hexane, 2,5-hexanedione (2,5-HD), carbon disulfide (CS2), beta, beta-iminodipropionitrile (IDPN), an acrylamide. These agents produce cytoskeletal disorganization and NF accumulation within susceptible nerve fibers. The NF neurotoxins represent a significant public health hazard, and in view of their great chemical and structural diversity, it is important to identify common steps in their respective mechanisms. Such information would be of great value in predicting similar effects from untested or newly synthesized chemicals. Specific aims include: i) Characterization and quantitation of the binding of 2,5-HD, CS2, IDPN (or its metabolites), and acrylamide to axonal cytoskeletal proteins; ii) Determination of whether such derivatization is limited to specific sites within the NF proteins or is randomly distributed; iii) Elucidation of the effects of in vitro and in vivo neurotoxin exposure on NF-NF and NF-microtubule (MT) interactions and on axonal cytoskeletal structures; and iv) Determination of the differential susceptibility of the distal vs. proximal axon to uptake of and covalent derivatization by NF neurotoxins. In vitro axonal cytoskeletal and whole animal model systems will be employed. Binding studies will utilize high specific activity [14C]-labelled NF neurotoxins. Analytical techniques will include gel electrophoresis of cytoskeletal proteins, fluorography, and scintillation counting of gel slices. Sites of protein binding will be assessed by chemical and enzymatic cleavage followed by HPLC peptide mapping and automated peptide sequencing. Protein adducts will be characterized by mass spectrometry. NF-NF interactions will be assessed by measuring the kinetics of NF protein reassembly and NF network formation using native NFs from treated rats. NF-MT affinity will be studied with blotting assays utilizing isolated NFs and [32P]-labelled MTs. Cytoskeletal reorganization will be examined in organotypic nerve cultures using video-enhanced and differential interference contrast light microscopy, immunofluorescence microscopy, and high voltage electron microscopy. Regional uptake and covalent binding of radiolabelled NF neurotoxins will be assessed along the optic nerve of treated rats and in organotypic nerve cultures. Findings will provide substantial progress towards defining mechanisms of action for this important class of neurotoxins.

该项目的广泛，长期目标是阐明 环境和职业作用的分子机制 神经毒素诱导实验动物的轴突变性和 男人。 该提案研究了该组的重要子类 神经丝（NF）神经毒素，其中包括N-己烷，2,5-己二酮 （2,5-HD），二硫化碳（CS2），β-iminodipropopionitrile（IDPN）， 丙烯酰胺。 这些药物会产生细胞骨架混乱和NF 易感神经纤维内的积累。 NF神经毒素代表 鉴于其伟大的化学物质和 结构多样性，重要的是要确定他们的共同步骤 各自的机制。 这样的信息将具有很大的价值 预测未经测试或新合成化学物质的类似作用。 具体目的包括：i）结合的表征和定量 2,5-HD，CS2，IDPN（或其代谢产物）和丙烯酰胺至轴突 细胞骨架蛋白； ii）确定这种衍生化是否为 仅限于NF蛋白内的特定位点或随机 分布式; iii）阐明体外和体内的影响 NF-NF和NF-Microubule（MT）相互作用的神经毒素暴露以及 轴突细胞骨骼结构； iv）确定差分 远端与近端轴突对吸收和共价的敏感性 NF神经毒素衍生化。 体外轴突细胞骨架和整体 动物模型系统将被采用。 绑定研究将使用高 特定活性[14C]标记的NF神经毒素。 分析技术 将包括细胞骨架蛋白的凝胶电泳，荧光图， 和凝胶切片的闪烁计数。 蛋白质结合的位点将是 通过化学和酶促切割评估，然后是HPLC肽 映射和自动化肽测序。 蛋白质加合物将是 以质谱为特征。 NF-NF互动将由 测量NF蛋白重新组装和NF网络形成的动力学 使用来自治疗大鼠的天然NF。 NF-MT亲和力将研究 使用孤立的NFS和[32p]标记的MTS的墨水测定法。 细胞骨架重组将在器官型神经培养物中进行检查 使用视频增强和差分干扰对比度 显微镜，免疫荧光显微镜和高压电子 显微镜。 放射性标记NF的区域吸收和共价结合 神经毒素将沿处理的大鼠的视神经和 器官培养物。 调查结果将带来重大进展 为了定义这一重要类别的行动机制 神经毒素。