ENVIRONMENTAL NEUROTOXICANTS & THE AXONAL CYTOSKELETON
环境神经毒剂
基本信息
- 批准号:2153961
- 负责人:
- 金额:$ 7.4万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:1990
- 资助国家:美国
- 起止时间:1990-07-01 至 1995-06-30
- 项目状态:已结题
- 来源:
- 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
项目摘要
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.
这个项目的广泛、长期的目标是阐明
环境和职业作用的分子机制
诱导实验动物轴突变性的神经毒素和
天哪。这项提议考察了这一组的一个重要子类,即
神经丝(NF)神经毒素,包括正己烷、2,5-己二酮
(2,5-HD)、二硫化碳(CS2)、β,β-亚氨基二丙腈(IDPN)、
一种丙烯酰胺。这些制剂会产生细胞骨架紊乱和核因子
在易感神经纤维内堆积。核因子神经毒素代表
一种重大的公共健康危害,鉴于它们的巨大化学和
结构多样性,重要的是确定它们的共同步骤
各自的机制。这样的信息将在
预测未经测试或新合成的化学品的类似效果。
具体目标包括:一)约束性的表征和量化
2,5-HD、CS2、IDPN(或其代谢物)和丙烯酰胺至轴突
细胞骨架蛋白;二)确定这种衍生化是否
仅限于核因子蛋白内的特定位置或随机
分布;iii)阐明体外和体内的影响
神经毒素暴露对核因子-核因子和核因子-微管相互作用的影响
轴突细胞骨架结构;以及iv)差异的测定
远端轴突与近端轴突对摄取和共价的敏感性
核因子神经毒素的衍生化。体外培养的轴突细胞骨架和整体
将采用动物模型系统。约束性研究将利用高
比活度[14C]标记的神经毒素。分析技术
将包括细胞骨架蛋白的凝胶电泳,荧光照相,
以及凝胶片的闪烁计数。蛋白质结合的部位将是
用化学和酶裂解后的高效液相多肽进行评估
测绘和自动多肽测序。蛋白质加合物将是
以质谱学为特征。将通过以下方式评估核因子-核因子相互作用
测定核因子蛋白质重组和核因子网络形成的动力学
使用处理过的大鼠的原生神经营养因子。将研究核因子-MT亲和力
印迹分析使用分离的NFS和[32P]标记的MT。
细胞骨架重组将在器官型神经培养中进行检测。
使用视频增强和差分干涉对比光
显微镜、免疫荧光显微镜和高压电子
显微镜。放射性标记神经营养因子的区域摄取和共价结合
神经毒素将沿着接受治疗的大鼠的视神经和在
器官型神经培养。调查结果将提供实质性的进展
为这一重要类别确定行动机制
神经毒素。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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