CELLULAR AND MOLECULAR APPROACHES TO NEUROTOXICOLOGY

神经毒理学的细胞和分子方法

• 批准号: 4696881
• 负责人: S J MORRIS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/4696881
• 关键词: adenosinetriphosphatase; adrenal medulla; calcium channel blockers; catecholamines; chromaffin cells; electron microscopy; exocytosis; fluorescence; fluorescent dye /probe; freeze etching; heavy metals; membrane activity; membrane fusion; membrane lipids; membrane model; metal poisoning; myelin; neurochemistry; neurotoxins; neurotransmitter metabolism; photochemistry; pinocytosis; sarcoplasmic reticulum; tissue /cell culture; toxicant screening

The calcium-requlated activity of chromaffin cells provide a well-studied system for investigating molecular and cell-surface mediated mechanisms of neurotoxin action. The storage granules of these cells, chromaffin granules, accumulate large concentrations of catecholamines and ATP which are eventually released by exocytosis. Isolated chromaffin granules will aggregate and fuse in the presence of calcium. We have been exploring the molecular basis of these activities. Fluorescent-labelled lipid probes have been successfully inserted into chromaffin granule membranes in vitro without altering the storage properties of the particles. Resonance energy transfer studies of calcium-promoted fusion of these membranes show that, unlike artificial phospholipid vesicles, fusion runs 5-10 fold slower than aggregation. These results support the previous findings that substantial rearrangement of the protein and lipid components of the membrane is required for fusion to occur. This in vitro fusion is inhibited by both organic and inorganic monovalent anions and cations and is insensitive to the presence of Mg-ATP. It is abolished by lysis and resealing the granules. Neurotoxins are known to disrupt the structure of myelin. Myelin basic protein (MBP), which accounts for 30 percent of CNS meylin proteins, has no known physiological function, although injection of purified MBP will cause experimental Ascending Encephalomyelities (EAE), considered by some as a model for Multiple Sclerosis. A molecular model for the structure of MBP generates a series of testable predictions. We have been examining the structural properties of MBP using fluorescence and optical spectroscopy. Contrary to many reports, we find evidence for extensive long range structural specificity of myelin basic protein in agreement with the model. These studies may lead to a rapid, more precise functional assay for MBP than induction of EAE.

嗜铬细胞的钙调节活性提供了一个研究充分的 用于研究分子和细胞表面介导的 神经毒素作用 这些细胞的储存颗粒，嗜铬细胞 颗粒，积累大量的儿茶酚胺和ATP， 最终通过胞吐作用释放出来。 孤立的嗜铬颗粒 在钙的存在下聚集并融合。 我们一直在探索 这些活动的分子基础。 荧光标记脂质探针 已成功地插入到嗜铬颗粒膜在体外 而不改变颗粒的储存性能。 共振能量 钙促进这些膜融合的转移研究表明， 与人工磷脂囊泡不同，融合比人工磷脂囊泡慢5-10倍。 聚合来 这些结果支持了先前的发现， 膜的蛋白质和脂质成分的重排是 才能发生聚变 这种体外融合受到两种 有机和无机单价阴离子和阳离子，并且对 Mg-ATP的存在。 通过溶解和重新密封， 颗粒。 已知神经毒素会破坏髓鞘的结构。 髓鞘碱性 蛋白（MBP），占中枢神经系统胰蛋白酶蛋白的30%，没有 已知的生理功能，尽管注射纯化的MBP会引起 实验性上行性脑脊髓炎（EAE），被一些人认为是 多发性硬化症的模型。 MBP结构的分子模型 生成一系列可测试的预测。 我们一直在研究 使用荧光和光学光谱的MBP的结构特性。 与许多报道相反，我们发现了广泛的长距离 髓鞘碱性蛋白的结构特异性与 模型 这些研究可能会导致一个快速，更精确的功能测定 而不是诱发EAE。