CHEMISTRY AND BIOLOGY OF ALZHEIMERS AMYLOID PROTEINS

描述：（改编自申请人的摘要） 本研究旨在确定淀粉样蛋白在病理学中的作用， 老年痴呆症（AD） 尽管许多人取得了进步， 在实验室里，淀粉样蛋白的确切化学性质 组件仍有待阐明。 它是假设 这些辅助β淀粉样蛋白（AB）在 淀粉样蛋白沉积和AD的发病机制。 因此本研究 具体目的是纯化和确定一级结构， 主要和次要淀粉样蛋白的翻译后修饰 成分来自神经炎斑块、血管壁和弥漫性沉积物。 将利用超结构免疫化学来确定 这些沉积物中次要淀粉样成分的位置。 的 神经炎斑、血管淀粉样蛋白、弥漫性淀粉样蛋白的生物学效应 将在神经元和神经胶质细胞上评估其分离的组分， 细胞培养。 为了实现这些目标，将从AD脑中纯化淀粉样蛋白 匀浆以及来自血管的，通过最近开发的 包括过滤，等度蔗糖分离， 超离心、胶原酶水解、去污剂裂解和 不连续蔗糖密度梯度。 淀粉样蛋白溶解 通过变性剂、离液剂和分散剂将蛋白质 然后使用多个高性能步骤对其进行纯化， 液相色谱法（HPLC）。 抗血清将针对 不同的分离蛋白质组分，用于光和电子 显微免疫化学（胶体金）研究，以确定其 在原位和纯化的淀粉样蛋白中的定位。相关蛋白和 衍生的酶和化学肽将进一步分级， 通过自动氨基酸分析表征， 微测序以及质谱法。 决定后， 翻译修饰将通过气相色谱法进行， 质谱法和二维高压电泳。 化学性质和生物作用的阐明 淀粉样蛋白将有助于理解 AD的发病机制和病理生理学。 这些知识也将有助于 在AD的分子模型的发展，并将有助于 实施治疗策略。

DESCRIPTION: (adapted from Applicant's Abstract) The long-term goal of this study is to establish the role that amyloid plays in the pathology of Alzheimer's Disease (AD). Despite the advances made by numerous laboratories, the exact chemical nature of the amyloid minor protein components still remains to be elucidated. It is hypothesized that these ancillary beta amyloid (AB) proteins are of great significance in amyloid deposition and in the pathogenesis of AD. Therefore, this study specifically aims to purify and determine the primary structures and post-translational modifications of the major and minor amyloid components from neuritic plaques vascular walls and diffuse deposits. Ultra structural immunochemistry will be utilized to ascertain the location of the minor amyloid components in these deposits. The biological effects of neuritic plaque, vascular amyloid, diffuse amyloid and their separated components will be assessed on neuronal and glial cells in culture. To achieve these goals, amyloid will be purified from AD brain homogenates as well as from blood vessels by recently developed protocols which include filtration, isocratic sucrose separations, ultracentrifugation, collagenase hydrolysis, detergent lysis and discontinuous sucrose density gradients. Solubilization of the amyloid proteins by denaturing, chaotropic and dispersing agents will be followed by their purification using multiple steps of high performance liquid chromatography (HPLC). Antisera will be generated against the different separated protein fractions and used in light and electron microscopic immunochemical (colloidal gold) studies to determine their location in situ and in purified amyloid.The relevant proteins and derived enzymic and chemical peptides will be further fractionated by HPLC and characterized by automatic amino acid analysis and microsequencing as well as by mass spectrometry. Determination of post- translational modifications will be carried out by gas chromatography- mass spectrometry and by two-dimensional high voltage electrophoresis. Elucidation of the chemical nature and of the biological actions of amyloid proteins will contribute to the understanding of the pathogenesis and pathophysiology of AD. This knowledge will also assist in the development of molecular models for AD and will aid in the implementation of therapeutic strategies.