MECHANISM OF PATHOGENESIS IN MYCOPLASMAL PNEUMONIA

该项目旨在提供有关生物化学的新信息， 肺炎支原体感染的毒力基础，特别是 原发性非典型肺炎。 我们将研究 通过分析受体位点相互作用和宿主 细胞核酸代谢 受体部位研究将从以下内容开始开始： 通过使用本发明的方法从人肺成纤维细胞制备膜级分， 锌/荧光素汞酸法。 将提取糖蛋白 用三种方法：二碘水杨酸锂、TRIS/EDTA和Triton X-100。 将采用梯度（7.5 - 20%）定量糖蛋白 聚丙烯酰胺凝胶电泳和银银/丹酰肼 染色 主要物质将通过柱色谱法（Bio 凝胶P-150）和制备型聚丙烯酰胺凝胶电泳。 双方都将 评价其抑制14 C标记的 支原体对肺成纤维细胞单层和仓鼠气管 外植体 受体将相对于分子量进行表征， 碳水化合物和氨基酸组成。 衣霉素对 将评估受体生物合成。 因为对宿主的主要影响 细胞贴壁后嘌呤合成减少，我们还将研究 受感染细胞的核酸生物化学。 高效液相 将使用高效液相色谱法（HPLC）和薄层色谱法（TLC） 定量对照和感染细胞（成纤维细胞）中的嘌呤核苷酸 和气管外植体）。 支原体感染对肺部的影响 用TLC评价成纤维细胞腺苷脱氨酶活性 比色法 我们还将评估脱氧科福霉素增强 细胞毒性通过抑制腺苷脱氨酶。 这些数据将表明 肺炎支原体破坏了新生儿的代谢途径， 嘌呤合成 关于附着和作用机制的信息 将被用来描绘感染过程，并提供一个新的 这是一个改进治疗和预防的基础。

This project is designed to provide new information on the biochemical basis for virulence in Mycoplasma pneumoniae infections, particularly Primary Atypical Pneumonia. We will study the molecular mechanisms of pathogenesis through an analysis of receptor site interactions and host cell nucleic acid metabolism. Receptor site studies will begin with membrane fractions prepared from human lung fibroblasts by using the zinc/fluorescein mercuric acid technique. Glycoproteins will be extracted by 3 methods: lithium diiodosalycilate, TRIS/EDTA, and Triton x-100. Glycoproteins will be quantitated with gradient (7.5 to 20 %) polyacrylamide gel electrophoresis and silver nitrate/dansyl hydrazine staining. The major species will be isolated by column chromatography (Bio Gel P-150) and preparative polyacrylamide gel electrophoresis. Each will be evaluated for its ability to inhibit attachment of 14C-labelled mycoplasmas to lung fibroblast monolayers and to hamster tracheal explants. The receptor will be characterized relative to molecular weight, carbohydrate and amino acid composition. The effect on tunicamycin on receptor biosynthesis will be evaluated. Because a major effect on host cells post-attachment is a decrease in purine synthesis, we will also study the nucleic acid biochemistry of infected cells. High-performance liquid chromatography (HPLC) and thin layer chromatography (TLC) will be used to quantitate purine nucleotides in control and infected cells (fibroblasts and tracheal explants). The influence of mycoplasma infection on lung fibroblast adenosine deaminase activity will be evaluated with a TLC assay. We will also evaluate the ability of deoxycorformycin to potentiate cytotoxicity by inhibiting adenosine deaminase. These data will indicate where Mycoplasma pneumoniae disrupts the metabolic pathway of de novo purine synthesis. This information on attachment and mechanism of action will be used to delineate the infective process, and provide a new perspective on which to base improved therapy and prophylaxis.