MICROTUBULES ROLE IN MACROPHAGE RESPONSE TO LPS

这项建议的目标是进一步了解所涉及的机制 在内毒素介导的白细胞活动中，重点关注 LPs和小鼠巨噬细胞的微管网络(Mphi)。LP是 已知对Mphis有深远的、多重的影响。初步调查结果 已知影响的药物(如秋水仙碱和紫杉醇) 微管组织可模拟内毒素诱导(A)下调 Mphi TNFpha受体，(B)TNFpha的释放，和(C)分泌 亚硝酸盐，而内毒素低反应的C3H/HeJ小鼠缺乏这些反应 紫杉醇，提高MT可能在内毒素中发挥重要作用的可能性- 调解的行动。这项研究的具体目的是回答内毒素是如何 行动是否与MT组织有关，是否有任何结构性的 或MT与内毒素结合蛋白之间的功能关联(S)。这个 提出的实验包括：(1)确定功能相互作用 通过检测内毒素对MT修饰的影响探讨内毒素与MT之间的关系 (酪氨酸化和磷酸化)，以及MT激动剂的作用 秋水仙碱、诺可达唑、紫杉醇对脂多糖介导的Mphis激活的影响 (释放IL-1、肿瘤坏死因子α、活性氧或氮中间体； 细胞内钙离子、Ia表达的变化； 蛋白激酶c)。(2)检查是否有任何My组件可以 作为内毒素受体或与内毒素受体结合，通过共定位 MT和内毒素结合位点双重免疫荧光染色，通过 测定标记的内毒素与分离的MT的结合，并用共聚焦显微镜观察 在紫杉醇存在的情况下，用MT沉淀内毒素受体。(3)至 确定细菌内毒素和紫杉醇反应性之间的遗传联系 通过检查这两个响应在Mphis中的接近程度 C5/BL/6J和C3H/HeJ杂交F2代，可能导致 C3H/HeJ小鼠缺陷蛋白(S)的鉴定

The goal of this proposal is to further understand the mechanisms involved in LPS-mediated actions in leukocytes, focusing on the interaction between LPS and the microtubule (MT) network of murine macrophages (Mphi). LPS is known to have profound, multiple effects on Mphis. Preliminary findings that drugs (e.g. colchicine and taxol) which are known to affect microtubule organization can mimic LPS in inducing (a) down-regulation of Mphi TNFalpha receptors, (b) release of TNFalpha, and (c) secretion of nitrite, and that LPS-hyporesponsive C3H/HeJ mice lack these responses to taxol, raise the possibilities that MT may play an important role in LPS- mediated actions. The specific aims of this study are to answer how LPS's actions are related to MT organization, and whether there is any structural or functional association between MT and LPS-binding protein(s). The proposed experiments include: (1) To determine the functional interaction between LPS and MT by examining the effect of LPS on MT modification (tyrosinolation and phosphorylation), and the effect of MT-active agents (i.e., colchicine, nocodazole, taxol) on lPS-mediated activation of Mphis (release of IL-1, TNFalpha, reactive oxygen or nitrogen intermediates; changes in intracellular calcium, Ia expression; and translocation of protein kinase c). (2) To examine whether any of the MY components can serve as an LPS-receptor or associate with an LPS receptor, by colocalizing MT and LPS binding sites with double immunofluorescent staining, by measuring the binding of labeled LPS to isolated MT, and by co- precipitating an LPS receptor with MT in the presence of taxol. (3) To determine the genetic linkage between LPS- and taxol-responsiveness in the mouse by examining the closeness of these two responses in Mphis from the F2 generation of a cross between C5/BL/6J and C3H/HeJ, possibly leading to the identification of the defective protein(s) in C3H/HeJ mice.