MECHANISM OF BOTULINUM TOXIN ACTION

拟议研究的长期目标是确定 肉毒杆菌毒素的作用机制，负责 肉毒杆菌中毒作为这一目标的一部分，拟议的研究将 寻求预防和治疗疾病的方法。 大多数肉毒杆菌中毒病例是由于摄入了被 预先形成的毒素或摄入被细菌污染的食物， 会在肠道内产生毒素无论哪种情况肉毒杆菌毒素 从胃肠道系统逃逸到全身 循环（血液和淋巴）。血液中的毒素然后被分配到 外周胆碱能神经末梢，这是靶细胞， 毒素作用。毒素进入这些细胞，在那里产生 胞吐作用的阻断。 典型的肉毒杆菌中毒病例中的事件进展要求， 肉毒杆菌毒素穿过两个主要的细胞屏障。首先，毒素必须 从肠粘膜向肠内皮细胞的浆膜侧交叉。 接下来，毒素必须穿透细胞膜和内体膜， 到达胆碱能神经的胞质溶胶。因此， 拟议的研究将是表征机制， 毒素会结合并穿透肠道和神经细胞。 拟议的研究将由三个主要部分组成。第一、 将使用蛋白质化学和分子生物学技术 以产生完整的毒素或毒素片段。接下来，人类T-84细胞将 可以用作分析毒素在肠道细胞中移动的模型。 同时，将小鼠膈神经-hermidiapragm制剂 用作评估毒素穿过神经细胞的模型。结果 这些实验将用于：1）比较毒素结合和 肠和神经细胞中的转运，以及2）确定 阻断毒素在两种细胞中的运输。

The long-term objective of the proposed research is to determine the mechanism of action of botulinum toxin, the agent responsible for the disease botulism. As part of this objective, the proposed research will seek ways to prevent the disease and to treat the disease. Most cases of botulism are due to ingestion of food contaminated with pre-formed toxin or to ingestion of food contaminated with bacteria that can produce the toxin in the gut. In either case, botulinum toxin escapes from the gastrointestinal system to reach the general circulation (blood and lymph). Toxin in the blood is then distributed to peripheral cholinergic nerve endings, which are the target cells for toxin action. The toxin enters these cells, where it acts to produce blockade of exocytosis. The progression of events in a typical case of botulism requires that botulinum toxin cross two major cell barriers. First, the toxin must cross from the mucosal to the serosal side of gut endothelial cells. Next, the toxin must penetrate cell membranes and endosomal membranes to reach the cytosol of cholinergic nerves. Therefore the specific aim of the proposed research will be to characterize the mechanisms by which the toxin binds to an penetrates gut and nerve cells. The proposed research will be composed of three major elements. First, the techniques of protein chemistry and molecular biology will be used to generate intact toxin or toxin fragments. Next, human T-84 cells will be used as a model to analyze toxin movement across gut cells. Simultaneously, mouse phrenic nerve-hermidiapragm preparations will be used as a model to assess toxin movement across nerve cells. The results of these experiments will be used to: 1) compare toxin binding and transport in gut and nerve cells, and 2) identify mechanisms for blocking toxin transport in both types of cells.