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.
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