The mycotoxin talaromycin is a putative inducer of a voltage dependent Ca2+ conductance. It is now possible to totally synthesize structural analogs of talaromycin and determine its molecular activity. The proposed project will facilitate cross communication among investigators from diverse scientific disciplines. Our objectives are grouped below by PI and quartile of the total project: Taylor - long-term objectives are (i) to determine the relations among chemical structure and biological effects of agents that alter excitation, contraction, or excitation-contraction coupling, (ii) to understand the transducer mechanisms by which target cells are activated and how receptor-mediated cell behavior is normally regulated (e.g., the relations among the size, time course, and distribution in a cell of voltage dependent ionic currents measured by on-cell patch clamping, intracellular Ca2+ transients monitored via the photoprotein aequorin, bond formation (cross-bridge attachment) between contractile filaments, force generation, striation spacing, and myofibrillar orientation measured with fast time resolution), and (iii) to understand how abnormal receptor function relates to diminished activation, impaired regulation, or disease states (e.g., the molecular changes associated with flaccid paralysis induced by talaromycin and drug-induced or energy metabolism myopathies; the slow inward TTX-resistant current induced by talaromycin and its similarity to Ca2+ conductances associated with the paroxysmal depolarization shifts). Powis - the objective is to determine the acute and long term activity and toxicity of talaromycin analogs on cultured target cells from laboratory animals and humans. Excitable or contractile cells are included because of known effects on excitation in muscle cells (Appendix III) and flaccid paralysis in animals. But several additional cell lines will be studied to aid understanding overall effects observed in intact animals. Activity and toxicity is measured by dye exlusion, 51Cr release, cytosolic enzyme release, intracellular glutathione, thymidine incorporation, colony formation in soft agarose, and L5178Y/TKJ gene mutation assay. Lynn - objectives are (i) to synthesize numerous structural analogs of the only spiroketal containing metabolite known to be elaborated by fungi (i.e., talaromycin), and (ii) to determine the molecular structure of each analog by NMR and mass spectrometry and aid understanding the relation between chemical structure and activity of this toxin on target cells. Cole - the objective is to determine the activity and toxicity of structural analogs of talaromycin in intact laboratory animals.
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