The primary objective of this proposal is to isolate and characterize peptides contained in the skin of various amphibians (frogs and toads). Specific emphasis will be given to the structural and biological characterization of two important classes of peptides (i. e., the antimicrobial and bombesin/GRP peptide families). These two families have been chosen because (i) they are present in most, if not all, amphibian species, (ii) they possess important bioactivites relevant to drug development for human cancer and antimicrobial therapies, and (iii) they represent the two major amphibian skin peptide groups; the lytic/antimicrobial and the peptide hormone-like families. Given the recent demise of amphibian populations and species worldwide, this study will attempt to lay the framework for cataloging what may be a vanishing natural products resource. Specifically, we propose to carry out a natural products characterization on several species of amphibians (Bombina orientalis, Bombina maxima, Rana aurora, Rana cascadae, Phyllomedusa sauvagei, Bufo boreas, Leptodactylus rugosa and Hyla regilla) as described below: 1. Collect, purify and structurally characterize peptide components of the skin secretion of the selected amphibians. As previously mentioned, specific attention will be placed on peptides that belong to the antimicrobial and bombesin/GRP families, although peptides not belonging to these two classes may undergo limited structural analyses. Advanced chromatographic and mass spectrometric technologies will be used to complete what would otherwise be a difficult and time-consuming task. 2. Construct cDNA libraries from amphibian skin mRNAs and isolate and sequence the cDNA encoding the corresponding precursor polypeptides to the targeted antimicrobial and bombesin/GRP peptides. 3. Evolutionary comparison of peptide structures and their corresponding precursor and cDNA sequences. 4. Limited biological testing of the targeted antimicrobial and bombesin/GRP peptides to define more specific biological activities. By combining a natural products search with a biological assessment, we hope to exploit the natural structural diversity of peptides in the skin of amphibians to determine the specific chemical features of these two important families of peptides that confers their specific activities.
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