See following pages for detailed abstracts for each of the eight compounds labelled during this User visit. Details of the Chemical Synthesis Facility (CSF), which co-ordinated these projects into a single User visit to the NTLF, may be found at: http://biotech.genetics.utah.edu/CSF/ User Details: Experiment Details: User Number: 1708 Tritiation City, State: Salt Lake City, UT NMR Funding Source: NIH 5 R01 NS29632-08 5 days USDA 9601859 8 compounds NIH 1 R01 DC 03320-01A1 (PI: Lois Rasmussen) University of Utah Center for Chemical Synthesis Charge: $5218.74 Program Income: $5218.74 (projected) Secondary Users: Compound #1 Radiolabelled Cyclomusalenol as Substrate for Cyclopropyl Isomerase Principal Investigator: Seiichi Matsuda Department of Chemistry and Department of Biochemistry and Cell Biology Rice University Animals construct the sterol ring system using lanosterol synthase, whereas plants and many protozoa arrive at the same ring system with two enzymes: cycloartenol synthase and cyclopropyl isomerase. Cycloartenol synthase and cyclopropyl isomerase are potential targets for antiprotozoal drugs because of this dichotomy. Cyclopropyl isomerase is a particularly attractive target because humans have no known homolog of this enzyme. Cyclopropyl isomerase inhibitors are promising as antiprotozoal drugs with a broad therapeutic window. A current difficulty in testing cyclopropyl isomerase inhibitors is that assays require measuring NMR spectra of reaction mixtures and integrating substrate and product peaks. GC quantitation methods are impractical because sterols are insufficiently volatile without derivatization. Although substrates (such as the 4-methyl cyclopropyl sterol cyclomusalenol) separate readily from their isomerized products using silver HPLC, these methods are unreliable be cause the substrate and product lack workable chromophores. An appropriate radiolabelled cyclopropyl sterol substrate would make possible automated radiochromatographic assays, and would greatly aid this research. Cyclomusalenol is an available sterol that would be readily radiolabelled by reduction with tritiated hydrogen gas. Secondary Users: Compound #2 Isolation of Molecular Receptors for the Odorant Androstenone and a Functional Analog Principal Investigator: Charles J. Wysocki Monell Chemical Senses Center and Department of Animal Biology School of Veterinary Medicine University of Pennsylvania Philadelphia, PA 19104 Two inbred strains of mice have been used to explore sensitivity to androstenone (AND); one is quite sensitive to AND while the other is insensitive. Additional studies have determined that the insensitive strain can become behaviorally sensitized to AND by repeated exposures to it and that the increase in sensitivity is correlated with an increase in neuronal activity of the olfactory epithelium. Hence, a probe for potential changes in the molecular receptor of AND would be most useful. To this end, we propose to use diazo-androstenone and a diazo-modified functional analog of AND to """"""""fish out"""""""" the AND receptor from the olfactory epithelium. To extract the putative receptor from the epithelium, the diazo molecules must also be tritiated. This will allow us to track the molecule during isolation. Compound #3 Isolation of Molecular Receptors for the Odorant Androstenone and a Functional Analog Principal Investigator: Charles J. Wysocki Monell Chemical Senses Center and Department of Animal Biology School of Veterinary Medicine University of Pennsylvania Philadelphia, PA 19104 Two inbred strains of mice have been used to explore sensitivity to androstenone (AND); one is quite sensitive to AND while the other is insensitive. Additional studies have determined that the insensitive strain can become behaviorally sensitized to AND by repeated exposures to it and that the increase in sensitivity is correlated with an increase in neuronal activity of the olfactory epithelium. Hence, a probe for potential changes in the molecular receptor of AND would be most useful. To this end, we propose to use diazo-androstenone and a diazo-modified functional analog of AND to """"""""fish out"""""""" the AND receptor from the olfactory epithelium. To extract the putative receptor from the epithelium, the diazo molecules must also be tritiated. This will allow us to track the molecule during isolation. For humans, the bicyclohexyl compound is a functional analog of androstenone -- it smells just like androstenone, perhaps because of its backbone structure and the theoretical way in which androstenone (and therefore the bicyclohexyl compound), interacts with its receptor. Both parent compounds were tested in mice to determine whether they could smell each - they can. Both diazo compounds also were tested to determine whether the mice smelled each - they did. We know from other work that mice exposed to one of the parent compounds will become more sensitive to that compound and to the other compound. It is our hope to sensitize some mice to one of the parent compounds and compare binding with olfactory receptors in these animals to that seen in non-sensitized mice. This of course will be performed for each parent compound. Secondary Users: Compound #4 Transport of Hydrocarbons by Lipophorin: Selective Sorting of Hydrocarbons to the Cuticle and Pheromone Gland Principal Investigator: Coby Schal Blanton J. Whitmire Professor Department of Entomology Gardner Hall, Box 7613 North Carolina State University Raleigh, NC 27695-7613 Our long-term goals are to understand the dynamics and regulation of hydrocarbon transport through the hemolymph and to apply this information towards the development of new tactics for insect control. Hydrocarbons play critical roles in preventing water loss, as semiochemicals, and in penetration of hydrophobic xenobiotics through the cuticle. However, the mechanism of deposition of hydrocarbons has not been investigated in any tissue. The current paradigm is that hydrocarbons are transported directly from the oenocytes to the epicuticle, but our recent results have failed to support this view. Rather, we have shown that cuticular lipids are transported from the oenocytes that synthesize them by lipophorin (a hemolymph protein), to storage and deposition sites. Different lipid ligands are deposited by lipophorin at different tissues. It appears that a unique sorting mechanism exists for hydrocarbons whereby different hydrocarbons can be
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