application) This core will measure tissue, cellular, and mitochondrial levels of ceramide, sphingosine, sphingosine-1-phosphate (S-1-P), diacylglycerol (DAG), and other lipids involved in the regulation of apoptosis. The core will concentrate mostly on the quantitative determination of total ceramide and DAG via DAG-kinase phosphorylation. In selected situations, LC-MS spectroscopic method will be used to obtain both qualitative and quantitative determination of the molecular species of ceramides present in specimens. Determination of the cellular levels of D-erythro-sphingosine and D-erythro-dihydrosphingosine will be based on the BPLC technique developed for their fluorescent analogs. A Synthetic Unit will provide homogenous sphingolipid standards corresponding to the naturally occurring lipids. This core will also synthesize radiolabeled analogs, which will allow the study of sphingolipid metabolic pathways. Key molecules to be synthesized include: stereoisomers of sphingosine, dihydrosphingosine, ceramides, dihydroceramide, sphingosine-1-phosphate and their modified analogs. Also, the core will provide inhibitors of sphingolipid metabolic pathways (e.g., D-e-MAPP).
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