Long-chain (sphingoid) bases, as the """"""""free"""""""" sphingoid base, the N-acyl-derivatives (ceramides), 1-phosphates, and other simple and complex derivatives affect cell growth, differentiation, and diverse cell functions (including apoptosis). Moreover, these compounds not only serve as intra- and extra-cellular messengers in normal cell regulation, but also, cause cellular dysfunction in diseases where sphingolipid biosynthesis or turnover are abnormal. The plan for this competitive renewal is to provide fundamental information about the regulation of sphingolipid biosynthesis using techniques and findings developed during the previous period of support. The proposed studies will: 1) characterize the regulation of de novo sphingolipid biosynthesis by sphingolipids and sphingolipid analogs using a variety of tools, including luciferase promoter constructs for the mouse and human SPT2 genes; 2) provide a comprehensive analysis of the regulation of sphingolipid biosynthesis and turnover in NIH3T3 and HepG2 cells by growth factors, cytokines, and other factors using HPLC/electrospray tandem mass spectrometry, which allows definitive structural elucidation and quantitation of all of these metabolites in each sample; and, 3) identification of the origin and function(s) of a novel inhibitor of sphingolipid metabolism that we have isolated from conditioned medium from cells in culture: 2,6-bis-(w-aminobutyl)-3,5-diimino-piperazine (betrachamine). The better understanding of sphingolipid metabolism that will be provided by these studies will benefit not only other """"""""sphingolipidologists"""""""" but also the many investigators who are studying various aspects of sphingolipid signaling in cell regulation.
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