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Publications (3)11.87 Total impact

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    ABSTRACT: Activation of RAW264.7 cells with a lipopolysaccharide specific for the TLR4 receptor, Kdo(2)-lipid A (KLA), causes a large increase in cellular sphingolipids, from 1.5 to 2.6 × 10(9) molecules per cell in 24 h, based on the sum of subspecies analyzed by "lipidomic" mass spectrometry. Thus, this study asked the following question. What is the cause of this increase and is there a cell function connected with it? The sphingolipids arise primarily from de novo biosynthesis based on [U-(13)C]palmitate labeling, inhibition by ISP1 (myriocin), and an apparent induction of many steps of the pathway (according to the distribution of metabolites and microarray analysis), with the exception of ceramide, which is also produced from pre-existing sources. Nonetheless, the activated RAW264.7 cells have a higher number of sphingolipids per cell because KLA inhibits cell division; thus, the cells are larger and contain increased numbers of membrane vacuoles termed autophagosomes, which were detected by the protein marker GFP-LC3. Indeed, de novo biosynthesis of sphingolipids performs an essential structural and/or signaling function in autophagy because autophagosome formation was eliminated by ISP1 in KLA-stimulated RAW264.7 cells (and mutation of serine palmitoyltransferase in CHO-LYB cells); furthermore, an anti-ceramide antibody co-localizes with autophagosomes in activated RAW264.7 cells versus the Golgi in unstimulated or ISP1-inhibited cells. These findings establish that KLA induces profound changes in sphingolipid metabolism and content in this macrophage-like cell line, apparently to produce sphingolipids that are necessary for formation of autophagosomes, which are thought to play important roles in the mechanisms of innate immunity.
    Journal of Biological Chemistry 09/2010; 285(49):38568-79. · 4.65 Impact Factor
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    ABSTRACT: CHO-LY-B cells have been useful in studies of sphingolipid metabolism and function because they lack serine palmitoyltransferase (SPT) activity. Cloning and sequencing of the SPT1 transcript of LY-B cells identified the mutation as a guanine to adenine change at nucleotide 738, causing a G246R transformation. Western blots revealed low expression of the mutant SPT1 peptide, but activity was not detectable by mass spectrometric analysis of [(13)C]-palmitate incorporation into sphinganine, sphingosine, 1-deoxysphinganine, or 1-desoxymethylsphinganine. Treatment of LY-B cells with chemical chaperones (DMSO or glycerol) increased the amounts of mutant SPT1 as well as SPT2, but SPT activity was not restored. This study has established that G246R mutation in hamster SPT1 results in the loss of SPT activity.
    Lipids 07/2009; 44(8):725-32. · 2.56 Impact Factor
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    ABSTRACT: Serine palmitoyltransferase (SPT) has been localized to the endoplasmic reticulum (ER) by subcellular fractionation and enzymatic assays, and fluorescence microscopy of epitope-tagged SPT; however, our studies have suggested that SPT subunit 1 might be present also in focal adhesions and the nucleus. These additional locations have been confirmed by confocal microscopy using HEK293 and HeLa cells, and for focal adhesions by the demonstration that SPT1 co-immunoprecipitates with vinculin, a focal adhesion marker protein. The focal adhesion localization of SPT1 is associated with cell morphology, and possibly cell migration, because it is seen in most cells before they reach confluence but disappears when they become confluent, and is restored by a standard scratch-wound healing assay. Conversely, elimination of SPT1 using SPTLC1 siRNA causes cell rounding. Thus, in addition to its "traditional" localization in the ER for de novo sphingolipid biosynthesis, SPT1 is present in other cellular compartments, including focal adhesions where it is associated with cell morphology.
    Biochimica et Biophysica Acta 05/2009; 1791(8):746-56. · 4.66 Impact Factor