Chapter 9 TLC and HPTLC of Phospholipids and Glycolipids in Health and Disease

Ando, Susumu; Saito, Megumi

doi:10.1016/s0301-4770(08)60603-x

Cited by 16 publications

(7 citation statements)

References 168 publications

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“…In some analyses, to achieve a better resolution between glucosyl-and galactosyl-ceramide, neutral GSLs were resolved on HPTLC plates dipped in 2.5% (w/v) methanolic boric acid and activated by heating at 110°C for 30 min [18]. Neutral GSLs were visualized by the a-naphthol reagent [16]. For phospholipid analysis, aliquots of the Folch's lower phase were developed in chloroform/methanol/acetic acid/water (60:27:8:2, v/v/v/v).…”

Section: Lipid Analysismentioning

confidence: 99%

“…For ganglioside separation, the Folch's upper phase was dried under vacuum, redissolved in distilled water and desalted by gel filtration. Gangliosides recovered in the void volume were dried under vacuum, resuspended in chloroform/methanol (2:1, v/v), spotted onto HPTLC plates, developed in chloroform/methanol/0.22% CaCl 2 (60:40:9, v/v/ v) and visualized by staining with the resorcinol reagent [16]. For analysis of neutral GSLs and ceramide, the Folch's lower phase was dried, lipids resuspended in 0.1 M methanolic KOH, subjected to mild alkaline hydrolysis for 1 h at 37°C and re-extracted by the Folch's method.…”

Section: Lipid Analysismentioning

confidence: 99%

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Evidence for a role of glycosphingolipids in CXCR4‐dependent cell migration

et al. 2007

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Chemotaxis induction is a major effect evoked by stimulation of the chemokine receptor CXCR4 with its sole ligand CXCL12. We now report that treatment of CHP-100 human neuroepithelioma cells with the glucosylceramide synthase (GCS) inhibitor DL-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol inhibits CXCR4-dependent chemotaxis. We provide evidence that the phenomenon is not due to unspecific effects of the inhibitor employed and that inhibition of GCS neither affects total or plasmamembrane CXCR4 expression, nor CXCL12-induced Ca 2+ mobilization. The effects of the GCS inhibitor on impairment of CXCL12-induced cell migration temporally correlated with a pronounced downregulation of neutral glycosphingolipids, particularly glucosylceramide, and with a delayed and more moderate downregulation of gangliosides; moreover, exogenously administered glycosphingolipids allowed resumption of CXCR4-dependent chemotaxis. Altogether our results provide evidence, for the first time, for a role glycosphingolipids in sustaining CXCL12-induced cell migration.

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Section: Lipid Analysismentioning

confidence: 99%

Section: Lipid Analysismentioning

confidence: 99%

Evidence for a role of glycosphingolipids in CXCR4‐dependent cell migration

et al. 2007

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“…The extracts were applied to DEAE-Toyopearl columns to separate the neutral lipids and acidic lipids (39).…”

Section: Preparation Of the Detergent-insoluble Low-density Membranementioning

confidence: 99%

Mutant Presenilin 2 Transgenic Mice

Sawamura¹,

Morishima-Kawashima²,

Waki³

et al. 2000

Journal of Biological Chemistry

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A subset of early-onset familial Alzheimer's disease (FAD) 1 is inherited as an autosomal dominant trait, and presenilin (PS) 1 and 2 genes in addition to ß-amyloid precursor protein (APP) gene were identified as the causative genes. PS1 is mapped to chromosome 14 (1,2), while PS2 is mapped to chromosome 1 (3-5). These PS1 and PS2 genes encode multispanned transmembrane proteins showing high degrees of homology (4,5).Both proteins are located predominantly in the endoplasmic reticulum (ER), and partly in the Golgi apparatus and other compartments (6-8), but their physiological functions remain unclear. More than 50 different mutations have thus far been identified in PS1 (9), while only two mutations have been found in PS2 (10). The residue at position 141 (Asn) in PS2, which is conserved in human and mouse PS1 and PS2, is substituted by Ile (N141I) in the Volga German kindred. Another missense mutation (M239V) in PS2 has been found in Italian FAD families (5).Although the pathogenetic mechanism how Alzheimer's disease (AD) is developed by PS mutations remains unknown, mutations of PS1 and PS2 are known to have similar effects on the production of amyloid ß-protein (Aß) 42, the initially deposited Aß species in senile plaques (11)(12)(13)(14): While Aß42 is normally secreted in much lower quantities than Aß40, these aforementioned mutations induce elevation of the Aß42 levels in cultured cells and transgenic mice (15)(16)(17)(18)(19)(20). It has also been reported that in primary neuronal cultures derived from PS1-knockout mouse embryos, Aß secretion was remarkably decreased, concomitant with the accumulation of the C-terminal fragment of APP (21). The mutation in the two particular Asp residues in the PS1-transmembrane domains induced a profound decrease in the Aß production and an increase in the levels of the C-terminal fragment of APP (22). These observations indicate that PS1 may have a direct or indirect role in the γ -cleavage of APP.We previously reported that the mutant PS2 transgenic mice showed increases in the Aß42 levels in the Tris-saline (TS)-soluble fractions in an age-dependent manner during 2 to 8 months of age (20). On the other hand, a series of Aß quantitation studies of autopsied human tissues has clearly shown that Aß42 already accumulates to a significant extent in the TS- In the present study, we sought to obtain further insight into the effects of mutant PS2on the Aß levels in the TS-insoluble, guanidine hydrochloride-solubilized fraction of the mouse brain, and to characterize the intracellular compartmentalization of insoluble Aß. EXPERIMENTAL PROCEDURESTransgenic mice -The heterozygous PS2 transgenic mice used in this study were from the previously established lines W2 (wild-type PS2 transgenic mice), and M1 and M2(N141I mutant PS2 transgenic mice) (20). Each line of transgenic mice was backcrossed to the C57BL/6J strain, and those mice carrying the PS2 transgene were selected using a transgene-specific PCR assay (20). Littermates without PS2 transgenes were used as the nontransge...

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“…Unless otherwise mentioned, lipids were visualized by dipping the plates in a solution of 3% cupric acetate in 8% phosphoric acid and heating in an oven for 15 min at 1808C, according to Macala et al (1983). For glycosphingolipid detection, plates were sprayed with a solution made of 10.5 ml methanolic a-naphthol (5%, w/v), 6.5 ml concentrated sulphuric acid, 40.5 ml ethanol and 4 ml water, followed by hearing for 10 min at 908C, as reported (Ando and Saito, 1987). Fatty acids were transmethylated and analyzed by a Perkin Elmer 8310 gas chromatograph, equipped with a Supelcowax 10 fused silica capillary column, as previously described (Spinedi et al, 1987).…”

Section: Lipid Extraction and Separationmentioning

confidence: 99%

Apoptosis induced by N-hexanoylsphingosine in CHP-100 cells associates with accumulation of endogenous ceramide and is potentiated by inhibition of glucocerebroside synthesis

1998

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We report that apoptosis induced by N-hexanoylsphingosine (C 6 -Cer) in CHP-100 human neuroepithelioma cells associates with accumulation of monohexosylsphingolipids produced not only by short-chain ceramide glycosylation but also through glycosylation of a ceramide pool endogenously produced. By high-performance thin layer chromatography on borate silica gel plates, newly formed monohexosylsphingolipids were identified as glucosylceramides (GluCer); however, accumulation of lactosylceramide or higher-order glycosphingolipids was not observed. GluCer accumulation was fully suppressed by D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol; moreover, while this inhibitor had no effect on cell viability when administered alone, it markedly potentiated the apoptotic effect of C 6 -Cer. These results provide evidence that activation of GluCer synthesis is an important mechanism through which CHP-100 cells attempt to escape ceramide-induced apoptosis.

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Chapter 9 TLC and HPTLC of Phospholipids and Glycolipids in Health and Disease

Cited by 16 publications

References 168 publications

Evidence for a role of glycosphingolipids in CXCR4‐dependent cell migration

Evidence for a role of glycosphingolipids in CXCR4‐dependent cell migration

Mutant Presenilin 2 Transgenic Mice

Apoptosis induced by N-hexanoylsphingosine in CHP-100 cells associates with accumulation of endogenous ceramide and is potentiated by inhibition of glucocerebroside synthesis

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