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PubMed1980

Application of "high-performance" liquid chromatography to the study of sphingolipidoses.

Quantitative high-performance liquid chromatographic analysis of perbenzoylated sphingolipids has been used to study the correlations of body chemistry to clinical phenomena. Plasma sphingolipids were isolated from 32 Gaucher (beta-glucosidase deficiency) and six Fabry (alpha-galactosidase deficiency) patients by solvent partition and chromatographic separation on silicic acid columns. Plasma sphingolipids from a patient undergoing plasma-exchange were separated from interfering lipids with reversed-phase columns. Liquid-chromatographic analysis of sphingolipids provides useful supportive information for diagnoses because affected individuals are shown to possess increased circulating concentrations of the pathognomonic sphingolipid. We also used this technique to monitor sphingolipid concentrations in plasma and urine sediment during plasma exchange of a p atient with Fabry's disease. Regular plasma exchanges produced and maintained decreased concentrations of sphingolipids in plasma, but near pre-exchange concentrations were observed within days after the therapy was terminated.

Chromatography, High Pressure Liquid↗
PubMed1997

Identification and initial characterizations of free, glycosylated, and phosphorylated ceramides of Paramecium.

Paremecium tetraurelia contains high concentrations of six ethanolamine sphingolipids in the cell surface membrane surrounding somatic cilia. Three have phosphoryl groups and the other three have phosphonyl groups, and each contains either dihydrosphingosine, sphingosine, or phytosphingosine. In the present study, free, phosphorylated, and three major glycosylated ceramides were identified in the neutral sphingolipid fraction of this organism when it was grown on a crude medium. Individual lipids were characterized by specific staining on thin-layer chromatographic plates and the fatty acids derived from them were identified by gas-liquid chromatography and mass spectrometry. Unlike the esterified fatty acid composition of the ciliate's phospholipids, which have only small amounts of fatty acids greater than 20 carbons in length, the neutral sphingolipids mainly contain fatty acids greater than 22 carbons in length as well as high concentrations of long chain hydroxy fatty acids. The sugars, glucose, galactose, and fructose, were identified in total neutral sphingolipid fraction. The long chain bases in the neutral sphingolipid fraction were mainly C18 compounds and were identified as C18 dihydrosphingosine, C20 sphingosine, and isomers of C18 sphingosine. Phytosphingosine was not detected in the neutral sphingolipid fraction obtained from whole cells. Because most of the cell's phytosphingosine-containing ethanolamine sphingolipids are in cilia, the inability to detect phytosphingosine as part of putative precursor ceramide compounds suggests that conversions of the long chain base moiety of ethanolamine phospho- and phosphonosphingolipids occur in situ in the ciliary membrane.

Animals↗
PubMed2001

G(s) signaling is intact after disruption of lipid rafts.

Membrane microdomains enriched in cholesterol and sphingolipids modulate a number of signal transduction pathways and provide a residence for heterotrimeric G proteins, their receptors, and their effectors. We investigated whether signaling through G(s) was dependent on these membrane domains, characterized by their resistance to detergents, by depleting cells of cholesterol and sphingolipids. For cholesterol depletion, rat salivary epithelial A5 cells were cultured under low-cholesterol conditions, and then treated with the cholesterol chelator methyl-beta-cyclodextrin. For sphingolipid depletion, LY-B cells, a mutant CHO cell line that is unable to synthesize sphingolipids, were incubated under low-sphingolipid conditions. Depletion of cholesterol or sphingolipid led to a loss or decrease, respectively, in the amount of Galpha(s) from the detergent-resistant membranes without any change in the cellular or membrane-bound amounts of Galpha(s). The cAMP accumulation in response to a receptor agonist was intact and the level slightly increased in cells depleted of cholesterol or sphingolipids compared to that in control cells. These results indicate that localization of Galpha(s) to detergent-resistant membranes was not required for G(s) signaling. Analysis of the role of lipid rafts on the kinetics of protein associations in the membrane suggests that compartmentalization in lipid rafts may be more effective in inhibiting protein interactions and, depending on the pathway, ultimately inhibit or promote signaling.

Animals↗
PubMed1996

Cholesterol-induced interfacial area condensations of galactosylceramides and sphingomyelins with identical acyl chains.

The interfacial interactions occurring between cholesterol and either galactosylceramides (GalCers) or sphingomyelins (SMs) with identical acyl chains have been investigated using Langmuir film balance techniques. Included among the synthesized GalCers and SMs were species containing palmitoyl (16:0), stearoyl (18:0), oleoyl [18:1 delta 9(c)], nervonoyl [24:1 delta 15(c)], or linoleoyl [18:2 delta 9,12(c)] acyl residues. The cholesterol-induced condensations in the average molecular areas of the monolayers were determined by classic mean molecular area vs composition plots as well as by expressing the changes in terms of sphingolipid cross-sectional area reduction over the surface pressure range from 1 to 40 mN/m (at 1 mN/m intervals). The results show that, at surface pressures approximating bilayer conditions (30 mN/m), acyl heterogeneity in naturally occurring SMs (bovine of egg SM) enhanced the area condensation induced by cholesterol compared with their predominant molecular species (e.g. 18:0 SM in bovine SM; 16:0 SM in egg SM). Nonetheless, cholesterol always had a greater condensing effect on SM compared to GalCer when these sphingolipids were acyl chain matched and in similar phase states (prior to mixing with cholesterol). Also, the cholesterol-induced area changes for a given sphingolipid type (e.g. SM or GalCer) were similar whether the acyl chains were saturated, cis-delta 9-monounsaturated, or cis-delta 9,12-diunsaturated if the sphingolipids were in similar phase states (prior to mixing with cholesterol) and compared at equivalent surface pressures. These results indicate that, under conditions were hydrocarbon structure is matched, the sphingolipid head group plays a dominant role in determining the extent to which cholesterol reduces sphingolipid cross-sectional area. Despite the larger cholesterol-induced area condensations observed in SMs compared to those in GalCers, the molecular-packing densities showed that equimolar GalCer-cholesterol films were generally packed as tight as or slightly tighter than those of the SM-cholesterol films. The results are discussed in terms of a molecular model for sphingolipid-cholesterol interactions. Our findings also do only raise questions as to whether cholesterol-induced condensation data provide a reliable measure of the affinity, i.e. interaction strength, between cholesterol and different lipids but also provide insight regarding the stability of sterol/sphingolipid 1-1 rich microdomains thought to exist in caveolae and other cell membrane regions.

Animals↗
PubMed1990

Evaluation of synthetic sphingosine, lysosphingolipids and glycosphingolipids as inhibitors of functional responses of human neutrophils.

Human neutrophils, when exposed to soluble stimuli, aggregate, release oxygenated products of arachidonic acid and generate active oxygen species. Sphingolipid-derived products such as sphingosine and lysosphingolipids have been shown to exert selective actions on a variety of cell types, including neutrophils. Therefore, to determine the structural basis for selective inhibition of neutrophil responses by naturally occurring sphingolipids, seven compounds were prepared by total organic synthesis, and their impact on neutrophils in suspension has been studied. The compounds synthesized included sphingosine, psychosine, lactosyl lysosphingolipid, globotriaosyl (Gb3) lysosphingolipid, galactosyl cerebroside, lactosyl ceramide and Gb3 ceramide. The neutrophil responses studied were aggregation, leukotriene generation and superoxide anion production. When exposed to non-cytotoxic levels of the synthetic compounds, as monitored by exclusion of Trypan Blue, none of the synthetic sphingolipids inhibited A23187-induced aggregation of neutrophils. Only lactosyl lysosphingolipid, at a concentration of 1 microM, significantly inhibited aggregation induced by fMetLeuPhe; the other compounds in this series including sphingosine were without effect at equal molar concentrations (1 microM). Aggregation induced by phorbol 12-myristate 13-acetate (PMA) (0.1 microM) was significantly blocked by only two of the synthetic sphingolipids (1 microM). At concentrations below 1 microM, these inhibitory actions were not evident, nor was it possible to assign a structure-activity relationship for this series of compounds. None of the synthetic sphingolipids effectively inhibited the generation of superoxide anions induced by PMA. In addition, neither synthetic sphingosine nor psychosine affected either the formation or metabolism of leukotriene B4. Taken together, the results provide further evidence that sphingolipids, when added to intact cells, are not potent selective inhibitors of functional responses of human neutrophils.

Antigens, CD↗
PubMed2002

The uracil transporter Fur4p associates with lipid rafts.

Sphingolipids are abundant components of eucaryotic membranes, where they perform essential functions. To uncover new roles for sphingolipids, we studied Saccharomyces cerevisiae lcb1-100 cells, which have a temperature-sensitive block in the first step in sphingolipid synthesis. We find that the level of all five species of the sphingoid long chain base intermediates is reduced 2-7-fold in cells grown at a permissive temperature, and the level of complex sphingolipids is reduced 50%. In addition, lcb1-100 cells make no detectable phosphorylated sphingoid bases. After transfer to a restrictive temperature (a heat shock), the level of the major sphingoid bases drops rather than transiently rising, as in wild type cells. These changes affect lcb1-100 cells in multiple ways. Basal uracil transport by Fur4p is reduced 25%, and when cells are heat-shocked, uracil transport activity falls rapidly and is not restored as it is in wild type cells. Restoration requires a functional secretory pathway and synthesis of complex sphingolipids, leading us to hypothesize that Fur4p associates with lipid rafts. The finding that Fur4p is insoluble in TritonX-100 at 4 degrees C and behaves like a raft-associated protein on a density gradient supports this hypothesis. Raft association may be essential for regulating breakdown of Fur4p in response to stresses and other factors that govern uracil transport activity. Our results show that long chain bases do not contribute to the inactivation of Fur4p transport activity after heat stress, but they are essential for some later, but unknown, process that leads to degradation of the protein. Further studies using lcb1-100 cells should reveal new roles of sphingolipids in nutrient uptake and other membrane-dependent processes.

Alleles↗
PubMed1992

Characterization of a novel, potent, and specific inhibitor of serine palmitoyltransferase.

We have examined the mechanism of action of two natural products identified as broad spectrum antifungal agents (VanMiddlesworth, F., Dufresne, C., Wincott, F. E., Mosley, R. T., and Wilson, K. E. (1992) Tetrahedron Lett., in press; VanMiddlesworth, F., Giacobbe, R. A., Lopez, M. Garrity, G., Bland, J. A., Bartizal, K., Fromtling, R. A., Polishook, J., Zweerink, M. M., Edison, A. M., Rozdilsky, W., Wilson, K. E., and Monaghan, R. L. (1992) J. Antibiot. (Tokyo) 45, 861-867), designated sphingofungin B (2S-amino-3R,4R,5S,14-tetrahydroxyeicos-6-enoic acid) and sphingofungin C (2S-amino-5S-acetoxy-3R,4R,14-trihydroxyeicos-6-enoic acid), and find they are potent specific inhibitors of serine palmitoyltransferase, which catalyze the committed step of sphingolipid biosynthesis. We used Saccharomyces cerevisiae as a model to investigate the mechanism of the antifungal activity of these compounds. Macromolecular synthesis was not immediately affected by either sphingofungin B or C, synthesis continued for 60-90 min following the addition of drug to growing cultures. Significant loss of viability with sphingofungins required growing cultures and began only after several hours, with greater than 99.9% of drug-treated cells non-viable after 24 h. No lysis or other gross changes in cell morphology were observed in drug-treated cells. The structural similarity of sphingofungin B and C to sphingosine and phytosphingosine prompted us to investigate their effects on sphingolipid synthesis. Nanomolar levels of the drugs inhibited the incorporation of [3H]inositol into sphingolipid before incorporation into the sphingolipid precursor, phosphatidylinositol was affected, suggesting specific inhibition of sphingolipid synthesis. This hypothesis was confirmed by experiments in which the growth inhibitory activity of both drugs was completely ablated by the addition of phytosphingosine, dihydrosphingosine, or ketodihydrosphingosine to the culture medium. Reversal of antifungal activity by ketodihydrosphingosine suggested that serine palmitoyltransferase could be the actual target of these compounds. Direct evidence for this hypothesis was the observation of inhibition of serine palmitoyltransferase activity in crude membrane preparations at nanomolar concentrations of each drug. The potent inhibition of serine palmitoyltransferase coupled with the apparent lack of effect of these compounds on other cellular functions suggests that sphingofungin B and C will prove to be important new tools for studying the role of sphingolipids in yeast and perhaps in other organisms.

Acyltransferases↗
PubMed1983

Human stratum corneum lipids: characterization and regional variations.

The lipids of mammalian stratum corneum are known to be important regulators of skin permeability. Since the human stratum corneum displays remarkable regional variations in skin permeability, we assessed the total lipid concentration, the distribution of all major lipid species, and the fatty acid composition in Bligh-Dyer extracts from four skin sites (abdomen, leg, face, and sole) that are known to display widely disparate permeability. Statistically significant differences in lipid weight were found at the four sites that were inversely proportional to their known permeability. In all four sites, among the polar lipids, the stratum corneum contained negligible phospholipids, but substantially more cholesterol sulfate (1-7%) than previously appreciated. As in the stratum corneum from other mammals, the bulk of the lipids consisted of neutral (60-80%) and sphingolipids (15-35%). Of the neutral lipids, free sterols (4- to 5-times greater than esterified sterols), free fatty acids, triglycerides, and highly nonpolar species (n-alkanes and squalene) predominated. n-Alkanes, which were present in greater quantities than previously appreciated, comprised a homologous series of odd- and even-chained compounds ranging from C19 to C34. The sphingolipids comprised over 80% ceramides vs. lesser quantities of glycosphingolipids. In all four sites, the sphingolipids were the major repository of long-chain, saturated fatty acids. The neutral lipid:sphingolipid ratio generally was proportional to the known permeability of each site: higher neutral lipids and lower sphingolipids generally were associated with superior barrier properties. These studies provide: 1) the first detailed, quantitative analysis of human stratum corneum lipids and 2) information about the variability in lipid composition at four skin sites with known differences in permeability. The latter results suggest that variations in neutral lipids, rather than sphingolipids, may underlie local variations in skin permeability.

Alkanes↗
PubMed1998

UVB irradiation up-regulates serine palmitoyltransferase in cultured human keratinocytes.

The enzyme serine palmitoyltransferase (SPT; EC 2.3.1.50), which catalyzes the first committed and rate-limiting step in sphingolipid synthesis, is up-regulated in the epidermis as part of the homeostatic repair in response to permeability barrier perturbation. Moreover, UVB exposure, which also perturbs the barrier, up-regulates sphingolipid synthesis, but the basis for this increase is not known. The recent isolation of cDNAs for SPT (i.e., LCB1 and LCB2) allow molecular regulation studies to be performed. Therefore, we determined whether UVB exposure alters mRNA, protein, or activity levels for SPT in cultured human keratinocytes (CHKs) as a mechanism for regulation of epidermal sphingolipid synthesis. In CHK, transcripts for both LCB1 (3.0 kb) and LCB2 (2.3 kb) are evident by Northern blot analysis, and UVB exposure (23 mJ/cm2) induces a delayed 1.8 to 3.3-fold increase in LCB2 mRNA levels (P < 0.01) 48 h after treatment versus non-irradiated control cells. In contrast, neither LCB1 nor a second LCB2 transcript (8.0 kb) changed significantly. Likewise, Lcb2 protein levels (by Western blot analysis), as well as SPT activity, increase in parallel with the increased LCB2 mRNA. Finally, incorporation of [14C]-acetate into sphingolipids was increased significantly 48 h after UVB treatment. Together, these results demonstrate that CHKs respond to UVB by increasing sphingolipid synthesis, primarily through increases in both LCB2 mRNA and protein levels, leading to increased SPT activity. These results demonstrate one mechanism (UVB) whereby SPT is regulated at the molecular level, and suggest further that epidermis up-regulates sphingolipid synthesis at both the mRNA and protein levels in response to UVB.

Acetates↗
PubMed2000

Colonic cell proliferation and aberrant crypt foci formation are inhibited by dairy glycosphingolipids in 1, 2-dimethylhydrazine-treated CF1 mice.

Dietary sphingomyelin (SM) inhibits early stages of colon cancer (appearance of aberrant crypt foci, ACF) and decreases the proportion of adenocarcinomas vs. adenomas in 1,2-dimethylhydrazine (DMH)-treated CF1 mice. To elucidate the structural specificity of this inhibition, the effects of the other major sphingolipids in milk (glycosphingolipids) were determined. Glucosylceramide (GluCer), lactosylceramide (LacCer) and ganglioside G(D3) were fed individually to DMH-treated (six doses of 30 mg/kg body weight) female CF1 mice at 0.025 or 0.1 g/100 g of the diet for 4 wk. All reduced the number of ACF by > 40% (P < 0.001), which is comparable to the reduction by SM in earlier studies. Immunohistochemical analysis of the colons revealed that sphingolipid feeding also reduced proliferation, with the most profound effect (up to 80%; P < 0.001) in the upper half of the crypts. Since the bioactive backbones of the glycosphingolipids (i.e., ceramide and other metabolites) are the likely mediators of these effects, the susceptibility of these complex sphingolipids to digestion in the colon was examined by incubating 500 microgram of each sphingolipid with colonic segments from mice and analysis of substrate disappearance and product formation by tandem mass spectrometry. All of the sphingolipids (including SM) disappeared over time with a substantial portion appearing as ceramide. Partially hydrolyzed intermediates (such as GluCer from LacCer or G(D3)) were not detected, which suggests that the cleavage involves colonic (or microflora) endoglycosidases. In summary, consumption of dairy SM and glycosphingolipids suppresses colonic cell proliferation and ACF formation in DMH-treated mice; hence, many categories of sphingolipids affect these key events in colon carcinogenesis.

1,2-Dimethylhydrazine↗
PubMed1998

Human fibroblasts in culture metabolize differently exogenous G(M3) ganglioside species containing C18 and C20 sphingosine.

Preparation of radioactive GM3 species containing isotopically labeled C18 sphingosine or C20 sphingosine is reported and their use for studying some aspects of the sphingolipid biosynthesis in cells is discussed. Human fibroblasts in culture that have only C18 sphingolipids and GM3 as the major gangliosides, were fed with the two radioactive GM3 species. The radioactive gangliosides were taken up by the cells and metabolized. The analyses of the radioactivity metabolic fate, in this model provides the following information. i--About 70-80% of the total catabolic sphingosine is re-cycled for biosynthesis of complex sphingolipids. ii--A small amount of the catabolic C20 sphingosine was re-cycled for biosynthesis of C20 sphingolipids, thus yielding complex lipids that are not naturally present in fibroblast cells. iii--A regulatory step in the biosynthesis of sphingolipid species differring long chain base content, C18 or C20 sphingosine, is in some way involved in the first steps of sphingolipid biosynthesis, and thus plays a decisive role in the availability of the long chain bases.

Cells, Cultured↗
PubMed1993

Intracellular transport, organelle biogenesis and establishment of Golgi identity: impact of brefeldin A on the activity of lipid synthesizing enzymes.

1. The effect of brefeldin A (BFA) on generation of transport vesicles, synthesis of phosphoglycerides, sphingosine and ceramides, and utilization of the sphingolipid precursors in the formation of sphingomyelin and glycosphingolipids in Golgi was investigated. 2. In the presence of 5-10 micrograms/ml BFA, the incorporation of [3H]palmitate into glycerides, phosphoglycerides and sphingolipids decreased 45-60%, and the production of endoplasmic reticulum transport vesicles was reduced 30-50%. 3. In Golgi membranes, the presence of 5-10 micrograms/ml BFA in the mixture, assembled to generate Golgi vesicles, evoked inhibitory effect on the synthesis of sphingomyelin, glycosphingolipids and phosphatidylcholine. On average, the synthesis of the sphingolipids and phosphatidylcholine and production of Golgi transport vesicles declined to 30-40%. 4. Addition of 5-10 micrograms/ml BFA to the assay mixture prepared to measure the activity of cytidylyltransferase, phosphocholine diacylglyceroltransferase, and serine palmitoyltransferase, caused up to 50% inhibition of the enzymes involved in the synthesis of phosphatidylcholine and up to 70% inhibition of the enzyme generating 3-ketosphinganine. 5. The results suggest that BFA inhibits the synthesis of phosphoglycerides and sphingolipids. This, at first, is displayed in reduced production of endoplasmic reticulum and Golgi transport vesicles, while the depletion of sphingolipids abrogates the identity of Golgi membranes.

Acyltransferases↗
PubMed1988

Rapid turnover of sphingosine synthesized de novo from [14C]serine by Chinese hamster ovary cells.

It has been hypothesized that complex sphingolipids may serve as another "lipid second messenger" system via their hydrolysis to free sphingosine, which inhibits protein kinase C and affects multiple cellular functions. To investigate sphingolipid turnover, Chinese hamster ovary cells were pulse labelled with [14C]serine and the [14C]sphingosine in cellular sphingolipids was determined over time. Much of the radiolabelled sphingosine was initially seen in ceramides and was incorporated into sphingomyelin during the 5-hour chase. A major portion of the radiolabel that was initially seen in other sphingolipids disappeared over time. Overall, about half of the total long-chain bases made during this pulse were degraded within 2 to 5 h, depending on the method of analysis. Hence, a substantial portion of the sphingosine synthesized de novo by these cells is turned over fairly quickly. Since the doubling time of these cells is 12 h, this rapid turnover may reflect the remodelling of the cell surface, or the utilization of the free sphingosine derived from sphingolipid turnover, as part of the control of cell growth and division.

Cell Line↗
PubMedDate not supplied

Lipid membrane domains in cell surface and vacuolar systems.

Detergent insoluble sphingolipid-cholesterol enriched 'raft'-like membrane microdomains have been implicated in a variety of biological processes including sorting, trafficking, and signaling. Mutant cells and knockout animals of sphingolipid biosynthesis are clearly useful to understand the biological roles of lipid components in raft-like domains. It is suggested that raft-like domains distribute in internal vacuolar membranes as well as plasma membranes. In addition to sphingolipid-cholesterol-rich membrane domains, recent studies suggest the existence of another lipid-membrane domain in the endocytic pathway. This domain is enriched with a unique phospholipid, lysobisphosphatidic acid (LBPA) and localized in the internal membrane of multivesicular endosome. LBPA-rich membrane domains are involved in lipid and protein sorting within the endosomal system. Possible interaction between sphingolipids and LBPA in sphingolipid-storage disease is discussed.

Animals↗
PubMed2000

Role for de novo sphingoid base biosynthesis in the heat-induced transient cell cycle arrest of Saccharomyces cerevisiae.

The recent findings of sphingolipids as potential mediators of yeast heat stress responses led us to investigate their possible role in the heat-induced cell cycle arrest and subsequent recovery. The sphingolipid-deficient yeast strain 7R4 was found to lack the cell cycle arrest seen in the isogenic wild type. Furthermore, strain lcb1-100, which harbors a temperature-sensitive serine palmitoyltransferase, lacked increased de novo generated sphingoid bases upon heat stress. Importantly, this strain was found to lack the transient heat-induced G0/G1 arrest. These results indicate a role for sphingolipids and specifically those generated in the de novo pathway in the cell cycle arrest response to heat. To determine the bioactive sphingolipid regulating this response, an analysis of key mutants in the sphingolipid biosynthetic and degradation pathways was performed. Strains deleted in sphingoid base kinases, sphingoid phosphate phosphatase, lyase, or dihydrosphingosine hydroxylase were found to display the cell cycle arrest. Also, the knockout of a fatty acyl elongation enzyme, which severely attenuates ceramide production, displayed the arrest. These experiments suggested that the active species for cell cycle arrest were the sphingoid bases. In further support of these findings, exogenous phytosphingosine (10 microM) was found to induce transient arrest. Stearylamine did not induce an arrest, demonstrating chemical specificity, and L-erythro- was not as potent as D-erythro-dihydrosphingosine showing stereospecificity. To investigate a possible arrest mechanism, we studied the hyperstable Cln3 (Cln3-1) strain LDW6A that has been previously shown to be resistant to heat stress-induced cell cycle arrest. The strain containing Cln3-1 was found to be resistant to cell cycle arrest induced by exogenous phytosphingosine, indicating that Cln3 acts downstream of the sphingoid bases in this response. Interestingly, cell cycle recovery from the transient arrest was found to be dependent upon the sphingoid base kinases (LCB4, LCB5). Overall, this combination of genetic and pharmacologic results demonstrates a role for de novo sphingoid base biosynthesis by serine palmitoyltransferase in the transient G0/G1 arrest mediated through Cln3 via a novel mechanism.

Acyltransferases↗
PubMed1999

Microdomain-dependent regulation of Lck and Fyn protein-tyrosine kinases in T lymphocyte plasma membranes.

Src family protein-tyrosine kinases are implicated in signaling via glycosylphosphatidylinositol (GPI)-anchored receptors. Both kinds of molecules reside in opposite leaflets of the same sphingolipid-enriched microdomains in the lymphocyte plasma membrane without making direct contact. Under detergent-free conditions, we isolated a GPI-enriched plasma membrane fraction, also containing transmembrane proteins, selectively associated with sphingolipid microdomains. Nonionic detergents released the transmembrane proteins, yielding core sphingolipid microdomains, limited amounts of which could also be obtained by detergent-free subcellular fractionation. Protein-tyrosine kinase activity in membranes containing both GPI-anchored and transmembrane proteins was much lower than in core sphingolipid microdomains but was strongly reactivated by nonionic detergents. The inhibitory mechanism acting on Lck and Fyn kinases in these membranes was independent of the protein-tyrosine phosphatase CD45 and was characterized as a mixed, noncompetitive one. We propose that in lymphocyte plasma membranes, Lck and Fyn kinases exhibit optimal activity when juxtaposed to the GPI- and sphingolipid-enriched core microdomains but encounter inhibitory conditions in surrounding membrane areas that are rich in glycerophospholipids and contain additional transmembrane proteins.

Animals↗
PubMed1996

Development of a new method for the analysis of sphinganine and sphingosine in urine and tissues.

The fumonisins are inhibitors of de novo sphingolipid biosynthesis in vitro and in vivo and thus possibly interfere with the regulation of cell growth, differentiation, and neoplastic transformation. In addition, the ratio of free sphinganine (Sa) to free sphingosine (So) has been proposed as a marker of exposure for animals or humans consuming feed or food contaminated by these toxins. A method to analyze these sphingolipid bases has been proposed [Merrill et al., 1988: Anal Biochem 171:373-381; Riley et al., 1994a: JAOAC 77:533-540] but involves numerous steps and consequently is not ideally suited to the analysis of large numbers of samples, as is often required in epidemiological studies. A new method was therefore developed for the analysis of the Sa/So ratio in tissues as well as human and rat urine. Briefly, the method involves isolation of exfoliated cells from as little as 0.5 ml of rat urine or 2 ml of human urine followed by a rapid and efficient extraction of sphingolipid bases in ethyl acetate, an optimized derivatization step with o-phthaldialdehyde and a high-pressure liquid chromatography separation on a 250 mm x 4.6 mm. 5 microns Kromasil C18 column, with a 4-step phosphate buffer/methanol gradient. Fluorescence was monitored at 340 nm excitation, 455 nm emission, and retention times for So, Sa, and C-20 Sa were about 11, 14, and 22 min, respectively. The method was adapted to tissue analysis by partially digesting approximately 30 mg tissue with trypsin to permit isolation of a cell pellet before extraction of the sphingolipids as described above. The method was applied to the analysis of So and Sa in urines and tissues of fumonisin B1 (FB1) treated and untreated male BDIV rats. The Sa/So ratio in urine of untreated rats varied from 0.1 to 0.7, and for treated rats (between 1-5 mg FB1/kg body weight daily by gavage), the ratio was between 1.2-10. In kidney, the ratio was 0.1 in control rats and varied from 4 to 10.3 in treated rats. In human urine, measurements could easily be made in 2 ml of urine in females, but in males much larger volumes were required due to the low levels of sphingolipid bases.

Adolescent↗
PubMed1997

Ceramide signalling: regulatory role in cell proliferation, differentiation and apoptosis in human epidermis.

The stratum corneum of vertebrates is a major structural compartment that provides mechanical protection and prevents skin desiccation. The water barrier function of the stratum corneum was first reported in 1944, and this was shown later to be associated with multilayered lipid lamellae localized in the extracellular spaces. The major lipid components isolated from the cornified epidermal layers are ceramides, which belong to the class of sphingolipids, cholesterol and free fatty acids; their biosynthesis is in tight relationship with the cutaneous barrier function. In studies in which the barrier is artificially disturbed, lipid biosynthesis is found to be directly regulated by barrier permeability. As mentioned above, the ceramides involved in this process are located in the extracellular spaces of the upper epidermal layers, whereas sphingomyelin, the most common sphingolipid, is an integral part of the bilayer plasma membrane of the keratinocytes. During the last few years, however, increasing evidence has shown that sphingolipids may also take part in cell signalling, and the term 'sphingomyelin cycle' has been coined to describe this novel path-way of signal transduction. Intracellular messengers of the sphingomyelin cycle are ceramides as the products of an agonist-stimulated sphingomyelin hydrolysis. Increased levels of intracellular ceramides induce cell differentiation and/or apoptosis and reduce cell proliferation. In contrast to the extracellular barrier-forming ceramides which are complex partly O-acylated species containing long-chain fatty acids, intracellular signal-transducing ceramides are not O-acylated and have acyl chain lengths of 16 and 18 carbon atoms. We present here a review of our present knowledge on the sphingomyelin cycle as a possible signal transduction pathway in the human epidermis. We discuss the common origin of extracellular ceramides constituting the lipid barrier and of intracellular ceramides generated by agonist-stimulated sphingomyelin hydrolysis and serving as second messengers. A summary of alterations in sphingolipid metabolism and lipid composition of the epidermis in diseased skin is also given and the possible use of different sphingolipids for therapy is envisaged.

Apoptosis↗