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At least 19 recordsLinked to original sources
PubMed1991

Sphingolipids are required for mammalian epidermal barrier function. Inhibition of sphingolipid synthesis delays barrier recovery after acute perturbation.

Stratum corneum lipids comprise an approximately equimolar mixture of sphingolipids, cholesterol, and free fatty acids, arranged as intercellular membrane bilayers that are presumed to mediate the epidermal permeability barrier. Prior studies have shown that alterations in epidermal barrier function lead to a rapid increase in cholesterol and fatty acid synthesis which parallels the early stages of the repair process. Despite an abundance of indirect evidence for their role in the barrier, the importance of sphingolipids has yet to be demonstrated directly. Whereas sphingolipid synthesis also increases during barrier repair, this response is delayed in comparison to cholesterol and fatty acid synthesis (Holleran, W.M., et al. 1991. J. Lipid Res. 32:1151-1158). To further delineate the role of sphingolipids in barrier homeostasis, we assessed the impact of inhibition of sphingolipid synthesis on epidermal barrier recovery. A single topical application of beta-chloro-L-alanine (beta-CA), an irreversible inhibitor of serine-palmitoyl transferase (SPT), applied to acetone-treated skin of hairless mice resulted in: (a) greater than 75% inhibition of SPT activity at 30 min (P less than 0.001); (b) a global decrease in sphingolipid synthesis between 1 and 3 h (P less than 0.02); (c) reduction of epidermal sphingolipid content at 18 h (P less than 0.01); (d) delayed reaccumulation of histochemical staining for sphingolipids in the stratum corneum; and (e) reduced numbers and contents of lamellar bodies in the stratum granulosum. Finally, despite its immediate, marked diminution of sphingolipid synthesis, beta-CA slowed barrier recovery only at late time points (greater than 6 h) after acetone treatment. This inhibition was overridden by coapplications of ceramides (the distal SPT product), indicating that the delay in repair was not due to non-specific toxicity. These studies demonstrate a distinctive role for epidermal sphingolipids in permeability barrier homeostasis.

Acyltransferases↗
PubMed1992

Sphingolipids are essential for the growth of Chinese hamster ovary cells. Restoration of the growth of a mutant defective in sphingoid base biosynthesis by exogenous sphingolipids.

We previously isolated a temperature-sensitive Chinese hamster ovary cell mutant (strain SPB-1) with thermolabile serine palmitoyltransferase, which is involved in the first step of sphingolipid synthesis (Hanada, K., Nishijima, M., and Akamatsu, Y. (1990) J. Biol. Chem. 265, 22137-22142). In this study, sphingolipid-deficient culture medium was used to examine the effect of exogenous sphingolipids on the cell growth of SPB-1. When cultivated in the sphingolipid-deficient medium, SPB-1 cells ceased growing at non-permissive temperatures. Under these conditions, de novo sphingolipid synthesis ceased in the SPB-1 cells, resulting in a decrease in levels of sphingomyelin and ganglioside sialyl lactosylceramide (GM3), whereas the parental CHO-K1 cells grew logarithmically with normal sphingolipid synthesis. Exogenous sphingosine restored the contents of both sphingomyelin and GM3 in the SPB-1 cells near to the parental levels through metabolic utilization and allowed the mutant cells to grow even at the non-permissive temperature. Similarly, exogenous sphingomyelin restored the sphingomyelin levels and only partly the GM3 levels and also suppressed the temperature-sensitivity of the SPB-1 cell growth. In contrast, exogenous glucosylceramide, which restored the GM3 levels but not the sphingomyelin levels, failed to suppress the temperature sensitivity of the SPB-1 cell growth. Combination of exogenous sphingomyelin with ceramide, glucosylceramide, GM3, or sphingoid bases did not show any synergistic or additive effect on the SPB-1 cell growth enhancement, compared with sphingomyelin alone. The results indicated that the temperature sensitivity of the SPB-1 cell growth was due to the lack of cellular sphingolipids, possibly that of sphingomyelin.

Acyltransferases↗
PubMed1992

Use of N-([1-14C]hexanoyl)-D-erythro-sphingolipids to assay sphingolipid metabolism.

An advantage of using N-([1-14C]hexanoyl)sphingolipids to assay sphingolipid metabolism is their ability to rapidly and spontaneously transfer into biological membranes without destroying membrane integrity. This property allows analysis of the activity of enzymes of sphingolipid metabolism under conditions in which the rate of product formation is not limited by availability of substrate, as is often the case with naturally occurring lipids whose rates of spontaneous transfer are extremely slow. Thus, the use of N-([1-14C]hexanoyl)sphingolipids provides an alternative means for studying sphingolipid metabolism in vitro.

Animals↗
PubMed1975

Further study on cerebral sphingolipids including gangliosides in two cases of juvenile amaurotic family idiocy (Spielmeyer-Vogt type) using a new analytical procedure of sphingolipids.

Sphingolipids isolated from cerebral grey and white matter of two patients with Juvenile Amaurotic Idiocy (Spielmeyer-Vogt Type) were studied. A new analytical procedure was attempted for the determination of sphingolipids, i.e., cerebroside, sulfatide, sphingomyelin and gangliosides were subjected to ozonolysis and reduced with NaBH4. Fatty alcohols thus derived from the double bond-containing long chain bases of the sphingolipids were analyzed by GLC as their TMS-derivatives using an internal standard. The new procedure was suitable for the analysis of small amounts of sphingolipids and could determine the amounts of C18 and C20 sphingosines. It was found that all individual gangliosides in both cases gave lower proportions of C20 sphingosine to the total long chain bases. Sphingomyelin in normal human grey matter contained a small but significant amount of C20 sphingosine, while the sphingomyelin of the two patient brains indicated a much lower proportion of C20 sphingosine in comparison with those of age-matched controls. Thus, this disease seemed to be related to a genetical defect in the metabolic regulation of the long chain bases of gangliosides and grey matter sphingomyelin. On the other hand, it was noted that the ganglioside pattern of grey matter in case-1 was entirely different from that of case-2. The grey matter gangliosides in case-1 were composed of 1.78% of GM2, 81.19% of GM1 and 17.02% of GD1b by the amounts of long chain bases, while the grey matter gangliosides in case-2 seemed to be similar to those of normal human brains. Also, unusual fatty acid compositions of galactosphingolipids (cerebrosides and sulfatides) were observed to a somewhat extent in the grey matter of case-1.

Adult↗
PubMed1989

Functions of sphingolipids and sphingolipid breakdown products in cellular regulation.

The discovery that breakdown products of cellular sphingolipids are biologically active has generated interest in the role of these molecules in cell physiology and pathology. Sphingolipid breakdown products, sphingosine and lysosphingolipids, inhibit protein kinase C, a pivotal enzyme in cell regulation and signal transduction. Sphingolipids and lysosphingolipids affect significant cellular responses and exhibit antitumor promoter activities in various mammalian cells. These molecules may function as endogenous modulators of cell function and possibly as second messengers.

Animals↗
PubMed1989

Human sphingolipid activator protein-1 and sphingolipid activator protein-2 are encoded by the same gene.

Mixed oligonucleotide primers complementary to the translation product of the sphingolipid activator protein (SAP)-2 were used to generate a 144-base pair (bp) complementary DNA (cDNA). This cDNA probe was used to isolate a 2,649-nucleotide-long cDNA that was sequenced and found to contain coding sequences for two known activators of lysosomal enzymes, namely, the sphingolipid activator protein (SAP)-1 and SAP-2. The cDNA contains an open reading frame of 1,482 nucleotides and 1,167 nucleotides of 3'-nontranslated region, followed by a stretch of 24 residues of adenylic acid. At 20 nucleotides upstream from the poly(A) tail there is a consensus AATAAA polyadenylation signal that is preceded by another potential polyadenylation signal. The cDNA, designed SAP-1/SAP-2 cDNA, hybridizes with two human mRNA species of approximately 3 kb in length, which most probably arise from polyadenylation at different sites. There are higher amounts of steady-state RNA levels of the SAP-1/SAP-2 mRNA in skin fibroblasts in comparison to B cells. The steady-state SAP-1/SAP-2 mRNA levels in Gaucher B cells are higher than in their normal counterparts. There is one human SAP-1/SAP-2 gene that has been cloned and is localized on two approximately 5 kb BamHI fragments.

Amino Acid Sequence↗
PubMed1992

Phenotypes of sphingolipid-dependent strains of Saccharomyces cerevisiae.

To study sphingolipid function(s) in Saccharomyces cerevisiae, we have investigated the effects of environmental stress on mutant (SLC) strains (R. C. Dickson, G. B. Wells, A. Schmidt, and R. L. Lester, Mol. Cell. Biol. 10:2176-2181, 1990) that either contain or lack sphingolipids, depending on whether they are cultured with a sphingolipid long-chain base. Strains lacking sphingolipid were unable to grow at low pH, at 37 degrees C, or with high salt concentrations in the medium; these environmental stresses are known to inhibit the growth of some S. cerevisiae strains with a defective plasma membrane H(+)-ATPase. We found that sphingolipids were essential for proton extrusion at low pH and furthermore found that cells lacking sphingolipid no longer exhibited net proton extrusion at normal pH after a 1-min exposure to pH 3. Cells lacking sphingolipid appeared to rapidly become almost completely permeable to protons at low pH. The deleterious effects of low pH could be partially prevented by 1 M sorbitol in the suspension of cells lacking sphingolipid. Proton extrusion at normal pH (pH 6) was significantly inhibited at 39 degrees C only in cells lacking sphingolipid. Thus, the product of an SLC suppressor gene permits life without sphingolipids only in a limited range of environments. Outside this range, sphingolipids appear to be essential for maintaining proton permeability barriers and/or for proton extrusion.

Fungal Proteins↗
PubMed1990

Isolation of mutant Saccharomyces cerevisiae strains that survive without sphingolipids.

Sphingolipids comprise a large, widespread family of complex eucaryotic-membrane constituents of poorly defined function. The yeast Saccharomyces cerevisiae is particularly suited for studies of sphingolipid function because it contains a small number of sphingolipids and is amenable to molecular genetic analysis. Moreover, it is the only eucaryote in which mutants blocked in sphingolipid biosynthesis have been isolated. Beginning with a nonreverting sphingolipid-defective strain that requires the addition of the long-chain-base component of sphingolipids to the culture medium for growth, we isolated two strains carrying secondary, suppressor mutations that permit survival in the absence of exogenous long-chain base. Remarkably, the suppressor strains made little if any sphingolipid. A study of how the suppressor gene products compensate for the lack of sphingolipids may reveal the function(s) of these membrane lipids in yeast cells.

Blotting, Southern↗
PubMed1991

Regulation of epidermal sphingolipid synthesis by permeability barrier function.

A mixture of sphingolipids, cholesterol, and free fatty acids forms the intercellular membrane bilayers of the stratum corneum which are presumed to regulate epidermal barrier function. Prior studies have shown that both cholesterol and fatty acid synthesis are rapidly regulated by epidermal barrier requirements. In contrast, the importance of sphingolipids in barrier function has not been directly demonstrated. Here, we have assessed both sphingolipid synthesis by [3H]H2O incorporation and serine palmitoyl transferase (SPT) activity in relation to modulations in barrier function. Incorporation of [3H]H2O into sphingolipids increased after barrier disruption with acetone, with maximal increase (170%) occurring 5-7 h after treatment (P less than 0.005). As barrier function returned to normal over 24 h, incorporation of tritium into sphingolipids normalized. SPT activity also increased after barrier disruption, peaking at 6 h (150%) (P less than 0.05), and returning towards normal by 24 h. Artificial restoration of the barrier with a water vapor-impermeable membrane prevented the increases in both [3H]H2O incorporation into sphingolipids and enzyme activity. Finally, SPT activity was increased in two other models of barrier dysfunction, cellophane tape-stripping and essential fatty acid deficiency. Occlusion normalized SPT activity in both of these models as well. These studies: a) demonstrate a distinctive, delayed increase in epidermal sphingolipid synthesis in response to barrier requirements that contrasts with the immediate responses of cholesterol and fatty acid synthesis; and b) suggest that sphingolipids are important for the maintenance of the epidermal permeability barrier.

Acetone↗
PubMed1992

Stratum corneum sphingolipids and free amino acids in experimentally-induced scaly skin.

Stratum corneum sphingolipids are of particular importance in maintaining the water permeability barrier of mammalian epidermis. Free amino acids also play an important role in water retention in the stratum corneum. To clarify the way in which these substances affect scaly skin, stratum corneum sphingolipids and free amino acids collected from artificially-induced scaly skin were analysed. Scaly skin was induced by tape stripping. The total amount of sphingolipids was quantified by gas chromatography and five of sphingolipid fractions were isolated and quantified by thin-layer chromatography. Free amino acids were analysed using a high-speed amino analyser. The total amount of sphingolipid in scaly skin did not differ statistically from that in control skin. However, a significant change in the distribution of the five sphingolipid species was observed in scaly skin and the total amount of amino acids was decreased in scaly skin. These results suggest that the distribution of these five types of sphingolipid and the total amount of amino acids are responsible for scaly skin.

Adult↗
PubMed1991

The phosphoinositol sphingolipids of Saccharomyces cerevisiae are highly localized in the plasma membrane.

To investigate the vital function(s) of the phosphoinositol-containing sphingolipids of Saccharomyces cerevisiae, we measured their intracellular distribution and found these lipids to be highly localized in the plasma membrane. Sphingolipids were assayed in organelles which had been uniformly labeled with [3H]inositol or 32P and by chemical measurements of alkali-stable lipid P, of long chain bases, and of very long chain fatty acids. We have developed an improved method for the preparation of plasma membranes which is based on the procedure of Duran et al. (Proc. Natl. Acad. Sci. USA 72:3952-3955, 1975). On the basis of marker enzyme and DNA assays carried out with a number of preparations, the plasma membranes contained less than 10% vacuolar membranes (alpha-mannosidase) and nuclei (DNA); the contamination by the endoplasmic reticulum (NADPH-cytochrome c reductase) varied from 0 to 20%. The plasma membrane preparations showed a 13-fold increase in the specific activity of vanadate-sensitive ATPase, compared with that in the homogenate, with a yield ranging from 50 to 80%. A comparison of the distribution of the ATPase with that of sphingolipids assayed by a variety of methods showed that 80 to 100% of the sphingolipids are localized in the plasma membrane; the sphingolipids constitute about 30% of the total phospholipid content of the plasma membrane. Minor amounts of sphingolipids that were found in isolated mitochondria and nuclei can be attributed to the presence of small amounts of plasma membrane in these fractions. These results suggest that one or more essential functions of these lipids is in the plasma membrane. Furthermore, sphingolipids may be useful chemical markers of the plasma membrane of S. cerevisiae.

Adenosine Triphosphatases↗
PubMed1992

Increases in serum sphingosine and sphinganine and decreases in complex sphingolipids in ponies given feed containing fumonisins, mycotoxins produced by Fusarium moniliforme.

Consumption of food contaminated with Fusarium moniliforme causes leucoencephalomalacia and hepatotoxicity in horses, pulmonary edema in pigs and liver cancer in rats, and has been correlated with esophageal cancer in humans. The causative agents are thought to be a family of compounds called fumonisins, which have recently been shown to be potent inhibitors of sphingosine (sphinganine) N-acyltransferase. Because inhibition at this step blocks the formation of complex sphingolipids while leading to accumulation of sphinganine, we hypothesized that exposure of animals to fumonisin-contaminated feed might be detected by analyses of serum sphingolipids. Within days of giving ponies feed contaminated with 15 to 44 micrograms/g fumonisin B1, there was an increase in the amount of free sphinganine (and sometimes sphingosine) and a reduction in complex sphingolipids. Free sphinganine and sphingosine decreased when ponies consumed less of the contaminated feed, and increased again when they consumed more fumonisin. When toxicosis was evident as indicated by other serum markers, complex sphingolipids as well as free sphingosine and sphinganine were elevated, probably due to loss of sphingolipids from dying cells. These findings establish that consumption of fumonisin-contaminated feed disrupts sphingolipid metabolism. Because the changes in sphinganine and sphingosine were seen before liver enzymes were noticeably elevated, they may be an early marker of exposure to fumonisins.

Animal Feed↗
PubMed1992

Fumonisin inhibition of de novo sphingolipid biosynthesis and cytotoxicity are correlated in LLC-PK1 cells.

Fumonisins are a group of structurally related compounds produced by Fusarium moniliforme. Recently, it has been shown that fumonisins B1 and B2 are the first naturally occurring inhibitors of sphingosine and sphinganine N-acyltransferase (ceramide synthase) in rat primary hepatocytes (Wang et al. J. Biol. Chem. 266, 14, 486-14, 490, 1991). These enzymes are key components in the pathways for de novo sphingolipid biosynthesis and sphingolipid turnover. The results of the present study show that fumonisins B1 and B2 inhibit proliferation and are cytotoxic to LLC-PK1 cells. Concentrations of fumonisin B1 and B2 between 10 and 35 microM inhibited cell proliferation, whereas higher concentrations (greater than 35 microM) killed cells. Inhibition of cell proliferation and cell death were preceded by a lag period of at least 24 hr during which cells appeared to be functioning normally. Cells exposed to fumonisin B1 exhibited normal growth kinetics and morphology soon after fumonisin B1 was removed; thus, the effects of fumonisin B1 were reversible. The EC50 for alterations in sphingolipid biosynthesis was 10 to 15 microM. Inhibition of de novo sphingolipid biosynthesis occurred before inhibition of cell proliferation or cytotoxicity, and the dose response for the decrease in the [3H]sphingosine to [3H]sphinganine ratio at 7 hr closely paralleled the dose response for effects on proliferation and cytotoxicity at 3-5 days. In addition, the level of free sphinganine, and to a lesser extent sphingosine, increased in fumonisin-treated cells in a dose-dependent manner. During the 24-hr lag period preceding inhibition of cell proliferation, the free sphinganine content increased by 12,800% in cells exposed to 35 microM fumonisin B1. Whereas a mechanistic relationship between the inhibition of de novo sphingolipid biosynthesis and inhibition of proliferation and cell death has not been demonstrated, the results of this study support the hypothesis that inhibition of de novo sphingolipid biosynthesis is an early event in the toxicity of fumonisins to LLC-PK1 cells.

Acyltransferases↗
PubMed1992

Effects of a sphingolipid synthesis inhibitor on membrane transport through the secretory pathway.

We investigated the effects of an inhibitor of sphingolipid biosynthesis, 1-phenyl-2-(decanoyl-amino)-3-morpholino-1-propanol (PDMP), on cells in culture. Two Golgi-associated enzymes were affected by incubation of cells with PDMP. The synthesis of glucosylceramide was inhibited at low concentrations of PDMP (2.5-10 microM), and in the presence of higher concentrations (greater than or equal to 25 microM), synthesis of sphingomyelin was also reduced. Transport of vesicular stomatitis virus G protein through the Golgi complex was progressively retarded by increasing concentrations of PDMP. In the presence of 75 microM PDMP, the half-times of VSV-G protein arrival at the cis, medial, and trans Golgi and the cell surface were increased 1.5-, 2.1-, 2.4-, and 2.8-fold, respectively, compared to control values. Transport of fluorescent sphingolipids, synthesized de novo at the Golgi complex from fluorescent ceramide precursors, to the cell surface was retarded by approximately 20% in the presence of 50 microM PDMP and by approximately 50% in the presence of 100 microM PDMP. Control experiments demonstrated that PDMP had minimal effects on cell morphology and physiology (including microtubule and endoplasmic reticulum structure, mitochondrial function, and endocytosis). Although incubation of cells with relatively high concentrations of PDMP was required to see the effects on protein and sphingolipid transport, use of a fluorescent analogue of PDMP demonstrated that most cell-associated PDMP was sequestered in lysosomes, while the concentration at the Golgi complex, the site of the target synthetic enzymes, was relatively low. Taken together, these results suggest that transport of proteins and sphingolipids through the secretory pathway may be coupled to sphingolipid synthesis.

Animals↗
PubMed1991

Mannose 6-phosphate-independent membrane association of cathepsin D, glucocerebrosidase, and sphingolipid-activating protein in HepG2 cells.

The membrane association of the lysosomal enzymes cathepsin D and glucocerebrosidase and its naturally occurring sphingolipid activating protein was studied in HepG2 cells. We differentially permeabilized cells with low concentrations of saponin, at which secretory proteins rinsed out completely, whereas integral membrane proteins were not released. All relevant intracellular compartments were shown to be permeabilized by saponin. Metabolic labeling showed that early precursors of cathepsin D, sphingolipid activating protein, and glucocerebrosidase were completely released from the cells, whereas more than 80% of the high molecular mass intermediates were retained by the cells. Treatment of permeabilized cells with 10 mM mannose 6-phosphate released only 50% of the cell-associated cathepsin D. Glucocerebrosidase remained membrane-associated, but cathepsin D and sphingolipid activating protein were released from the cells after proteolytic processing. Sphingolipid activating proteins and cathepsin D behaved similarly during biosynthesis and showed similar sensitivity to mannose 6-phosphate. The membrane association of the intermediate form of cathepsin D was independent of the presence of N-linked oligosaccharides. Subcellular fractionation on sucrose gradients showed that the lysosomal proteins became membrane-associated probably in the Golgi complex, and that both mannose 6-phosphate-dependent and mannose 6-phosphate-independent membrane association occur in the same compartments. We conclude that, in HepG2 cells, cathepsin D, sphingolipid activating protein, and glucocerebrosidase exhibit MPR-independent membrane association which is acquired in the same compartments beyond the rough endoplasmic reticulum.

Carcinoma, Hepatocellular↗
PubMed1990

A temperature-sensitive mammalian cell mutant with thermolabile serine palmitoyltransferase for the sphingolipid biosynthesis.

We devised an in situ assay method for the activity of serine palmitoyltransferase (SPT) that catalyzes the first step in sphingolipid biosynthesis and isolated a temperature-sensitive mutant of Chinese hamster ovary cells with thermolabile SPT. This mutant stopped growing at 40 degrees C after several generations, although the cells grew at 33 and 37 degrees C at rates similar to those of the parent. The SPT activity in cell homogenates of the mutant grown at low temperatures was 4-8% of that in the parent homogenates. When the cells were cultured for several generations at 40 degrees C, the activity in the mutant homogenate became negligible. When cell homogenates were incubated at 45 degrees C before enzyme assay, mutant SPT was more markedly inactivated than parental SPT, indicating that mutant SPT had become thermolabile. The rates of de novo synthesis of sphingolipids in the mutant were much slower at 40 degrees C than at lower temperatures, in contrast to those in the parent. The sphingomyelin content in the mutant cultivated at 40 degrees C for several generations was also less than that at low temperatures. These results indicate that SPT functions in the main pathway for sphingolipid biosynthesis. The temperature-sensitive growth of the mutant defective in sphingolipid synthesis suggests that sphingolipid(s) plays an essential role in cell growth.

Acyltransferases↗
PubMed1985

Characterization of neutral sphingolipids from chicken erythrocytes.

The neutral sphingolipids from chicken erythrocytes were characterized. The total concentration of neutral sphingolipids was found to be 480 nmol/g of dry stroma. They were isolated and purified by droplet counter-current chromatography, Iatrobeads column chromatography, and preparative thin-layer chromatography. The major neutral sphingolipids were free ceramide, ceramide monohexoside, ceramide dihexoside, and ceramide pentahexoside, which represented 43%, 23.5%, 10.0%, and 3.6% of the long chain bases, respectively. Thus, free ceramide was the most abundant neutral sphingolipid in chicken erythrocytes. Ceramide monohexoside was composed of more galactosylceramide than glucosylceramide. Galabiosylceramide was found in the ceramide dihexoside fraction together with lactosylceramide. Ceramide pentahexoside was a Forssman glycolipid. There were two groups of neutral sphingolipids; one had mainly C16 fatty acid and the other had C22 and C24 fatty acids. In both groups sphingosine (d18:1) was predominant as a long chain base. 2-Hydroxy-C16 fatty acid was a major component of one of the ceramide monohexosides.

Animals↗
PubMed1992

Activator proteins and topology of lysosomal sphingolipid catabolism.

The lysosomal degradation of several sphingolipids by acid hydrolases is dependent on small non-enzymic cofactors, called sphingolipid activator proteins some of which have been identified as sphingolipid binding proteins. This review summarizes the information available on the structure, function, biosynthesis, gene organization and pathobiochemistry of the known sphingolipid activator proteins. It also offers models for their mode of action and for the topology of lysosomal digestion of glycolipids.

Animals↗