Lipids in the American Alligator stratum corneum provide insights into the evolution of vertebrate skin.

In terrestrial vertebrates, the outermost layer of the skin, the stratum corneum (SC), provides a durable and flexible interface with the environment and is comprised of corneocytes embedded in lipids. However, the morphology and lipid composition of the SC varies throughout evolutionary history. Because crocodilians and birds phylogenetically bracket the Archosaurian clade, lipid composition in crocodilian SC may be compared with that of birds and other vertebrates to make inferences about broader phylogenetic patterns within Archosaurs while highlighting adaptations in vertebrate skin. We identified and quantified lipid classes in the SC of the American Alligator (Alligator mississippiensis) from three skin regions varying in mobility. Our results find similarities in lipid composition between alligator and avian SC, including a high percentage of cerebrosides, a polar lipid previously found only in the SC of birds and bats. Furthermore, polar lipids were more abundant in the most mobile region of the SC. Because polar lipids bind with water to increase skin hydration and therefore its pliability under physical stress, we hypothesize that selection for lipids in Archosaurian SC was driven by the unique distribution of proteins in the SC of this clade, and cerebrosides may have served as pre-adaptations for flight.

A Lipidomics Atlas of Selected Sphingolipids in Multiple Mouse Nervous System Regions.

Many lipids, including sphingolipids, are essential components of the nervous system. Sphingolipids play critical roles in maintaining the membrane structure and integrity and in cell signaling. We used a multi-dimensional mass spectrometry-based shotgun lipidomics platform to selectively analyze the lipid species profiles of ceramide, sphingomyelin, cerebroside, and sulfatide; these four classes of sphingolipids are found in the central nervous system (CNS) (the cerebrum, brain stem, and spinal cord) and peripheral nervous system (PNS) (the sciatic nerve) tissues of young adult wild-type mice. Our results revealed that the lipid species profiles of the four sphingolipid classes in the different nervous tissues were highly distinct. In addition, the mRNA expression of sphingolipid metabolism genes-including the ceramidase synthases that specifically acylate the N-acyl chain of ceramide species and sphingomyelinases that cleave sphingomyelins generating ceramides-were analyzed in the mouse cerebrum and spinal cord tissue in order to better understand the sphingolipid profile differences observed between these nervous tissues. We found that the distinct profiles of the determined sphingolipids were consistent with the high selectivity of ceramide synthases and provided a potential mechanism to explain region-specific CNS ceramide and sphingomyelin levels. In conclusion, we portray for the first time a lipidomics atlas of select sphingolipids in multiple nervous system regions and believe that this type of knowledge could be very useful for better understanding the role of this lipid category in the nervous system.

Lipid composition of the stratum corneum in different regions of the body of Kuhl's pipistrelle from the Negev Desert, Israel.

The most superficial epidermal layer in endotherms is the stratum corneum (SC), which is composed of dead corneocytes embedded in a lipid matrix with free fatty acids, cholesterol, ceramides, and cerebrosides; the lipid composition of the SC determines its permeability to water vapor. Lipids that are more polar, have longer hydrocarbon chains, and are less bulky are often packed in more ordered phase states to slow cutaneous evaporative water loss (CEWL); these lipids also resist transitions to more disordered phases at high ambient temperatures (Ta). In bats, wing and tail membranes (wing patagia and tail uropatagium, respectively) allow powered flight, but increase surface area, and hence CEWL, with implications for survival in arid environments. We captured Pipistrellus kuhlii from an arid habitat and measured the lipid composition of the SC of the plagiopatagium in the wing, the uropatagium, and the non-membranous region (NMR) of the body using thin layer chromatography and reversed phase high performance liquid chromatography coupled with atmospheric pressure photoionization mass spectrometry. The patagia contained more cholesterol and shorter-chained ceramides, and fewer cerebrosides than the NMR, indicating that the lipid phase transition temperature in the patagia is lower than in the NMR. Thus, at moderate Ta the lipids in the SC in all body regions will remain in an ordered phase state, allowing water conservation; but as Ta increases, the lipids in the SC of the patagia will more easily transition into a disordered phase, resulting in increased CEWL from the patagia facilitating efficient heat dissipation in hot environments.

Editorial to the Special Issue "Lipidomics and Neurodegenerative Diseases".

The contribution of dysregulation of lipid signaling and metabolism to neurodegenerative diseases including Alzheimer's and Parkinson's is the focus of this special issue. Here, the matter of three reviews and one research article is summarized.

New Cytotoxic Natural Products from the Red Sea Sponge Stylissa carteri.

Bioactivity-guided isolation supported by LC-HRESIMS metabolic profiling led to the isolation of two new compounds, a ceramide, stylissamide A (1), and a cerebroside, stylissoside A (2), from the methanol extract of the Red Sea sponge Stylissa carteri. Structure elucidation was achieved using spectroscopic techniques, including 1D and 2D NMR and HRMS. The bioactive extract's metabolomic profiling showed the existence of various secondary metabolites, mainly oleanane-type saponins, phenolic diterpenes, and lupane triterpenes. The in vitro cytotoxic activity of the isolated compounds was tested against two human cancer cell lines, MCF-7 and HepG2. Both compounds, 1 and 2, displayed strong cytotoxicity against the MCF-7 cell line, with IC50 values at 21.1 ± 0.17 µM and 27.5 ± 0.18 µM, respectively. They likewise showed a promising activity against HepG2 with IC50 at 36.8 ± 0.16 µM for 1 and IC50 30.5 ± 0.23 µM for 2 compared to the standard drug cisplatin. Molecular docking experiments showed that 1 and 2 displayed high affinity to the SET protein and to inhibitor 2 of protein phosphatase 2A (I2PP2A), which could be a possible mechanism for their cytotoxic activity. This paper spreads light on the role of these metabolites in holding fouling organisms away from the outer surface of the sponge, and the potential use of these defensive molecules in the production of novel anticancer agents.

Compound Porcine Cerebroside and Ganglioside Injection (CPCGI) Attenuates Sevoflurane-Induced Nerve Cell Injury by Regulating the Phosphorylation of p38 MAP Kinase (p38MAPK)/Nuclear Factor kappa B (NF-κB) Pathway.

BACKGROUND Compound porcine cerebroside and ganglioside injection (CPCGI) has been widely applied in clinical practice in China to treat functional confusion caused by brain diseases. Sevoflurane, a frequently-used inhalational anesthetic, was discovered to have neurotoxicity that can cause neurological damage in patients. The present study was performed to investigate the protective effect of CPCGI on sevoflurane-induced nerve damage and to reveal the neuroprotective mechanisms of CPCGI. MATERIAL AND METHODS Firstly, the hippocampal neurons were separated from Sprague-Dawley embryonic rats, and were stimulated by 3% sevoflurane for different times (0, 2, 4, and 6 h). Then, cell viability and cell apoptosis were assessed by thiazolyl blue tetrazolium bromide (MTT) and flow cytometry (FCM), respectively. Western blot analysis was used to determine the apoptosis-related protein expression levels. RESULTS The results demonstrated that 3% sevoflurane significantly inhibited cell viability but induced cell apoptosis in neurons in a time-dependent manner. Treatment with 3% sevoflurane also promoted the Bax (B cell leukemia/lymphoma 2​ (Bcl2)-associated X protein) and cleaved caspase3 protein expressions, and suppressed Bcl-2 and pro-caspase3 expressions in hippocampal neurons. In addition, phosphorylated (p)-p38 and p-p65 expression and the ratio of p-p38/p38 and p-p65/p65 were upregulated in a time-dependent manner after 3% sevoflurane treatment. Further analysis indicated that all the effects of 3% sevoflurane on hippocampal neurons were reversed by CPCGI pre-treatment. CONCLUSIONS We demonstrated the neuroprotective role of CPCGI in sevoflurane-stimulated neuronal cell damage via regulation of the MAPK/NF-kappaB signaling pathway.

Mixing brain cerebrosides with brain ceramides, cholesterol and phospholipids.

The properties of bilayers composed of pure brain cerebroside (bCrb) or of binary mixtures of bCrb with brain ceramide, cholesterol, egg phosphatidylcholine or brain sphingomyelin have been studied using a combination of physical techniques. Pure bCrb exhibits a rather narrow gel-fluid transition centred at ≈65 °C, with a half-width at half-height T1/2 ≈ 3 °C. bCrb mixes well with both fluid and gel phospholipids and ceramide, and it rigidifies bilayers of egg phosphatidylcholine or brain sphingomyelin when the latter are in the fluid state. Cholesterol markedly widens the bCrb gel-fluid transition, while decreasing the associated transition enthalpy, in the manner of cholesterol mixtures with saturated phosphatidylcholines, or sphingomyelins. Laurdan and DPH fluorescence indicate the formation of fluid ordered phases in the bCrb:cholesterol mixtures. Macroscopic phase separation of more and less fluid domains is observed in giant unilamellar vesicles consisting of bCrb:egg phosphatidylcholine or bCrb:sphingomyelin. Crb capacity to induce bilayer permeabilization or transbilayer (flip-flop) lipid motion is much lower than those of ceramides. The mixtures explored here contained mostly bCrb concentrations >50 mol%, mimicking the situation of cell membranes in Gaucher's disease, or of the Crb-enriched microdomains proposed to exist in healthy cell plasma membranes.

Engineered Pichia pastoris production of fusaruside, a selective immunomodulator.

BACKGROUD: Fusaruside is an immunomodulatory fungal sphingolipid which has medical potentials for treating colitis and liver injury, but its poor natural abundance limits its further study. RESULTS: In this study, we described a synthetic biology approach for fusaruside production by engineered Pichia pastoris that was based on polycistronic expression. Two fusaruside biosynthesis genes (Δ3(E)-sd and Δ10(E)-sd), were introduced into P. pastoris to obtain fusaruside producing strain FUS2. To further enhance the yield of fusaruside, three relevant biosynthetic genes (Δ3(E)-sd, Δ10(E)-sd and gcs) were subsequently introduced into P. pastoris to obtain FUS3. All of the biosynthetic genes were successfully co-expressed in FUS2 and FUS3. Compared to that produced by FUS2, fusaruside achieved from FUS3 were slightly increased. In addition, the culture conditions including pH, temperature and methanol concentration were optimized to improve the fusaruside production level. CONCLUSIONS: Here a novel P. pastoris fusaruside production system was developed by introducing the biosynthetic genes linked by 2A peptide gene sequences into a polycistronic expression construct, laying a foundation for further development and application of fusaruside.

Distinguishing the differences in ß-glycosylceramidase folds, dynamics, and actions informs therapeutic uses.

Glycosyl hydrolases (GHs) are carbohydrate-active enzymes that hydrolyze a specific ß-glycosidic bond in glycoconjugate substrates; ß-glucosidases degrade glucosylceramide, a ubiquitous glycosphingolipid. GHs are grouped into structurally similar families that themselves can be grouped into clans. GH1, GH5, and GH30 glycosidases belong to clan A hydrolases with a catalytic (ß/α)8 TIM barrel domain, whereas GH116 belongs to clan O with a catalytic (α/α)6 domain. In humans, GH abnormalities underlie metabolic diseases. The lysosomal enzyme glucocerebrosidase (family GH30), deficient in Gaucher disease and implicated in Parkinson disease etiology, and the cytosol-facing membrane-bound glucosylceramidase (family GH116) remove the terminal glucose from the ceramide lipid moiety. Here, we compare enzyme differences in fold, action, dynamics, and catalytic domain stabilization by binding site occupancy. We also explore other glycosidases with reported glycosylceramidase activity, including human cytosolic ß-glucosidase, intestinal lactase-phlorizin hydrolase, and lysosomal galactosylceramidase. Last, we describe the successful translation of research to practice: recombinant glycosidases and glucosylceramide metabolism modulators are approved drug products (enzyme replacement therapies). Activity-based probes now facilitate the diagnosis of enzyme deficiency and screening for compounds that interact with the catalytic pocket of glycosidases. Future research may deepen the understanding of the functional variety of these enzymes and their therapeutic potential.

Cerebrosides, extracellular glycolipids secreted by the selective lignin-degrading fungus Ceriporiopsis subvermispora.

Ceriporiopsis subvermispora is a selective white-rot fungus that degrades lignin at a site far from the hyphae and extracellular enzymes, without intensive damage to the cellulose. In selective ligninolysis, low molecular mass metabolites play a principal role and amphipathic substances are involved to control the degradation and transport of hydrophobic aromatic molecules, including lignin and lipids; however, secretion of the amphipathic substances by this fungus has not been well understood, except for alk(en)yl itaconates called ceriporic acids, which have a weak amphiphilicity. Herein, we report for the first time that the fungus secretes cerebrosides that are classified as glycosphingolipids. By using liquid chromatography electron spray ionization mass spectrometry (LC-ESI-MS) and nuclear magnetic resonance (NMR) spectroscopy coupled with stable isotope feeding experiments with 13C-glucose and 15N-ammonium sulfate, the cerebrosides were determined to be N-hydroxyoctadecanoyl-1-O-ß-d-glucopyranosyl-4E,8E-sphingadienine, N-hydroxyoctadecanoyl-1-O-ß-d-glucopyranosyl-4E,8Z-sphingadienine, and N-hydroxyoctadecanoyl-1-O-ß-d-glucopyranosyl-9-methyl-4E,8E-sphingadienine. The cerebrosides are strong amphipathic substances and potential metabolites for regulating difference and symbiosis within the microbial community.

Dietary Cerebroside from Sea Cucumber (Stichopus japonicus): Absorption and Effects on Skin Barrier and Cecal Short-Chain Fatty Acids.

Sphingolipids from marine sources have attracted more attention recently because of their distinctive structures and expected functions. In this study, the content and components of cerebroside from sea cucumber Stichopus japonicus were analyzed. The absorption of cerebroside from S. japonicus was investigated with an in vivo lipid absorption assay. The result revealed that S. japonicus is a rich source of cerebroside that contained considerable amounts of odd carbon chain sphingoid bases. The cumulative recoveries of d17:1- and d19:2-containing cerebrosides were 0.31 ± 0.16 and 0.32 ± 0.10%, respectively, for 24 h after administration. To the best of the authors' knowledge, this is the first work that shows sphingolipids from a marine source could be absorbed in vivo and incorporated into ceramides. In addition, dietary supplementation with sea cucumber cerebroside to hairless mouse improved the skin barrier function and increased short-chain fatty acids in cecal contents, which have shown beneficial effects on the host.

Transport and uptake effects of marine complex lipid liposomes in small intestinal epithelial cell models.

Nowadays, marine complex lipids, including starfish phospholipids (SFP) and cerebrosides (SFC) separated from Asterias amurensis as well as sea cucumber phospholipids (SCP) and cerebrosides (SCC) isolated from Cucumaria frondosa, have received much attention because of their potent biological activities. However, little information is known on the transport and uptake of these lipids in liposome forms in small intestinal cells. Therefore, this study was undertaken to investigate the effects of these complex lipid liposomes on transport and uptake in Caco-2 and M cell monolayer models. The results revealed that SFP and SCP contained 42% and 47.9% eicosapentaenoic acid (EPA), respectively. The average particle sizes of liposomes prepared in this study were from 169 to 189 nm. We found that the transport of the liposomes across the M cell monolayer model was much higher than the Caco-2 cell monolayer model. The liposomes consisting of SFP or SCP showed significantly higher transport and uptake than soy phospholipid (soy-PL) liposomes in both Caco-2 and M cell monolayer models. Our results also exhibited that treatment with 1 mM liposomes composed of SFP or SCP for 3 h tended to increase the EPA content in phospholipid fractions of both differentiated Caco-2 and M cells. Moreover, it was also found that the hybrid liposomes consisting of SFP/SFC/cholesterol (Chol) revealed higher transport and uptake across the M cell monolayer in comparison with other liposomes. Furthermore, treatment with SFP/SFC/Chol liposomes could notably decrease the trans-epithelial electrical resistance (TEER) values of Caco-2 and M cell monolayers. The present data also showed that the cell viability of differentiated Caco-2 and M cells was not affected after the treatment with marine complex lipids or soy-PL liposomes. Based on the data in this study, it was suggested that marine complex lipid liposomes exhibit prominent transport and uptake in small intestinal epithelial cell models.

Δ10(E)-Sphingolipid Desaturase Involved in Fusaruside Mycosynthesis and Stress Adaptation in Fusarium graminearum.

Sphingolipids are biologically important and structurally distinct cell membrane components. Fusaruside (1) is a 10,11-unsaturated immunosuppressive fungal sphingolipid with medical potentials for treating liver injury and colitis, but its poor natural abundance bottlenecks its druggability. Here, fusaruside is clarified biosynthetically, and its efficacy-related 10,11-double bond can be generated under the regioselective catalysis of an unprecedented Δ10(E)-sphingolipid desaturase (Δ10(E)-SD). Δ10(E)-SD shares 17.7% amino acid sequence similarity with a C9-unmethylated Δ10-sphingolipid desaturase derived from a marine diatom, and 55.7% with Δ8(E)-SD from Fusarium graminearum. Heterologous expression of Δ10(E)-SD in Pichia pastoris has been established to facilitate a reliable generation of 1 through the Δ10(E)-SD catalyzed desaturation of cerebroside B (2), an abundant fungal sphingolipid. Site directed mutageneses show that the conserved histidines of Δ10(E)-SD are essential for the 10,11-desaturation catalysis, which is also preconditioned by the C9-methylation of the substrate. Moreover, Δ10(E)-SD confers improved survival and faster growth to fungal strains at low temperature and high salinity, in parallel with to higher contents of 1 in the mycelia. Collectively, the investigation describes a new Δ10(E)-sphingolipid desaturase with its heterologous expression fundamentalizing a biotechnological supply of 1, and eases the follow-up clarification of the immunosuppression and stress-tolerance mechanism.

AAV-mediated gene delivery in a feline model of Sandhoff disease corrects lysosomal storage in the central nervous system.

Sandhoff disease (SD) is an autosomal recessive neurodegenerative disease caused by a mutation in the gene for the ß-subunit of ß-N-acetylhexosaminidase (Hex), resulting in the inability to catabolize ganglioside GM2 within the lysosomes. SD presents with an accumulation of GM2 and its asialo derivative GA2, primarily in the central nervous system. Myelin-enriched glycolipids, cerebrosides and sulfatides, are also decreased in SD corresponding with dysmyelination. At present, no treatment exists for SD. Previous studies have shown the therapeutic benefit of adeno-associated virus (AAV) vector-mediated gene therapy in the treatment of SD in murine and feline models. In this study, we treated presymptomatic SD cats with AAVrh8 vectors expressing feline Hex in the thalamus combined with intracerebroventricular (Thal/ICV) injections. Treated animals showed clearly improved neurologic function and quality of life, manifested in part by prevention or attenuation of whole-body tremors characteristic of untreated animals. Hex activity was significantly elevated, whereas storage of GM2 and GA2 was significantly decreased in tissue samples taken from the cortex, cerebellum, thalamus, and cervical spinal cord. Treatment also increased levels of myelin-enriched cerebrosides and sulfatides in the cortex and thalamus. This study demonstrates the therapeutic potential of AAV for feline SD and suggests a similar potential for human SD patients.

Description of the sphingolipid content and subspecies in the diabetic cornea.

PURPOSE: Diabetes mellitus (DM) is characterized by high blood sugar levels over a prolonged period. Long term complications include but not limited heart disease, stroke, kidney failure, and ocular damage. An estimated 382 million people are diagnosed with Type 2 DM accounting for 90% of the cases. Common corneal dysfunctions associated with DM result in impaired vision due to decreased wound healing, corneal edema, and altered epithelial basement membrane. Lipids play a fundamental role in tissue metabolism and disease states. We attempt to determine the role of sphingolipids (SPL) in human Type I and Type II diabetic corneas. MATERIALS AND METHODS: Cadaver corneas from healthy (non-diabetic/no ocular trauma), Type I (T1DM), and Type II diabetic (T2DM) donors were obtained and processed for lipidomics using LC-MS/MS. RESULTS: Our data show significant differences in the SPL composition between control, T1DM and T2DM corneas. Both T1DM and T2DM showed a 10-folddownregulation of sphingomyelin(SM), 5-fold up regulation of Ceramides (Cer) and 2-fold upregulation of monohexosylceramides (MHC). Differences were also seen in total amounts of SPL where Cer was increased by approximately 3 fold in both T1DM and T2DM where SM decreased by 50% in both T1DM and T2DM when compared to healthy controls. No differences were seen in MHC amounts. CONCLUSIONS: Overall, our data indicate major differences in SPL distribution in human diabetic corneas. Information on the sphingolipids role in cornea, corneal cell physiology, and diseases are very limitedwhich highlights the importance of these findings.

Spermatozoa as a transport system of large unilamellar lipid vesicles into the oocyte.

In addition to their role as man-made membranes, vesicles continue to be investigated as carriers for drug delivery. While most research focuses on their injectable properties, here a new delivery strategy is proposed. It is shown that spermatozoa can transport vesicles of variable composition. For human spermatozoa, the vesicles started to show binding after 20 mol% of the nonbinding vesicle backbone lipids were substituted with positive, negative, cerebroside or ganglioside lipids. Vesicle binding is a dynamic process with constant 'on' and 'off' binding. The physiological and motility attributes of the spermatozoa are not affected by the attached vesicles. Sperm swimming characteristics changed only marginally. Also, the activation status of the acrosomal membrane, tested with the fluorescent probe Pisum sativum agglutinin, was not affected by vesicle binding. Moreover, the hyaluronic acid-binding test showed that viable, fully developed spermatozoa will attach and remain bound to hyaluronic acid-coated slides regardless of vesicle binding. Therefore a new 'hybrid' delivery system was created with human spermatozoa, and tested with a mouse IVF system. Large unilamellar vesicles physisorbed to mouse spermatozoa can not only penetrate the mouse oocytes in these proof-of-principle experiments, but also deliver the cargo placed within the vesicles.

Eicosapentaenoic acid pre-treatment reduces biochemical changes induced in total brain and myelin of weanling Wistar rats by cuprizone feeding.

Recently, we investigated the effects of eicosapentaenoic acid (EPA), a fatty acid which modulates immune response and stimulates myelin gene expression, in an established model of multiple sclerosis (MS): the experimental autoimmune encephalomyelitis (EAE) induced in Dark Agouti rats. As scientific evidences and our previous studies have suggested that EPA could directly affect oligodendrocytes, we have now evaluated the effects of EPA in the non-immune mediate MS model characterized by selective oligodendrocytes damage induced by cuprizone (CPZ). We found that feeding weanling rats diets containing 0.6% CPZ for 2 weeks induced variation of whole brain and myelin biochemical composition representative of a severe myelin damage. We thus administered daily and by gavage EPA or PBS to 2-day old rats up to 21 days. Afterwards, rats were fed CPZ diet for 9 days. The results show that compared to PBS/CPZ fed rats, the whole brain cerebroside content in EPA pre-treated rats was statistically increased as well as there was an overall trend of increase of all other biochemical components.

Sulfocerebrosides upregulate liposome uptake in human astrocytes without inducing a proinflammatory response.

Astrocytes are involved in the pathogenesis of demyelinating diseases, where they actively regulate the secretion of proinflammatory factors, and trigger the recruitment of immune cells in the central nervous system (CNS). Antigen presentation of myelin-derived proteins has been shown to trigger astrocyte response, suggesting that astrocytes can directly sense demyelination. However, the direct response of astrocytes to lipid-debris generated during demyelination has not been investigated. The lipid composition of the myelin sheath is distinct, presenting significant amounts of cerebrosides, sulfocerebrosides (SCB), and ceramides. Studies have shown that microglia are activated in the presence of myelin-derived lipids, pointing to the possibility of lipid-induced astrocyte activation. In this study, a human astrocyte cell line was exposed to liposomes enriched in each myelin lipid component. Although liposome uptake was observed for all compositions, astrocytes had augmented uptake for liposomes containing sulfocerebroside (SCB). This enhanced uptake did not modify their expression of human leukocyte antigen (HLA) molecules or secretion of chemokines. This was in contrast to changes observed in astrocyte cells stimulated with IFNγ. Contrary to human monocytes, astrocytes did not internalize beads in the size-range of liposomes, indicating that liposome uptake is lipid specific. Epifluorescence microscopy corroborated that liposome uptake takes place through endocytosis. Soluble SCB were found to partially block uptake of liposomes containing this same lipid. Endocytosis was not decreased when cells were treated with cytochalasin D, but it was decreased by cold temperature incubation. The specific uptake of SCB in the absence of a proinflammatory response indicates that astrocytes may participate in the trafficking and regulation of sulfocerebroside metabolism and homeostasis in the CNS.

Invariant Natural Killer T-cell anergy to endogenous myelin acetyl-glycolipids in multiple sclerosis.

To extend our studies on glycolipid-reactive invariant Natural Killer T-cell (iNKT-cell) function in multiple sclerosis (MS), we investigated the stimulatory activities of two myelin-derived glycolipids that are poly-acetylated derivatives of ß-galactosylceramide designated as fast-migrating cerebrosides (FMC) by thin-layer chromatography. In healthy subjects, FMC stimulation of peripheral blood cells significantly expanded iNKT-cells similar to α-GalCer and induced significant increases in Th1, Th2 and Th17 cytokines. In marked contrast, MS patients failed to respond to FMCs or to α-GalCer stimulation indicating an anergic response. We propose that myelin-derived FMC glycolipids stimulate iNKT-cell responses in vivo and this is blocked in MS.

Regulation of integrin αV subunit expression by sulfatide in hepatocellular carcinoma cells.

Integrin is important in migration and metastasis of tumor cells. Changes of integrin expression and distribution will cause an alteration of cellular adhesion and migration behaviors. In this study, we investigated sulfatide regulation of the integrin αV subunit expression in hepatoma cells and observed that either exogenous or endogenous sulfatide elicited a robust upregulation of integrin αV subunit mRNA and protein expression in hepatoma cells. This regulatory effect occurred with a corresponding phosphorylation (T739) of the transcription factor Sp1. Based on the electrophoretic mobility shift assay, sulfatide enhanced the integrin αV promoter activity and strengthened the Sp1 complex super-shift. The results of chromatin immunoprecipitation analysis also indicated that sulfatide enhanced Sp1 binding to the integrin αV promoter in vivo. Silence of Sp1 diminished the stimulation of integrin αV expression by sulfatide. In the early stage of sulfatide stimulation, phosphorylation of Erk as well as c-Src was noted, and inhibition of Erk activation with either U0126 or PD98059 significantly suppressed Sp1 phosphorylation and integrin αV expression. We demonstrated that sulfatide regulated integrin αV expression and cell adhesion, which was associated with Erk activation.