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NKT cells, a unique type of regulatory T cells, respond to structurally diverse glycolipids presented by CD1d. Although it was previously thought that recognition of glycolipids such as α-galactosylceramide (α-GalCer) by the NKT cell TCR (NKTCR) obeys a key–lock principle, it is now clear this interaction is much more flexible. In this article, we report the structure–function analysis of a series of novel 6′′-OH analogs of α-GalCer with more potent antitumor characteristics. Surprisingly, one of the novel carbamate analogs, α-GalCer-6′′-(pyridin-4-yl)carbamate, formed novel interactions with the NKTCR. This interaction was associated with an extremely high level of Th1 polarization and superior antitumor responses. These data highlight the in vivo relevance of adding aromatic moieties to the 6′′-OH position of the sugar and additionally show that judiciously chosen linkers are a promising strategy to generate strong Th1-polarizing glycolipids through increased binding either to CD1d or to NKTCR.

A sticky situation: Domain-dependent recognition of the glycosphingolipid galactosylceramide by norovirus-like particles (see picture; red/yellow) is shown using supported lipid bilayers (purple) as model membranes. Optimal ligand presentation is found to promote strong binding to GalCer. This presentation can be found at the edges of the glycosphingolipid-enriched domains (green) and binding is repressed in the absence of these domains. Copyright © 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim.

Els glicolípids i particularment els glicoesfingolípids són compostos d’interès perquè poden interaccionar amb biofactors tot inhibint o interferint en processos fisiològics de les cèl•lules. Per exemple, els glicoesfingolípids que recobreixen les membranes cel•lulars poden interaccionar en processos de reconeixement amb bactèries, virus i toxines com per exemple la toxina del Còlera la qual inicia el procés d’infecció pel reconeixement de glicolípids com el GM1. Tot i que l’ús d’antibiòtics és el tractament més emprat, la resistència als antibiòtics a zones endèmiques fa necessària la recerca en síntesi d’inhibidors basats en derivats de carbohidrats. Donat que la síntesi de compostos glicoconjugats que presenten multivalència ha resultat competitiva en la preparació d’inhibidors contra patògens en aquest treball s’ha estudiat la síntesi de nous mimètics basats en -galactosilceramides; l’acoblament Sukuki-Miyaura en 2-iodoglicals per obtenir nous precursors de carbohidrats i l’anclat de -galactosilceramides en suports com polímers hiperramificats per tal d’avaluar la seva inhibició front la toxina del Còlera., Los glicolípidos y particularmente los glicoesfingolípidos son compuestos de interés porque pueden interaccionar con biofactores inhibiendo o interfiriendo en procesos fisiológicos de las células. Por ejemplo, los glicoesfingolípidos que recubren las membranas celulares pueden interaccionar en procesos de reconocimiento con bacterias, virus y toxinas como por ejemplo la toxina del Cólera la cual inicia el proceso de infección a través del reconocimiento de glicolípidos como el GM1. Aunque el uso de antibióticos es el tratamiento más empleado, la resistencia a los antibióticos en zonas endémicas hace necesaria la investigación en síntesis de inhibidores basados en derivados de carbohidratos. Dado que la síntesis de compuestos glicoconjugados que presentan multivalencia ha resultado competitiva en la preparación de inhibidores contra patógenos, en este trabajo se ha estudiado la síntesis de nuevos miméticos basados en -galactosilceramidas; el acoplamiento Sukuki-Miyaura en 2-yodoglicales para obtener nuevos precursores de carbohidratos y el anclado de -galactosilceramidas en suportes como polímeros hiperramificados con el fin de avaluar su inhibición frente la toxina del Cólera., Glycolipids such as glycosphingolipids are interesting compounds because they can interact with biofactors by inhibiting or interfering in physiological processes on cells. For instance, the glycolipids which present on cellular membranes can interact with bacteria, virus and toxins. In deed, Cholera toxin starts its infective process once it has recognized glycolipids such as GM1. Although the use of antibiotics is the commonest treatment against this disease, the antibiotic resistance in endemic areas makes the investigation in the synthesis of inhibitors based on carbohydrate derivatives necessary. Due to the synthesis of multivalent glycoconjugated compounds have been competitive in order to prepare inhibitors against these pathogens, in the present work we have studied: the synthesis of new mimetics based on -galactosylceramides; the Suzuki-Miyaura cross coupling in 2-iodoglycals in order to obtain new carbohydrate precursors and the anchoring of -galactosylceramides in scaffolds such as hyperbranched polymers in order to evaluate their inhibition binding against to Cholera toxin

The binding of a galactosylceramide analog to proteins in normal human cerebrospinal fluid was examined by photoaffinity labeling using a radioiodulabeled 2-（p-azidosalicylamido） ethyl-1, 3-dithiopropionate derivative of galactosylsphingosine as a probe. The affinity-bound peptides appeared at 66, 36 and 28 kDa as radiolabeled bands. The latter two peptides, 36 and 28 kDa, were independently identified by immunostaining of the isolated peptides using biotinylated galactosylsphingosine, the chemical structure of which was confirmed by nuclear magnetic resonance and mass- spectrometry, and immobilized Avidin, and by immunoprecipitation of the photolabeled peptide, to apolipoproteins E and A-I, respectively. The direct binding of the galactosylceramide analog to these apopeptides suggested that the existence in the body fluids or the transfer between the fluids and cells of the glycolipid was related to high density lipoprotein constructions.

Inspired by the anti-human immunodeficiency virus (HIV) activity of analogues of beta-galactosylceramide (GalCer), a set of mono- and di-saccharide fatty acid esters were designed as GalCer mimetics and their binding to the V3 loop peptide of HIV-1 and anti-HIV activity evaluated. 1,1-linked Gal-Man and Glu-Man disaccharides with an ester on the Man subunit bound the V3 loop peptide and inhibited HIV infectivity in single round infection assays with the TZM-bl cell line. IC(50)'s were in the 50 mu M range with no toxicity to the cells at concentrations up to 200 mu M. These compounds appear to inhibit virus entry at early steps in viral infection since they were inactive if added post viral entry. Although these compounds were found to bind to the V3 loop peptide of gp120, it is not clear that this interaction is responsible for their anti-HIV activity because the relative binding affinity of closely related analogues did not correlate with their antiviral behavior. The low cytotoxicity of these 1,1-linked disaccharide fatty acid esters, combined with the easy accessibility to structurally diverse analogues make these molecules attractive leads for new topical anti-viral agents. (C) 2011 Elsevier Ltd. All rights reserved.

alpha-Galactosyl ceramide (alpha-GalCer, also known as KRN 7000) is known as the prototypical antigen for invariant natural killer T (NKT) Celts. Stimulation of NKT Celts by Old-mediated alpha-GalCer presentation leads to rapid release of Th1 and Th2 cytokines. Since Th1 and Th2 cytokines antagonize each other's effects, alpha-GalCer analogues that induce a biased Th1/Th2 response are highly awaited. With the exception of a C-glycoside (alpha-C-GalCer), most of the known alpha-GalCer analogues able to induce polarized cytokine responses are characterized by modifications of the phytosphingosine or fatty acyl chains, expected to alter the affinity for Old. Herein we describe the synthesis of 6'-modified alpha-GalCer analogues with an intact phytoceramide moiety that are capable to skew the cytokine release profile to Th1, white maintaining strong antigenic activity. These analogues are characterized by the presence of an aromatic moiety that is connected via an amide or an urea linkage to C'-6 of the galactopyranose ring.

This thesis is aimed at extending the use of TOF-SIMS (time of flight secondary ion mass spectrometry) as an analytical tool in cell and tissue research. TOF-SIMS is a relatively new method that allows analysis of the chemical composition of sample surfaces. Originally, it was used for imaging the distribution of elements on surfaces in materials science. Technical improvements have made analysis of fragile biological samples possible through localization of relevant secondary ions, e.g. lipid fragments and inorganic ion patterns. TOF-SIMS has several advantages over alternative imaging methods e.g. its sensitivity to all elements, detection of all isotopes, and it allows composite imaging of the surface distribution of detected elements and molecules. The present work comprises new applications of cryomethods for preparation of TOF-SIMS tissue samples in an effort to obtain analytical results that reflect the vital situation as closely as possible. Lipid species (galactosylceramides, sulfatides or fatty acids) and ions (Na/K) were identified and localized in specimens from cerebellum, kidney and intestine with instruments equipped with a bismuth cluster primary ion source. Two sample preparation sequences were employed: (i) plunge freezing in liquid nitrogen and cryosectioning; (ii) high-pressure freezing followed by freeze-fracturing and freeze-drying. The two preparation methods resulted in similar distributions of lipids and fatty acids but only sample preparation with high-pressure freezing allowed demonstration of separate distributions of sodium and potassium in rat cerebellum and kidney. TOF-SIMS analysis revealed specific distributions of lipids cholesterol, phosphocholine, sulfatides, two galactosylceramide species and ions sodium and potassium in rat cerebellum. It could be shown that the white matter is separated into a “cytoplasm-rich” compartment containing phosphocholine, Na+, K+ and N-lignoceroylgalactosylceramide and a cholesterol-rich compartment containing cholesterol and N-stearoyl-galactosylceramide. Sulfatides with short chain fatty acids displayed a uniform distribution in the white matter and a patchy distribution for sulfatide with C24 fatty acids. Fatty acid signals showed high intensities for stearic acid in Purkinje cell bodies of the rat cerebellum; the fatty acids palmitic and oleic acid displayed most intense signals located to the molecular and granular layer. Fatty acid imaging in mouse intestine revealed highest palmitic acid signals in the secretory crypt cells and the intestinal lumen and highest oleic acid signals in intestinal villi. New data on the cellular and subcellular distribution of lipid molecular species in tissues have been presented that are not possible to achieve with other microscopical techniques. It is concluded that TOF-SIMS at the present state is a powerful tool in imaging of ions and organic compounds at a MW up to ≈ 1,000 in biological tissues. TOF-SIMS has a potential to provide very significant information for the understanding of physiological functions and pathological processes in biomedical research. Further technical development will extend the range of applications.

Earlier work from our laboratory provided evidence for myelin abnormalities (decreased quantities of proteins associated with myelin compaction, decreased sheath thickness) in cortex and hippocampus of Aldh5a1 −/− mice, which have a complete ablation of the succinate semialdehyde dehydrogenase protein [E.A. Donarum, D.A. Stephan, K. Larkin, E.J. Murphy, M. Gupta, H. Senephansiri, R.C. Switzer, P.L. Pearl, O.C. Snead, C. Jakobs, K.M. Gibson, Expression profiling reveals multiple myelin alterations in murine succinate semialdehyde dehydrogenase deficiency, J. Inher. Metab. Dis. 29 (2006) 143–156]. In the current report, we have extended these findings via comprehensive analysis of brain phospholipid fractions, including quantitation of fatty acids in individual phospholipid subclasses and estimation of hexose-ceramide in Aldh5a1 −/− brain. In comparison to wild-type littermates (Aldh5a1 +/+ ), we detected a 20% reduction in the ethanolamine glycerophospholipid content of Aldh5a1 −/− mice, while other brain phospholipids (choline glycerophospholipid, phosphatidylserine and phosphatidylinositol) were within normal limits. Analysis of individual fatty acids in each of these fractions revealed consistent alterations in n-3 fatty acids, primarily increased 22:6n-3 levels (docosahexaenoic acid; DHA). In the phosphatidyl serine fraction there were marked increases in the proportions of polyunsaturated fatty acids with corresponding decreases of monounsaturated fatty acids. Interestingly, the levels of hexose-ceramide (glucosyl- and galactosylceramide, principal myelin cerebrosides) were decreased in Aldh5a1 −/− brain tissue (one-tailed t test, p=0.0449). The current results suggest that lipid and myelin abnormalities in this animal may contribute to the pathophysiology. © 2007 Elsevier B.V. All rights reserved.

Molecular Dynamics (MD) simulations were performed on a two-component lipid bilayer system composed of a mixture of neutral galactosylceramide (GalCer) and charged dipalmitoylphosphatidylglycerol (DPPG) lipid molecules in a liquid crystalline phase in order to help understand the nature of the short-range interactions. Two lipid bilayers with the GalCer:DPPG ratios of 9.04:1 (system-1) and 3:1 (system-2) have been prepared and simulated. System-1 represents a 'collapsed' lipid bilayer state, with a narrow water space between the bilayers and system-2 represents an 'expanded' state with a wide (100 A?) water space between the bilayers. The simulations were validated by comparing with the experimental data for several important aspects of the bilayer structure and dynamics, such as the deuterium order parameters of the lipid chains, the area per lipid, the bilayer thickness, the thickness of the chain region and the diffusion constants of lipids and water. The interaction of water with the GalCer and DPPG oxygen atoms resulted in a strong water ordering via a spherical hydration shell and the formation of hydrogen bonds (H-bonds). The formation of intermolecular H-bonds was observed between hydroxyl groups from the opposing GalCer sugar units, giving the energy of adhesion in the range from -1.0 to -3.4 erg/cm2. We suggest that this value is the contribution of the H-bond component to the net adhesion energy between GalCer bilayers.

We describe a new method that permits quantification in the pmol to nmol range of three lyso-neutral glycosphingolipids (lyso-n-GSLs), glucosylsphingosine (GlcSph), galactosylsphingosine (GalSph), and lactosylsphingosine, in the same sample as neutral glycosphingolipids (n-GSLs). Lyso-n-GSLs and n-GSLs are initially obtained from a crude lipid extract using Sephadex G25 chromatography, followed by their isolation in one fraction, which is devoid of other contaminating lipids, by aminopropyl solid-phase chromatography. Lyso-n-GSLs and n-GSLs are subsequently separated from one another by weak cation exchange chromatography. N-GSLs are then deacylated by strong alkaline hydrolysis, and the N-deacylated-GSLs and lyso-n-GSLs are subsequently N-acetylated using [3H]acetic anhydride. An optimal concentration of 5 mM acetic anhydride was established, which gave95% N-acetylation. We demonstrate the usefulness of this technique by showing an approximately 40-fold increase of both GlcSph and glucosylceramide in brain tissue from a glucocerebrosidase-deficient mouse, as well as significant lactosylceramide accumulation. The application and optimization of this technique for lyso-n-GSLs and lyso-GSLs will permit their quantification in small amounts of biological tissues, particularly in the GSL storage diseases, such as Gaucher and Krabbe's disease, in which GlcSph and GalSph, respectively, accumulate.

6-Photocholesterol, a new photoactivatable analog of cholesterol in which a diazirine functionality replaces the 5,6-double bond in the steroid nucleus, was used recently to identify cholesterol-binding proteins in neuroendocrine cells [Thiele, C., Hannah, M.J., Farenholz, F. and Huttner, W.B. (2000) Nat. Cell Biol. 2, 42-49], to track the distribution and transport of cholesterol in Caenorhabditis elegans [Matyash, V., Geier, C., Henske, A., Mukherjee, S., Hirsh. D., Thiele, C., Grant, B., Maxfield, F.R. and Kurzehalia, T.V. (2001) Mol. Biol. Cell 12, 1725-1736], and to probe lipid-protein interactions in oligodendrocytes [Simons, M., Kramer, E.M., Thiele. C., Stoffel, W. and Trotter, J. (2000) J. Cell Biol. 151, 143-154]. To determine whether 6-photocholesterol is a faithful mimetic of cholesterol we analyzed the ability of this probe, under conditions in which it is not photoactivated to a carbene, to substitute for cholesterol in two unrelated assays: (1) to condense 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine monomolecular films and (2) to mediate the fusion of two alphaviruses (Semliki Forest and Sindbis) with liposomes. The results suggest that this analog is a suitable photoprobe of cholesterol. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

The glycolipid alpha-galactosylceramide (alpha-GalCer), ligand of NKT cells, has been recently shown to induce antitumor immunity in mice through the induction of IL-12 production by dendritic cells. In the present study we compared alpha-GalCer and rIL-12 antitumor activities in the treatment of hepatic metastases of the C-26 murine colon carcinoma. We show that in immunocompetent mice the two molecules display similar efficacy, whereas in mice knockout (KO) for beta2-microglobulin (beta2m), IFN-gamma or IL-12p40, alpha-GalCer antitumor activity is severely impaired. Conversely,in all such KO mice, rIL-12 retains its efficacy. In this context, the IL-12 effect relies on NK cell function since it is abrogated by antibodies to NK1.1, expressed by both NK and NKT cells, but not in beta2m KO mice that lack NKT and CD8 T cells, but have a perfectly functional NK cell population. Furthermore, in IFN-gamma and IL-12p40 double KO mice, exogenous rIL-12 completely loses antitumor efficacy, suggesting the existence of an IFN-gamma-independent IL-12 effect that does require the presence of endogenous IL-12p40 chain.

Modified galactosylceramide with a long-chain cyclic acetal at the sugar moiety, plasmalogalactosylceramide, was isolated from equine brain. To identify the isomeric stereostructure of the natural product, the plasmalo derivative was chemically synthesized from galactosylceramide through acetalization. The presence of cyclic acetal linkage, the linked position and length of the acetal chain of the synthesized and natural products were determined by proton nuclear magnetic resonance spectroscopy and fast-atom bombardment–mass spectrometry, as well as gas chromatography–mass spectrometry and gas–liquid chromatography. The orientation of the acetal chain linked to galactoside was characterized by connectivity between the cyclic acetal proton and ring proton(s) on the sugar moiety using the homonuclear Overhauser effect. This revealed that, of the two positional isomers of the acetal linkage with 4,6-O-acetal and 3,4-O-acetal derivatives obtained from the acetalization reaction, the former positional isomer, separated into two spots, was identified to 'endo'- and 'exo'-type acetal chains. In comparison to the NMR data of the synthesized derivative, equine brain acetalized lipid was found to be an 'endo'-type 4,6-O-acetal derivative.—Yachida, Y., M. Kashiwagi, T. Mikami, K. Tsuchihashi, T. Daino, T. Akino, and S. Gasa. Stereochemical structures of synthesized and natural plasmalogalactosylceramides from equine brain. J. Lipid Res. 1998. 39: 1039–1045.

In loading tests using galactosylceramide which had been labelled with tritium in the ceramide moiety, living skin fibroblast lines derived from the original prosaposin-deficient patients had a markedly reduced capacity to degrade galactosylceramide. The hydrolysis of galactosylceramide could be partially restored in these cells, up to about half the normal rate, by adding pure saposin A, pure saposin C, or a mixture of these saposins to the culture medium. By contrast, saposins B and D had little effect on galactosylceramide hydrolysis in the prosaposin-deficient cells. Cells from β-galactocerebrosidase-deficient (Krabbe) patients had a relatively high residual galactosylceramide degradation, which was similar to the rate observed for prosaposin-deficient cells in the presence of saposin A or C. An SV40-transformed fibroblast line from the original saposin C-deficient patient, where saposin A is not affected, showed normal degradation of galactosylceramide. The findings support the hypothesis, which was deduced originally from in vitro experiments, that saposins A and C are the in vivo activators of galactosylceramide degradation. Although the results with saposin C-deficient fibroblasts suggest that the presence of only saposin A allows galactosylceramide breakdown to proceed at a normal rate in fibroblasts, it remains to be determined whether saposins A and C can substitute for each other with respect to their effects on galactosylceramide metabolism in the whole organism.

International audience; Deca-bromo-diphenyl ether (DBDE) is one of the most efficient brominated flame retardant (BFR) available on the market. We recently demonstrated that when administered to female rat by oral route, DBDE is efficiently absorbed, with the highest residual concentrations found in two endocrine glands, namely the adrenal glands and the ovaries. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging, a technique usually used for the study of endogenous compounds, was applied for the first time to a persistent organic pollutant, allowing to detect and to precisely localize DBDE residues in these two target tissues. The detection of the bromide ion ((81)Br isotope) by TOF-SIMS mass spectrometry imaging allowed us to demonstrate a marked cortical tropism of DBDE residues for the adrenal glands in female rats dosed per os 2 mg·kg(-1) DBDE, daily, over 96 h. In ovaries, DBDE residues were found to be concentrated in spots corresponding to part of the corpora lutea. Hepatic residues of DBDE were found to be homogeneously distributed. Due to the intrinsic toxicity of DBDE, its accumulation in the adrenal glands and the ovaries may be connected to the mechanisms of actions by which DBDE could trigger endocrine disruption in mammals.

An inositol-phosphate-containing glycosphingolipid, not reported earlier, was isolated from human cauda equina. Structural characterization showed the glycosphingolipid to be inosital-phosphoryl-2(3) galactosylceramide. The concentration varied between 25 and 30 nmol/g fresh tissue.

A mouse monoclonal antibody (mAb) was developed against monohexaosylceramide. This mAb differentially reacted on thin-layer chromatograms with 3 types of galactosylceramide (GalCer) obtained from bovine brain. Structural analysis of the 3 glycolipids revealed that they consisted of the same galactose and sphingosine but of apparently different fatty acids. Among the GalCers, the mAb reacted with teh two GalCers which contained alpha-hydroxy fatty acids, but not with GalCer composed of nonhydroxy fatty acids. These findings suggest that not only that the mAb discriminated the fatty acid composition in the ceramide moiety of GalCer, but also that the ceramide structure defines the immunological epitope as it is known to do for the carbohydrate moiety of glycosphingolipid.