Comprehensive study of rodent olfactory tissue lipid composition.

The peripheral olfactory tissue (OT) plays a primordial role in the detection and transduction of olfactory information. Recent proteomic and transcriptomic studies have provided valuable insight into proteins and RNAs expressed in this tissue. Paradoxically, there is little information regarding the lipid composition of mammalian OT. To delve further into this issue, using a set of complementary state-of-the-art techniques, we carried out a comprehensive analysis of OT lipid composition in rats and mice fed with standard diets. The results showed that phospholipids are largely predominant, the major classes being phosphatidylcholine and phosphatidylethanolamine. Two types of plasmalogens, plasmenyl-choline and plasmenyl-ethanolamine, as well as gangliosides were also detected. With the exception of sphingomyelin, substantial levels of n-3 polyunsaturated fatty acids, mainly docosahexaenoic acid (22:6n-3; DHA), were found in the different phospholipid classes. These findings demonstrate that the rodent OT shares several features in common with other neural tissues, such as the brain and retina.

Computational Lipidomics with insane: A Versatile Tool for Generating Custom Membranes for Molecular Simulations.

For simulations of membranes and membrane proteins, the generation of the lipid bilayer is a critical step in the setup of the system. Membranes comprising multiple components pose a particular challenge, because the relative abundances need to be controlled and the equilibration of the system may take several microseconds. Here we present a comprehensive method for building membrane containing systems, characterized by simplicity and versatility. The program uses preset, coarse-grain lipid templates to build the membrane, and also allows on-the-fly generation of simple lipid types by specifying the headgroup, linker, and lipid tails on the command line. The resulting models can be equilibrated, after which a relaxed atomistic model can be obtained by reverse transformation. For multicomponent membranes, this provides an efficient means for generating equilibrated atomistic models. The method is called insane, an acronym for INSert membrANE. The program has been made available, together with the complementary method for reverse transformation, at http://cgmartini.nl/ . This work highlights the key features of insane and presents a survey of properties for a large range of lipids as a start of a computational lipidomics project.

Miglustat as a therapeutic agent: prospects and caveats.

A viable treatment for lysosomal storage disease has been very difficult to attain. One option is pharmacological inhibition of synthetic pathways to reduce substrate accumulations. Miglustat N-butyldeoxynojirimycin (NBDNJ), an inhibitor of glucosylceramide synthase, has shown much promise in clinical trials for the treatment of Type I Gaucher disease. The molecular events invoked by NBDNJ in cell culture and in animal models have not been so definitive. This review discusses the biochemical and molecular impact of NBDNJ as it relates to its potential as a therapeutic drug.

In vitro photothermal destruction of neuroblastoma cells using carbon nanotubes conjugated with GD2 monoclonal antibody.

Despite aggressive multimodality therapy, most neuroblastoma-bearing patients relapse and survival rate remains poor. Exploration of alternative therapeutic modalities is needed. Carbon nanotubes (CNTs), revealing optical absorbance in the near-infrared region, warrant their merits in photothermal therapy. In order to specifically target disialoganglioside (GD2) overexpressed on the surface of neuroblastoma stNB-V1 cells, GD2 monoclonal antibody (anti-GD2) was conjugated to acidified CNTs. To examine the fate of anti-GD2 bound CNTs after incubation with stNB-V1 cells, rhodamine B was labeled on carboxylated CNTs functionalized with and without anti-GD2. Our results illustrated that anti-GD2-linked CNTs were extensively internalized by neuroblastoma cells via GD2-mediated endocytosis. In addition, we showed that anti-GD2 bound CNTs were not ingested by PC12 cells without GD2 expression. After anti-GD2 conjugated CNTs were incubated with neuroblastoma cells for 6 h and endocytosed by the cells, CNT-laden neuroblastoma cells were further irradiated with an 808 nm near-infrared (NIR) laser with intensity ramping from 0.6 to 6 W cm(-2) for 10 min which was then maintained at 6 W cm(-2) for an additional 5 min. Post-NIR laser exposure, and after being examined by calcein-AM dye, stNB-V1 cells were all found to undergo necrosis, while non-GD2 expressing PC12 cells all remained viable. Based on the in vitro study, CNTs bound with anti-GD2 have the potential to be utilized as a therapeutic thermal coupling agent that generates heat sufficient to selectively kill neuroblastoma cells under NIR laser light exposure.

Inhibition of influenza virus infections by sialylgalactose-binding peptides selected from a phage library.

Influenza virus hemagglutinin recognizes sialyloligosaccharides of glycoproteins and glycolipids as cell surface receptors in the initial stage of the infection process. We demonstrate that pentadecapeptides that bind to a sialylgalactose structure (Neu5Ac-Gal) inhibited the infection of cells by influenza virus. The pentadecapeptides were identified through affinity selection from a phage-displayed random peptide library using a monolayer of the ganglioside Neu5Acalpha2-3Galbeta1-4Glcbeta1-1'Cer (GM3). The peptides were found to have affinity for GM3, and alanine scanning showed seven amino acid residues that contribute to carbohydrate recognition. The binding of peptides to the cell surface was significantly inhibited in the presence of sialic acid or by the digestion of cell surface sialyl residues by neuraminidase. Plaque assays indicated that a molecular assembly of alkylated peptides inhibited the infection of Madin-Darby canine kidney cells by influenza virus. Carbohydrate-binding peptides that inhibit carbohydrate-virus interaction showed inhibitory activity. These results may lead to a new approach to the design of antiviral drugs.

Identification of Escherichia coli enterotoxin inhibitors from traditional medicinal herbs by in silico, in vitro, and in vivo analyses.

ETHNOPHARMACOLOGICAL RELEVANCE: Glycyrrhiza uralensis has been used for the treatment of gastrointestinal disorders, such as diarrhea, in several ancient cultures. Glycyrrhizin is the principal component of liquorice and lots of pharmacological effects have been demonstrated. AIM OF THE STUDY: Heat-labile enterotoxin (LT), the virulence factor of enterotoxigenic Escherichia coli, induces diarrhea by initially binding to the GM1 on the surfaces of intestinal epithelial cells and consequently leading to the massive loss of fluid and ions from cells. Therefore, we evaluated the inhibitory effects of traditional medicinal herbs (TMH) on the B subunit of LT (LTB) and GM1 interaction. MATERIALS AND METHODS: The inhibitory effects of TMH on LTB-GM1 interaction were evaluated by GM1-enzyme-linked immunosorbent assay (ELISA). The likely active phytochemicals of these TMH were then predicted by in silico model (docking) and analyzed by in vitro (GM1-ELISA) and in vivo (patent mouse gut assay) models. RESULTS: We found that various TMH, which have been ethnomedically used for the treatment of diarrhea, inhibited the LTB-GM1 interaction. Docking data showed that triterpenoids were the most active phytochemicals and the oleanane-type triterpenoids presented better LTB-binding abilities than other types of triterpenoids. Moreover, by in vitro and in vivo models, we demonstrated that glycyrrhizin was the most effective oleanane-type triterpenoid that significantly suppressed both the LTB-binding ability (IC50=3.26+/-0.17 mM) and the LT-induced fluid accumulation in mice. CONCLUSIONS: We found an LT inhibitor, glycyrrhizin, from TMH by in silico, in vitro, and in vivo analyses.

Syntheses of new model compounds related to an antigenic epitope from Bupleurum falcatum L. and their distributions in various ganglioside-phospholipid monolayers.

6-N-[2-(Tetradecyl)hexadecanamido]hexyl beta-D-glucopyranosyluronic acid-(1-->6)-beta-D-galactopyranosyl-(1-->6)-beta-D-galactopyranoside (1) and its clustering compound (2) carrying a tetravalent sugar unit, which are new model compounds related to a major antigenic epitope from antiulcer pectic polysaccharide of Bupleurum falcatum L., were synthesized and the distributions of 1 and 2 in mixed ganglioside (GM1, GD1a or GT1b)/phospholipid (DPPC) monolayers were observed using atomic force microscopy (AFM). AFM images showed that 1 was distributed in the GM1, GD1a and GT1b region of the mixed monolayers, in which 1 was miscible with GD1a. Specific distribution of 1 was observed in the mixed GM1/DPPC monolayer. Compound 2 was miscible with GM1, while 2 formed associations with GD1a and GT1b in the mixed monolayers. The distribution mode of 1 and 2 was different among the mixed ganglioside/DPPC monolayers.

Mistletoe lectin I is a sialic acid-specific lectin with strict preference to gangliosides and glycoproteins with terminal Neu5Ac alpha 2-6Gal beta 1-4GlcNAc residues.

Mistletoe lectin I (ML-I) is a type II ribosome-inactivating protein, which inhibits the protein biosynthesis at the ribosomal level. ML-I is composed of a catalytically active A-chain with rRNA N-glycosidase activity and a B-chain with carbohydrate binding specificities. Using comparative solid-phase binding assays along with electrospray ionization tandem mass spectrometry, ML-I was shown to preferentially bind to terminally alpha2-6-sialylated neolacto series gangliosides from human granulocytes. IV(6)Neu5Ac-nLc4Cer, VI(6)Neu5Ac-nLc6Cer, and VIII(6)Neu5Ac-nLc8Cer were identified as ML-I receptors, whereas the isomeric alpha2-3-sialylated neolacto series gangliosides were not recognized. Only marginal binding of ML-I to terminal galactose residues of neutral glycosphingolipids with a Galbeta1-4Glc or Galbeta1-4GlcNAc sequence was determined, whereas a distal Galalpha1-4Gal, GalNAcbeta1-3Gal, or GalNAcbeta1-4Gal disaccharide did not bind at all. Among the glycoproteins investigated in Western blot and microwell adsorption assays, only those carrying Neu5Acalpha2-6Galbeta1-4GlcNAc residues, exclusively, predominantly, or even as less abundant constituents in an assembly with Neu5Acalpha2-3Galbeta1-4GlcNAc-terminated glycans, displayed high ML-I binding capacity. From our data we conclude that (i) ML-I has to be considered as a sialic acid- and not a galactose-specific lectin and (ii) neolacto series gangliosides and sialoglycoproteins with type II glycans, which share the Neu5Acalpha2-6Galbeta1-4GlcNAc terminus, are true ML-I receptors. This strict preference might help to explain the immunostimulatory potential of ML-I toward certain leukocyte subpopulations and its therapeutic success as a cytotoxic anticancer drug.

Preferential binding of the anticancer drug rViscumin (recombinant mistletoe lectin) to terminally alpha2-6-sialylated neolacto-series gangliosides.

Production of biochemically defined recombinant mistletoe lectin was achieved by cloning and separate expression of the single catalytically active A-chain and the B-chain with carbohydrate binding properties in Escherichia coli, yielding an active heterodimeric protein named rViscumin (Eck et al. [1999] Eur. J. Biochem., 265, 788-797). Employing solid phase binding assays, rViscumin was shown to preferentially bind to terminally alpha2-6-sialylated neolacto-series gangliosides IV(6)Neu5Ac-nLc4Cer, VI(6)Neu5Ac-nLc6Cer, and VIII(6)Neu5Ac-nLc8Cer isolated from human granulocytes. Only marginal binding of rViscumin to galactose-terminated neutral GSLs was determined, whereas reinvestigation of ricin specificity demonstrated this lectin as a galactose-binding protein. Human promyelotic HL-60 cells exhibited an IC(50) value (half maximum cytotoxicity) of 1.16 pM and human bladder carcinoma 5637 cells of 12.1 pM rViscumin; CHO-K1 cells were resistant to rViscumin treatment up to a concentration of 5.26 nM tested. Quantification of the predominant receptor ganglioside IV(6)Neu5Ac-nLc4Cer by means of a specific anti-Neu5Acalpha2-6Galbeta1-4GlcNAc-R antibody revealed 3.68 x 10(6) and 1.54 x 10(6) receptor molecules per HL-60 and 5637 cell, respectively; CHO-K1 cells were negative, lacking alpha2-6-sialylated gangliosides. The data imply a direct correlation of rViscumin cytotoxicity and the expression of receptor ganglioside. Moreover, CHO-K1 cells were rendered susceptible toward rViscumin cytotoxicity after exogenous application of human granulocyte gangliosides. Thus, (1) rViscumin has to be considered as a sialic acid-specific rather than a galactose-specific type II ribosome-inactivating protein, and (2) neolacto-series gangliosides with Neu5Acalpha2-6Galbeta1-4GlcNAc-terminus are true functional and physiologically relevant rViscumin receptors.

Studies of the chemical structure of gangliosides in deer antler, Cervus nippon.

The biological activity of deer antler has been considered to originate in the gangliosides, although the structures of gangliosides have not been well elucidated. The quality of deer antler as an Asian folk medicine has often been evaluated by the amount of gangliosides contained in the crude drug. We have completed the structural determination of five gangliosides isolated from deer antler in the present study. Five ganglioside fractions were isolated and purified from deer antler, Cervus nippon, by the Folch-Suzuki partition method, DEAE-Sephadex A-25, and further by silica gel column chromatography. High field proton nuclear magnetic resonance spectroscopy, gas chromatography/mass spectrometry, and fast atom bombardment-mass spectrometry studies characterized the isolated ganglioside fractions. GM3 and GD3 were present in the isolated ganglioside fractions. Samples were hydrolyzed in trifluoroacetic acid for direct compositional analysis and analyzed for sialic acid and neutral sugar without prior derivatization. Separation of the monosaccharides was achieved by HPLC on a Dionex CarboPac column eluted at a high pH. The resolved monosaccharides were identified using standard monosaccharides by pulsed amperometric detection. N-Acetyl GM3 (Neu5Ac), N-glycolyl GM3 (Neu5Gc), and N-acetyl GD3 (Neu5Ac) were present in the antler. The major ceramide moiety was composed of C16:0 or C22:0 fatty acids along with either C18 sphingosine or C20 eicosasphingosine.

Modulation by gangliosides of the lamellar-inverted micelle (hexagonal II) phase transition in mixtures containing phosphatidylethanolamine and dioleoylglycerol.

We studied the effect of gangliosides GD1a and GM1 on the lamellar-to-hexagonal II phase transition of mixtures of dioleoylphosphatidylethanolamine/dioleoylphosphatidyl choline, 3:1, and of transphosphatidylated phosphatidylethanolamine with dioleoylglycerol by high-sensitivity differential scanning calorimetry, 31P-NMR, and pyrene fluorescence of a phosphatidylcholine probe. Gangliosides had a dual effect. Below 1 mol % ganglioside the hexagonal II phase transition was affected but still occurred at lower temperature than in the absence of gangliosides. The presence of between 1 and 2 mol % gangliosides increased the temperature for formation of the hexagonal II phase and progressively decreased its cooperativity. Above 3 mol % gangliosides totally inhibited the formation of both the temperature-induced and composition-induced hexagonal phase, probably by opposing the geometric distortions necessary for the inverted micellar structures.

Study of lectin-ganglioside interactions by high-performance liquid affinity chromatography.

A high-performance affinity column containing immobilized modified GM1 (lyso-GM1) was used to study the binding of an endogenous human brain lectin (HBL) in comparison with other carbohydrate-binding proteins. The proteins are previously converted into biotinylated derivatives. Detection of biotinylated proteins in the eluates by a microtitre plate assay ensures good sensitivity. The maximum binding capacity of the adsorbent for HBL is obtained in Tris buffer supplemented with beta-mercaptoethanol. The binding is inhibitable by specific sugar. It is concluded that the use of immobilized glycolipids in analytical high-performance liquid affinity chromatographic methods may serve as models in the study of interactions between gangliosides and carbohydrate-binding proteins.

Lactational changes in the N-glycoloylneuraminic acid content of bovine milk gangliosides.

The N-glycoloylneuraminic acid (Neu5Gc) contents of milk and milk gangliosides from bovines were investigated during the different stages of lactation. The Neu5Gc content of milk is high in the colostrum (32% of the total sialic acid content of milk) and decreases thereafter until the end of the period considered (6% on day 30). When the Neu5Gc content of gangliosides was evaluated a similar profile to that of Neu5Gc in total sialic acids was found. Gangliosides from colostrum showed the highest Neu5Gc content (21-22% of the total sialic acid content of milk gangliosides). This content dropped towards the end of the period studied (8% on day 90). Our results indicate that a significant supply of Neu5Gc by the milk could be important for the newborn during the first days after parturition.