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Abstract Binding of cholera toxin subunit B (CTB) to its receptor and toxin transport into the intestinal epithelial cells are the causative events for the potentially lethal disease cholera. The five sugar mono-sialo ganglioside GM1 is the cell surface receptor for cholera toxin B-subunit. CTB binding was determined by use of immobilized GM1 to microtiter plates and by immunohistochemistry. Sections from the human colon and the human soft palate were incubated with FITC-conjugated CTB and with anti-MUC2. Both the luminal surface of the intestine and the secretory goblet cells exhibited strong binding. Addition of simple carbohydrates and milk to the incubation medium showed that a combination of lactose and non-fat dry milk was potent inhibitors of toxin- and mucin binding. Both CTB and ant-MUC2 stained to the cytoplasm (mucin granules) in the goblet cells from the human soft palate. In the colon CTB stained the entire cytoplasm of the goblet cells while anti-MUC2 detected only the supranuclear region of some cells, suggesting carbohydrate heterogeneity between goblet cell mucin granules in different regions of the human body. Both CTB- and MUC2 binding were inhibited when GM1 was added to the incubation medium. It is proposed that the human colonic goblet cells play a role in the secretory diarrhea in patients with cholera and that milk might have a prophylactic or therapeutic application in the management of cholera. Highlights • The human colonic goblet cells play a role in the secretory diarrhea in patients with cholera. Milk might have a prophylactic or therapeutic application in the management of cholera. [ABSTRACT FROM AUTHOR]

The B-subunits of heat-labile enterotoxins LT-I (LT-IB) and LT-IIa (LT-IIaB) are strong adjuvants that bind to cell-surface receptors, including gangliosides G M1 and GD1b, respectively. LT-IIaB also binds TLR-2. We demonstrate for the first time that co-incubation with the B-subunits induces significant clustering of B cells after only 4 h, and B and T cells in 24 h. Clustering was dependent on intact B-subunits, but not on the TLR-2 binding activity of LT-IIaB, indicating it was ganglioside-mediated. Treatment of B cells with LT-IB, a mixture of LT-IB + LT-IIaB, but not LT-IIaB alone, caused a delay in T cell division following ovalbumin endocytosis. B cell receptor-mediated uptake in presence of each treatment caused an arrest, but with increased production of IL-2. Further, treatments differentially increased the proportion of macrophages expressing MHC class-II. These results highlight the outcomes of interplay between signals involving different receptors and implicate a novel mechanism of adjuvanticity. [ABSTRACT FROM AUTHOR]

Abstract: In order to analyze the mechanisms for cancer metastasis, high metastatic sublines (H7-A, H7-Lu, H7-O, C4-sc, and C4-ly) were obtained by repeated injection of mouse Lewis lung cancer sublines H7 and C4 into C57BL/6 mice. These sublines exhibited increased proliferation and invasion activity in vitro. Ganglioside profiles exhibited lower expression of GM1 in high metastatic sublines than the parent lines. Then, we established GM1-Si-1 and GM1-Si-2 by stable silencing of GM1 synthase in H7 cells. These GM1-knockdown clones exhibited increased proliferation and invasion. Then, we explored genes that markedly altered in the expression levels by DNA microarray in the combination of C4 vs. C4-ly or H7 vs. H7 (GM1-Si). Consequently, pp-GalNAc-T13 gene was identified as up-regulated genes in the high metastatic sublines. Stable transfection of pp-GalNAc-T13 cDNA into C4 (T13-TF) resulted in increased invasion and motility. Then, immunoblotting and flow cytometry using various antibodies and lectins were performed. Only anti-trimeric Tn antibody (mAb MLS128), showed increased expression levels of trimeric Tn antigen in T13-TF clones. Moreover, immunoprecipitation/immunoblotting was performed by mAb MLS128, leading to the identification of an 80kDa band carrying trimeric Tn antigen, i.e. Syndecan-1. Stable silencing of endogenous pp-GalNAc-T13 in C4-sc (T13-KD) revealed that primary tumors generated by subcutaneous injection of T13-KD clones showed lower coalescence to fascia and peritoneum, and significantly reduced lung metastasis than control clones. These data suggested that high expression of pp-GalNAc-T13 gene generated trimeric Tn antigen on Syndecan-1, leading to the enhanced metastasis. [Copyright &y& Elsevier]

Abstract: Repression of human papillomavirus (HPV) E6 and E7 oncogenes in established cervical carcinoma cell lines causes senescence due to reactivation of cellular tumor suppressor pathways. Here, we determined whether ongoing expression of HPV16 or HPV18 oncogenes is required for the proliferation of primary human cervical carcinoma cells in serum-free conditions at low passage number after isolation from patients. We used an SV40 viral vector expressing the bovine papillomavirus E2 protein to repress E6 and E7 in these cells. To enable efficient SV40 infection and E2 gene delivery, we first incubated the primary cervical cancer cells with the ganglioside GM1, a cell-surface receptor for SV40 that is limiting in these cells. Repression of HPV in primary cervical carcinoma cells caused them to undergo senescence, but the E2 protein had little effect on HPV-negative primary cells. These data suggest that E6 and E7 dependence is an inherent property of human cervical cancer cells. [Copyright &y& Elsevier]

Abstract: Pharmacological regulation of gene expression was one of the top professional interests of Dr. Costa. He promoted the idea that drugs can improve the endogenous mechanisms of synaptic plasticity by modulating gene expression. In this article I reflect upon Dr. Costa''s leadership in projects undertaken at FGIN that were aimed at elucidating how neurotransmitter receptor activation could affect brain function by modulating genes and their products. I will be presenting examples of how pharmacological tools can change gene expression. These include the ability of drugs of abuse to alter the synthesis of opioid peptides or an endogenous ligand for GABAA receptor. I will conclude with a brief summary of intriguing discoveries about the regulation of nerve growth factor (NGF) and its receptors by beta-receptor agonists, adrenal steroids and cytokines. [Copyright &y& Elsevier]

Abstract: Cholera toxin B-subunit (CTB) treatment of K562 erythroleukemia cells increased their sensitivity to be killed by NK-92 cells with more than 10%, compared to untreated cells. A similar treatment of non-T, non-B acute lymphoblastic REH leukemia cells, known to be unsensitive to NK cell mediated cytotoxicity, did not have any impact at all. Visualization of the cross-linked gangliosideM1 (GM1) using fluorescent labeled CTB, indicated accumulation of the fluorescence to one cap and a few smaller patches in both type of cells. Additional cross-linking using anti-CTB antibodies further accentuated capping and increased lysis in the case of K562 cells. Blocking experiments performed with anti-MICA/B, ULBP-2 and/or CD59 antibodies could not inhibit the increased sensitivity mediated by CTB. [Copyright &y& Elsevier]

Abstract: The outer segment of rod photoreceptor cells is responsible for initiating visual signal transduction when light levels are low. It consists of stacked disk membranes surrounded by the plasma membrane and is under continuous renewal. Disk membranes are synthesized at the base of the outer segment and are progressively displaced toward the apical tip where they are phagocytosed by the pigment epithelium. This process takes approximately ten days during which time the lipid composition of the disk membrane is modified. Disk membranes become progressively lower in cholesterol and higher in unsaturated phospholipids during spatial displacement. The modification of disk composition is likely important for normal function of the rod cell. Flow cytometry is shown in this study to be a powerful tool to assess differences among rod outer segment disk membranes. First, the feasibility of flow cytometry to detect individual osmotically intact disks was established. Fluorescent beads of 1.0 μm, 0.5 μm, 0.2 μm, and 0.1 μm diameter were used to demonstrate that side-scatter intensity measured on a log scale corresponds to bead diameter. The intensity of disk side scatter predominantly corresponded to 0.1–0.2 μm diameter beads. We next applied this technique to detect caveolin-1, peripherin/rds, and GM1 in disk membranes. Caveolin-1 was detected with FITC-labeled anti-caveolin-1, peripherin/rds with Cy3 labeled anti-peripherin/rds, and GM1 with FITC-labeled cholera toxin subunit B. Fluorescence due to caveolin-1 and peripherin/rds binding was detected in 80% and 70% respectively of the events that corresponded to scattering attributed to disks. Fluorescence attributed to GM1 was detected in 60% of the events that corresponded to disk scattering. The intensity of fluorescence due to caveolin-1 and peripherin/rds labeling was directly proportional to the intensity of side scatter, indicating that both caveolin-1 and peripherin/rds concentrations are uniform among all the disks. Fluorescence intensity of GM1 labeled disks was independent of side-scatter intensity indicating GM1 is heterogeneously distributed among the disks. These results are consistent with integral membrane proteins such as caveolin-1, and peripherin/rds remaining in the disk bilayer as the disks are apically displaced. The GM1 distribution may be similar to the spatial distribution of cholesterol in the outer segment. [Copyright &y& Elsevier]

Abstract: We report an investigation of the self-assembly of the monosialoganglioside (GM1) at interfaces formed between aqueous solutions of 10μM GM1 (at 25°C) and micrometer-thick films of the nematic liquid crystal (LC) 4′-pentyl-4-cyanobiphenyl (5CB). We observe the process of spontaneous transfer of GM1 onto the interfaces to be accompanied by continuous ordering transitions within the micrometer-thick films of the LC. At saturation coverage, the GM1 orders the LC in an orientation that is perpendicular to the interface, an orientation that is similar to that caused by phospholipids such as dilauroylphosphatidylcholine (DLPC). This result suggests an interaction between the LC and GM1 that is dominated by the hydrophobic tails of the GM1. Relative to DLPC, however, we observe the dynamics of the LC ordering transition driven by GM1 to be slow (2h for DLPC versus 100h for GM1). To provide insight into the origins of the slow dynamics of the GM1-induced ordering transition in the LC, we performed two additional measurements. First, we quantified the time-dependent adsorption of GM1 at the LC interface by using fluorescently-labeled GM1. Second, we used the Langmuir–Schaefer method to transfer preorganized monolayers of GM1 from an air–water interface to the aqueous-LC interface. Results obtained from these two experiments are consistent with a physical picture in which the final stages of spontaneous adsorption/ordering of GM1 at the aqueous-LC interface dictate the dynamics of the LC ordering transition. This rate limiting process underlying the ordering transition was substantially accelerated by heating the system above the phase transition temperature of GM1(26°C), suggesting that the phase state of the GM1 micellar aggregates in bulk solution strongly influences the kinetics of the final stages of ordering/adsorption of GM1 at the LC interface. Overall, these results and others presented in this manuscript reveal that it is possible to decorate interfaces of a nematic LC with GM1, and that the assembly of GM1 at these interfaces impacts the dynamic and equilibrium ordering of the LC. [Copyright &y& Elsevier]

Abstract: A method to coat hydrophobic surfaces with lipid molecules in a reproducible manner and in which the lipid molecules are resistant to detergent washings, would benefit the development of new ELISA assays. This work presents different approaches to build 1,2-dioleolyl-sn-glycero-3-phosphocholine (DOPC) layers doped with a monosialoganglioside (GM1) supported on silica surfaces, which are stable toward buffer rinsing and washing with surfactant (Tween 20). The three methods employed were: method 1, coadsorption of DOPC:GM1 (0–10 mol%) with the surfactant n-dodecyl-β-D-maltoside (DDM) from micellar solutions, with successive adsorption and rinsing steps; method 2, vesicle fusion from DOPC: GM1 (0–10 mol%) liposomes; and method 3, deposition of GM1 from organic solvent (chloroform) and exposure to an aqueous environment (hydration method). The vesicle fusion method was also tested in polystyrene surfaces. Cholera toxin subunit B (CTB) was used to detect the presence of GM1 on the formed layers. The results indicated that the vesicle fusion was the only method that was successful in creating stable mono- and bilayers onto hydrophobized and hydrophilic silica, respectively. The mixed micellar solution method was suitable for creating pure lipid (DOPC) monolayers but the incorporation of GM1 in the micelles led to monolayers which were very unstable with respect to buffer rinsing. The hydration method led to monolayers of GM1 that were partly rinsed off by a continuous buffer flow. Adsorption of CTB was found to be proportional to the amount of GM1 present in the liposomes. The amount of CTB adsorbed onto the lipid bilayers was roughly the double as the one determined on the monolayers with the same liposome compositions. The vesicle fusion method was also able to create monolayers of pure DOPC and DOPC:10 mol% GM1 on the polystyrene surfaces. [Copyright &y& Elsevier]

Abstract: GM1 gangliosidosis is an inherited neurodegenerative disorder caused by lysosomal β-galactosidase deficiency, resulting in the storage of GM1 and GA1, primarily in the central nervous system. This disease typically afflicts infants and young children and there is currently no effective therapy. Substrate reduction therapy (SRT) could be of potential benefit. The imino sugars N-butyldeoxynojirimycin (NB-DNJ, miglustat, Zavesca™) and N-butyldeoxygalactonojirimycin (NB-DGJ) used for SRT inhibit glucosylceramide synthase (GlcCerS) that catalyses the first committed step in glycosphingolipid biosynthesis. We have compared the efficacy and tolerability of NB-DNJ and NB-DGJ in the β-galactosidase knockout mouse. NB-DGJ was better tolerated than NB-DNJ, due to intrinsic gastrointestinal tract dysfunction that was exacerbated by NB-DNJ. However, functional improvement was greatest with NB-DNJ treatment which may potentially be caused by novel anti-inflammatory properties of NB-DNJ. [Copyright &y& Elsevier]

Abstract: Membrane rafts are small and dynamic regions enriched in sphingolipids, cholesterol, ganglioside GM1 and protein markers like flotillins, forming the flatter domains or caveolins, which are characterized as stable flask-shape invaginations. We explored whether membrane rafts participate in the entry of Trypanosoma cruzi’s trypomastigotes into murine macrophages through transient depletion of macrophage membrane cholesterol with methyl-beta-cyclodextrin and treatment with filipin. These treatments led to a decrease in the trypomastigote invasion process. Macrophage pre incubated with increasing concentrations of cholera toxin B, that binds GM1, inhibited the adhesion and invasion of trypomastigote and amastigote forms. Immunofluorescence analysis demonstrated a colocalization of GM1, flotillin 1 and caveolin 1 in the T. cruzi parasitophorous vacuole. Taken together these data suggest that membrane rafts, including caveolae, are involved in the process of T. cruzi invasion of macrophages. [Copyright &y& Elsevier]

Abstract: Mutations at the attractin (Atrn) locus in mice result in altered pigmentation on an agouti background, higher basal metabolic rate and juvenile-onset hypomyelination leading to neurodegeneration, while studies on human immune cells indicate a chemotaxis regulatory function. The underlying biochemical defect remains elusive. In this report we identify a role for attractin in plasma membrane maintenance. In attractin''s absence there is a decline in plasma membrane glycolipid-enriched rafts from normal levels at 8 weeks to a complete absence by 24 weeks. The structural integrity of lipid rafts depends upon cholesterol and sphingomyelin, and can be identified by partitioning within of ganglioside GM1. Despite a significant fall in cellular cholesterol with maturity, and a lesser fall in both membrane and total cellular GM1, these parameters lag behind raft loss, and are normal when hypomyelination/neurodegeneration has already begun thus supporting consequence rather than cause. These findings can be recapitulated in Atrn-deficient cell lines propagated in vitro. Further, signal transduction through complex membrane receptor assemblies is not grossly disturbed despite the complete absence of lipid rafts. We find these results compatible with a role for attractin in plasma membrane maintenance and consistent with the proposal that the juvenile-onset hypomyelination and neurodegeneration represent a defect in attractin-mediated raft-dependent myelin biogenesis. [Copyright &y& Elsevier]

Abstract: Campylobacteriosis is frequently associated with Guillain–Barré syndrome. Poultry are frequently highly colonized with Campylobacter jejuni and are a major foodborne vehicle for campylobacteriosis. In this study, high titer anti-GM1 antibodies were found in the serum of a laboratory worker who developed campylobacteriosis. The microbiologically confirmed strain VLA2/18 (non-serotyped) was isolated from the worker and subsequently inoculated into chickens, resulting in high titers of serum antibodies to GM1. However, none of the immunized chickens in our study showed any noticeable neurological symptoms, such as paralysis or cramping. High titer anti-GM1 antibodies in chicken and human sera strongly inhibited spontaneous muscle action potential in an in vitro system of spinal cord and muscle cell co-culture. In addition, infection of chickens with C. jejuni strains 81116 (HS6) and 99/419 (HS21) or immunization with purified GM1, GM2, and GM3 resulted in elevation of serum anti-ganglioside antibodies with an inhibitory effect on spontaneous muscle action potential. Immunoabsorption studies demonstrated that this inhibitory activity is due to anti-ganglioside antibodies. On the other hand, anti-GM1 is the only specific human serum antibody to induce an inhibitory effect on neuromuscular junctions. Chicken anti-GM1 antibodies showed a strong inhibitory effect, but anti-GM2 and -GM3 had weaker activities. Taken together, our data suggest that campylobacteriosis in chickens may provide a strong link between infection and the development of anti-ganglioside antibody-mediated peripheral nerve dysfunctions. [Copyright &y& Elsevier]

Abstract: Monosialoganglioside (GM1) is a glycosphingolipid that protects against some neurological conditions, such as seizures and ischemia. Glutaric acidemia type I (GA-I) is an inherited disease characterized by striatal degeneration, seizures, and accumulation of glutaric acid (GA). In this study, we show that GA inhibits Na+,K+-ATPase activity and increases oxidative damage markers (total protein carbonylation and thiobarbituric acid-reactive substances—TBARS) production in striatal homogenates from rats in vitro and ex vivo. It is also shown that GM1 (50 mg/kg, i.p., twice) protects against GA-induced (4 μmol/striatum) seizures, protein carbonylation, TBARS increase, and inhibition of Na+,K+-ATPase activity ex vivo. Convulsive episodes induced by GA strongly correlated with Na+,K+-ATPase activity inhibition in the injected striatum but not with oxidative stress marker measures. Muscimol (46 pmol/striatum), but not MK-801 (3 nmol/striatum) and DNQX (8 nmol/striatum) prevented GA-induced convulsions, increase of TBARS and protein carbonylation and inhibition of Na+,K+-ATPase activity. The protection of GM1 and muscimol against GA-induced seizures strongly correlated with Na+,K+-ATPase activity maintenance ex vivo. In addition, GM1 (50–200 μM) protected against Na+,K+-ATPase inhibition induced by GA (6 mM) but not against oxidative damage in vitro. GM1 also decreased pentylenetetrazole (PTZ)-induced (1.8 μmol/striatum) seizures, Na+,K+-ATPase inhibition, and increase of TBARS and protein carbonyl in the striatum. These data suggest that Na+,K+-ATPase and GABAA receptor-mediated mechanisms may play important roles in GA-induced seizures and in their prevention by GM1. [Copyright &y& Elsevier]

Abstract: Lipid bilayers containing 5% nitrilotriacetic acid (NTA) lipids supported on SiO2 have been used as a template for immobilization of oligohistidine-tagged single-chained antibody fragments (scFvs) directed against cholera toxin. It was demonstrated that histidine-tagged scFvs could be equally efficiently coupled to an NTA–Ni2+-containing lipid bilayer from a purified sample as from an expression supernatant, thereby providing a coupling method that eliminates time-consuming protein prepurification steps. Irrespective of whether the coupling was made from the unpurified or purified antibody preparation, the template proved to be efficient for antigen (cholera toxin) detection, verified using quartz crystal microbalance with dissipation monitoring. In addition, via a secondary amplification step using lipid vesicles containing GM1 (the natural membrane receptor for cholera toxin), the detection limit of cholera toxin was less than 750pM. To further strengthen the coupling of scFvs to the lipid bilayer, scFvs containing two histidine tags, instead of just one tag, were also evaluated. The increased coupling strength provided via the bivalent anchoring significantly reduced scFv displacement in complex solutions containing large amounts of histidine-containing proteins, verified via cholera toxin detection in serum. [Copyright &y& Elsevier]

We report here the interaction of bradykinin with ganglioside GM1 by circular dichroism, steady-state fluorescence, and one-dimensional 1H NMR spectroscopy. Circular dichroism spectroscopy is indicative of a turn formation of bradykinin backbone in the presence of GM1 micelle. The fluorescence quenching efficiencies of iodide and acrylamide are substantially reduced, indicating a shielding of phenylalanine residue of bradykinin from aqueous environment. Significant line broadening of NMR resonances of bradykinin, suggestive of motional restriction, is observed. [Copyright &y& Elsevier]

The gangliosides of carp intestinal mucosa were isolated and analysed by thin-layer chromatography (TLC), TLC immunostaining test, and TLC/secondary ion mass spectrometry (TLC/SIMS). Four species of gangliosides, designated as G-1, G-2, G-3 and G-4, were separated on TLC. The TLC/SIMS analysis of the G-1 ganglioside of carp intestinal mucosa revealed a series of [M-H]−ions from m/z 1061 to m/z 1131 representing the molecular mass range of GM4-like ganglioside with NeuAc. G-2, G-3 and G-4 gangliosides were analysed by the TLC immunostaining test. G-2 ganglioside was recognised by the monoclonal antibody specific for ganglioside GM1 (AGM-1 monoclonal antibody). However, G-3 ganglioside migrating on TLC between GM3 and GM1 ganglioside was not recognised by anti-GM3 monoclonal antibody and by AGM-1 monoclonal antibody. Furthermore, G-4 ganglioside with a similar TLC mobility as GD1a ganglioside did not show the reactivity to the anti-GD1a monoclonal antibody. In addition using the AGM-1 monoclonal antibody, the expression of GM1 ganglioside in the carp intestinal tissue was studied. GM1 ganglioside was detected on the epithelial cell surface of carp intestinal mucosa. [Copyright &y& Elsevier]

Components of caveolae and lipid rafts are characterized by their buoyancy after detergent extraction. Using flotations in density gradients, we now show that non-raft membrane molecules are also associated with detergent-insoluble, buoyant assemblies. When Triton X-100 cellular extracts were spun to equilibrium in Nycodenz, only components of classical rafts floated. In contrast, with the zwitterionic detergent CHAPS, non-raft residents such as calnexin and APP also buoyed. When CHAPS extracts were spun in non-equilibrium (velocity) conditions, some raft components rapidly exited the input fractions while other raft markers and non-raft molecules remained relatively immobile. This pointed to size heterogeneities of CHAPS-insoluble complexes. Combined velocity/equilibrium gradients broadly divided CHAPS-insoluble membrane complexes into three size categories, which all contained cholesterol and the glycosphingolipid GM1. Large complexes were enriched in caveolin and ESA. Medium size complexes were enriched in PrP, whereas small complexes contained non-raft proteins, PrP, and some ESA. While Alzheimer’s APP was primarily confined to small assemblies, a portion of its glycosylated form did buoy with large complexes. Large CHAPS-insoluble complexes resemble, but are not equal to, classical rafts. These findings extend considerably the range of detergent-insoluble membranal domains. [Copyright &y& Elsevier]

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and Ras proteins are involved in signalling pathways originating at the plasma membrane. The localisation and metabolism of PI(4,5)P2 was studied in Jurkat T cells using fluorescence microscopic imaging with EGFP-tagged and antibody probes. Software was developed to objectively quantitate colocalisation and was used to show that plasma membrane PI(4,5)P2 was enriched in lipid raft-containing patches of GM1 ganglioside, formed by crosslinking cholera toxin B-subunit (CT-B). The PI(4,5)P2 metabolites phosphatidylinositol 3,4,5-trisphosphate and diacylglycerol appeared in plasma membrane CT-B-GM1 patches upon induction of signalling. Transferrin receptor and the CD45 tyrosine phosphatase did not colocalise with CT-B-GM1 patches, whereas the tyrosine kinase Lck, the scaffolding protein LAT, and endogenous Ras proteins did partially colocalise with CT-B-GM1 patches as did transfected EGFP-K-Ras(4B) and EGFP-H-Ras. The results demonstrate that T-cell PI(4,5)P2 metabolism is occurring in GM1-enriched domains and that Ras proteins are present in these domains in vivo. [Copyright &y& Elsevier]

Field emission scanning electron microscopy (FE SEM) was used to visualize the distribution of virus-associated components, the virus-attachment (G) protein, and the host-cell-derived lipid, GM1, in respiratory syncytial virus (RSV) filaments. RSV-infected cells were labeled in situ with a G protein antibody (MAb30) whose presence was detected using a second antibody conjugated to colloidal gold. No bound MAb30 was detected in mock-infected cells, whereas significant quantities bound to viral filaments revealing G protein clusters throughout the filaments. GM1 was detected using cholera toxin B subunit conjugated to colloidal gold. Mock-infected cells revealed numerous GM1 clusters on the cell surface. In RSV-infected cells, these gold clusters were detected on the filaments in low, but significant, amounts, indicating the incorporation of GM1 within the viral envelope. This report describes the first use of FE SEM to map the distribution of specific structural components within the envelope of a Paramyxovirus. [Copyright &y& Elsevier]

We report here the interaction of melittin with ganglioside GM1 by steady-state fluorescence, one-dimensional 1H NMR spectroscopy and molecular modeling. In the presence of GM1 the emission maximum of melittin is blue shifted and fluorescence quenching efficiencies of iodide and acrylamide are substantially reduced, indicating a shielding of tryptophan of melittin from aqueous environment. Significant line broadening of NMR resonances of melittin, suggestive of motional restriction, is observed. Molecular modeling indicates a melittin–GM1 complex with N-terminal hydrophobic stretch of melittin associating with the ceramide tail and C-terminal hydrophilic end of melittin having favorable electrostatic interaction with the carbohydrate head group of GM1. [Copyright &y& Elsevier]

Mice genetically engineered to lack complex gangliosides are improved hosts for raising antibodies against those gangliosides. We report the generation and characterization of nine immunoglobulin G (IgG)-class monoclonal antibodies (mAbs) raised against the four major brain gangliosides in mammals. These include (designated as ganglioside specificity-IgG subclass) two anti-GM1 mAbs (GM1-1, GM1-2b), three anti-GD1a mAbs (GD1a-1, GD1a-2a, GD1a-2b), one anti-GD1b mAb (GD1b-1), and three anti-GT1b mAbs (GT1b-1, GT1b-2a, GT1b-2b). Each mAb demonstrated high specificity, with little or no cross-reactivity with other major brain gangliosides. Enzyme-linked immunosorbent assay (ELISA) screening against 14 closely related synthetic and purified gangliosides confirmed the high specificity, with no significant cross-reactivity except that of the anti-GD1a mAbs for the closely related minor ganglioside GT1aα. All of the mAbs were useful for ELISA, TLC immunooverlay, and immunocytochemistry. Neural cells from wild-type rats and mice were immunostained to differing levels with the anti-ganglioside antibodies, whereas neural cells from mice engineered to lack complex gangliosides (lacking the ganglioside-specific biosynthetic enzyme UDP-GalNAc:GM3/GD3 N-acetylgalactosaminyltransferase) remained unstained, demonstrating that most of the mAbs react only with gangliosides and not with related structures on glycoproteins. These mAbs may provide useful tools for delineation of the expression and function of the major brain gangliosides and for probing the pathology of anti-ganglioside autoimmune diseases. [Copyright &y& Elsevier]