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PubMed1999

Structural characterization of gangliosides isolated from mullet milt using electrospray ionization-tandem mass spectrometry.

Electrospray ionization (ESI) coupled with tandem mass spectrometry has been used in conjunction with microwave-mediated saponification, periodate oxidation, and clostridial sialidase hydrolysis to enable detailed structural characterization of gangliosides and their derivatives present in mullet milt. The gangliosides extracted from mullet milt were determined to be GM3, GM3 lactone, GM3 methyl ester, and 9-O-acetyl GM3. For the major ganglioside GM3 and all GM3 derivatives, the ceramide composition was revealed to be C18:1/C16:0. GM3 with a C18:0/C16:0 ceramide was also found as a minor ganglioside. Both the ganglioside intramolecular ester and the ganglioside methyl ester (lacking carboxylic acid groups) showed dominant chloride attachment peaks (M + Cl)- in negative ion ESI-MS in addition to low intensity peaks corresponding to (M-H)-. GM3 and O-acetyl GM3 bearing carboxylic acid functions showed only (M-H)-. In positive ion ESI, GM3 and O-acetyl GM3 revealed (M + 2Na-H)+ peaks in addition to (M + Na)+, indicating free exchange of the carboxylic acid proton with a sodium cation, while the ganglioside intramolecular ester and ganglioside methyl ester with no acidic protons yielded only (M + Na)+. The strategy of employing ESI-MS to detect products of established wet chemical reactions represents a general approach for elucidation of ganglioside structural details.

Animals↗
PubMed2004

Involvement of gangliosides in glucosamine-induced proliferation decrease of retinal pericytes.

The hexosamine pathway (HP) is a biochemical hypothesis recently proposed explaining cellular alterations occurring during diabetic microvascular complications. Diabetic retinopathy is a common microvascular complication of diabetes, and it is known that cell proliferation is severely affected during the development of the disease. Particularly, early stages are characterized by death of the retinal microvascular cells, pericytes. Gangliosides have often been described to regulate cell growth; however, very few studies focused on the potential role of gangliosides in diabetic microvascular alterations. The aim of this article was to investigate the effect of the HP activation on pericyte proliferation and determine the potential implication of gangliosides in this process. Results indicate first that HP activation, mimicked by glucosamine treatment, decreased pericyte proliferation. Second, glucosamine treatment induced a modification of gangliosides pattern, particularly GM1 and GD3 were significantly increased. Next, results showed that exogenous addition of a-series gangliosides (GM3, GM2, GM1, GD1a) and b-series ganglioside (GD3) caused a decrease of pericyte proliferation, whereas nonsialylated precursors glucosylceramide and lactosylceramide were without effect. Furthermore, when ganglioside biosynthesis was blocked using PPMP, a glucosylceramide synthase inhibitor, the effects of glucosamine on pericyte proliferation were partially reversed. Our results suggest that in retinal pericytes, gangliosides and particularly GM1 and GD3 that are increased in response to glucosamine, are involved in the antiproliferative effect of glucosamine. These observations also underlie the potential involvement of gangliosides in a pathological context, such as diabetic microvascular complications.

Animals↗
PubMed1998

Further characterization of equine brain gangliosides: the presence of GM3 having N-glycolyl neuraminic acid in the central nervous system.

Equine brain gangliosides were isolated and their structures were characterized, to examine whether equine brain has N-glycolyl neuraminic acid in gangliosides, since other mammals predominantly possess N-acetyl neuraminic acid in brain gangliosides, and equine erythrocytes and organs except the brain have gangliosides exclusively containing N-glycolyl neuraminic acid. The gangliosides purified from the brain were identified by proton NMR spectroscopy and mass spectrometry, as well as GLC, resulting in their identification as GM4, GM3, GM2, GM1, GD1a, GD1b, and GT1b. Of these gangliosides, GM3 possessed N-glycolyl neuraminic acid as a minor component (18% of the total GM3), whereas other gangliosides exclusively contained N-acetyl neuraminic acid. The N-glycolyl neuraminic acid residue of the GM3 was confirmed by TLC immunostaining. The possibility of contamination of the GM3 by erythrocytes was eliminated based on the finding that the lipid compositions were characteristic of brain gangliosides. The presence, even as a minor component, of the N-glycolyl neuraminic acid in equine brain gangliosides is exceptional among the sialic acid species in mammalian central nervous system.

Animals↗
PubMed1983

Cellular localization of gangliosides in the developing mouse cerebellum: analysis using the weaver mutant.

The distribution of gangliosides was studied in the weaver (wv/wv) mutant mouse, where the vast majority of postmitotic granule cell neurons die prior to their differentiation. The wv mutation also shows a dosage effect, as granule cell migration is slowed or retarded in the +/wv heterozygotes. By correlating changes in ganglioside composition with the well-documented histological events that occur during cerebellar development in the normal (+/+), heterozygous (+/wv), and weaver (wv/wv) mutant mice, information was obtained on the cellular localization and function of gangliosides. Ganglioside GM1 may be enriched in granule cell growth cones and play an important role in neurite outgrowth. A striking accumulation of GM1, which may result from altered metabolism, occurred in the adult wv/wv mice. GD3 was heavily concentrated in undifferentiated granule cells, but was rapidly displaced by the more complex gangliosides during differentiation. GD1a became enriched in granule cells during formation of synaptic and dendritic membranes, whereas GT1a appeared enriched in Purkinje cell synaptic spines. A possible fucose-containing ganglioside was quantitated only in the wv/wv mice. Ganglioside GT1b became enriched in granule cells during synaptogenesis, whereas GQ1b became enriched in these cells after synaptogenesis. The concentrations of GT1b and especially GQ1b increased continuously with age. Our results provide further evidence for a differential cellular enrichment of gangliosides in the mouse cerebellum and also suggest that certain gangliosides may be differentially distributed within the membranes of these cells at various stages of development.

Aging↗
PubMed1998

Structure and function of a ganglioside receptor for porcine rotavirus.

A ganglioside fraction isolated from pooled intestines from newborn to 4-week-old piglets, which we previously partially characterized and showed to specifically inhibit the binding of porcine rotavirus (OSU strain) to host cells (M. D. Rolsma, H. B. Gelberg, and M. S. Kuhlenschmidt, J. Virol. 68:258-268, 1994), was further purified and found to contain two major monosialogangliosides. Each ganglioside was purified to apparent homogeneity, and their carbohydrate structure was examined by high-pH anion-exchange chromatography coupled with pulsed amperometric detection and fast atom bombardment mass spectroscopy. Both gangliosides possessed a sialyllactose oligosaccharide moiety characteristic of GM3 gangliosides. Compositional analyses indicated that each ganglioside was composed of sialic acid, galactose, glucose, and sphingosine in approximately a 1:1:1:1 molar ratio. Each ganglioside differed, however, in the type of sialic acid residue it contained. An N-glycolylneuraminic acid (NeuGc) moiety was found in the more polar porcine GM3, whereas the less polar GM3 species contained N-acetylneuraminic acid (NeuAc). Both NeuGcGM3 and NeuAcGM3 displayed dose-dependent inhibition of virus binding to host cells. NeuGcGM3 was approximately two to three times more effective than NeuAcGM3 in blocking virus binding. Inhibition of binding occurred with as little as 400 pmol of NeuGcGM3/50 ng of virus (approximately 2 x 10(7) virions) and 2 x 10(6) cells/ml. Fifty percent inhibition of binding was achieved with 0.64 and 1.5 microM NeuGcGM3 and NeuAcGM3, respectively. The free oligosaccharides 3'- and 6'-sialyllactose inhibited binding 50% at millimolar concentrations, which were nearly 1,000 times the concentration of intact gangliosides required for the same degree of inhibition. Direct binding of infectious, triple-layer rotavirus particles, but not noninfectious, double-layered rotavirus particles, to NeuGcGM3 and NeuAcGM3 was demonstrated by using a thin-layer chromatographic overlay assay. NeuGcGM3 and NeuAcGM3 inhibited virus infectivity of MA-104 cells by 50% at concentrations of 3.97 and 9. 84 microM, respectively. NeuGcGM3 (700 nmol/g [dry weight] of intestine) was found to be the predominant enterocyte ganglioside (comprising 75% of the total lipid-bound sialic acid) in neonatal piglets, followed by NeuAcGM3 (200 nmol/g [dry weight] of intestine). NeuGcGM3 and NeuAcGM3 together comprised nearly 100% of the lipid-bound sialic acid in the neonatal intestine, but their quantities rapidly diminished during the first 5 weeks of life. These data support the hypothesis that porcine NeuGcGM3 and NeuAcGM3 are physiologically relevant receptors for porcine rotavirus (OSU strain). Further support for this hypothesis was obtained from virus binding studies using mutant or neuraminidase-treated cell lines. Lec-2 cells, a mutant clone of CHO cells characterized by a 90% reduction in sialyllation of its glycoconjugates, bound less than 5% of the virus compared to control cell binding. In contrast, Lec-1 cells, a mutant CHO clone characterized by a deficiency in glycosylation of N-linked oligosaccharides, still bound rotavirus. Furthermore, exogenous addition of NeuGcGM3 to the Lec-2 mutant cells restored their ability to bind rotavirus in amounts equivalent to that of their parent (CHO) cell line. In the virus-permissive MA-104 cell line, NeuGcGM3 was also able to partially restore rotavirus infectivity in neuraminidase-treated cells. These data suggest that gangliosides play a major role in recognition of host cells by porcine rotavirus (OSU strain).

Animals↗
PubMed1993

Protein kinase C substrates and ganglioside inhibitors in bovine mammary nuclei.

In cow mammary gland, unlike in other tissues, gangliosides (putative physiologic regulators of protein kinase C) may be distributed in nuclei and on the cell surface. This study was designed to determine whether gangliosides and the protein kinase C system (the enzyme and its substrate proteins) are present in cow mammary gland nuclei and to examine the effect of gangliosides detected in nuclei on protein phosphorylation catalyzed by protein kinase C. Gangliosides GM3, GD3, and GT1b were detected in the highly purified nuclear fraction. The nuclear ganglioside pattern was different from those of whole tissue and cytosol, thereby suggesting the presence of the gangliosides in nuclei. Protein kinase C and its substrate proteins (120, 97, 56, 43, 38, and 36 kDa) were extracted by Triton X-100 treatment of nuclei. Both protein kinase C activity (histone phosphorylation) and the nuclear substrate phosphorylation were effectively inhibited by the three gangliosides. Of the gangliosides, GT1b was the most potent in inhibiting phosphorylation, followed by GD3 and GM3. These results suggest that signal transduction mediated by protein kinase C in cow mammary gland nuclei may be regulated by gangliosides.

Animals↗
PubMed1999

Tumor gangliosides inhibit the tumor-specific immune response.

Tumor gangliosides are highly immunosuppressive membrane glycosphingolipids that are shed into the tumor cell microenvironment. We directly tested the impact of shed gangliosides on the in vivo antitumor immune response in a syngeneic fully autochthonous system (FBL-3 erythroleukemia cells, C57BL/6 mice, and highly purified FBL-3 cell gangliosides). The major FBL-3 ganglioside was identified as GM1b by mass spectrometry. Substantial ganglioside shedding (90 pmol/108 cells/h), a requisite for their inhibition of the immune function of tumor-infiltrating leukocytes, was detected. Immunosuppression by FBL-3 gangliosides was potent; 5-20 microM inhibited the tumor-specific secondary proliferative response (80-100%) and suppressed the generation of tumor-specific CTLs (97% reduction of FBL-3 cell lysis at an E:T ratio of 100:1). In vivo, coinjection of 10 nmol of FBL-3 gangliosides with a primary FBL-3 cell immunization led to a reduced response to a secondary challenge (the increase in the draining popliteal lymph node mass, cell number, and lymphocyte thymidine incorporation were lowered by 70, 69, and 72%, respectively). Coinjection of gangliosides with a secondary tumor challenge led to a 61, 74, and 42% reduction of the increase in lymph node mass, cell number, and thymidine uptake and a 63-74% inhibition of the increase of draining lymph node T cells (CD3+), B cells (CD19+), and dendritic cells/macrophages (Mac-3+). Overall, the clear conclusion that tumor-derived gangliosides inhibit syngeneic antitumor immune responses implicates these molecules as a potent factor in promoting tumor formation and progression.

Animals↗
PubMed1999

Long-term treatment of chronic chagasic cardioneuropathy with a mixture of gangliosides: effect on evolution of circulating antiGM1 antibodies.

Chagas disease involves a cardiac impairment, being the first alterations of autonomic disorders which affect heart rate and blood pressure control. At this stage, diminished heart rate responses to atropine and propranolol are observed. Prior studies have shown that short term ganglioside treatment improves the responses to these agents, but there is no information about the long term effect of gangliosides and the evolution of antiGM1 titers. The effects of long term treatment with gangliosides on autonomic tests in patients with chagasic cardiodisautonomy and the evolution of antiGM1 titers were studied in 90 patients (57 men, 33 women, aged 25-60 years) with positive serology for Chagas disease and electrocardiogram showing sinusal bradycardia and incomplete right branch block, without cardiomegaly, with autonomic alterations by postural and Valsalva's tests. All patients were submitted to a test that consisted of intravenous injection of atropine 0.04 mg/kg followed 3 min later by intravenous injection of propranolol 0.2 mg/kg. During these tests heart rate and blood pressure were recorded continuously. Subsequently, 30 patients were treated with 100 mg/day of a mixture of gangliosides by intramuscular injection during 15 days in a row, followed by 40 mg/day during another 75 days. Another 30 patients received continuous treatment for 12 months. The remaining 30 patients were controls. The antiGM1 antibody circulating titers were determined before the treatment, at the third and 12th month. Seventy-four patients completed the study. Before treatment, the heart rate increased, though slightly, after the injection of atropine. After 3 months of ganglioside treatment a statistically significant increase in the response to atropine was recorded. In the controls at 12 months, the response to atropine remained increased without differences between the patients treated for 3 and 12 months. The control patients did not show any modification of the heart rate response during 12 months. Both ganglioside-treated groups showed an increase in the response to propranolol. The antiGM1 titer distribution was similar in both healthy subjects and chagasic patients. None of the patients had positive antiGM1 titers in basal conditions nor significant modifications after the ganglioside treatment. Chagasic cardioneuropathy was not associated in this study with high antiGM1 antibody titers. Chagasic patients showed a diminished heart rate response to atropine as well as to propranolol. Ganglioside treatment determined an increased heart rate response, particularly after atropine. Increased heart rate response was maintained until 1 year, without differences between the patients treated for 3 and 12 months. No changes in the antiGM1 titers were observed during the ganglioside treatment.

Adult↗
PubMed1990

Accumulation of gangliosides with N-acetylneuraminosyl(alpha 2-6)lactosamine structure in primary human hepatoma.

Gangliosides of hepatomas have been analyzed by using a monoclonal antibody directed to N-acetylneuraminosyl(alpha 2-6)lactoneotetraosylceramide (sialyl(alpha 2-6)paragloboside), which was prepared by injecting the monosialoganglioside fraction of human meconium into BALB/c mice. The monoclonal antibody, named MSG-15, was found to bind sialyl(alpha 2-6)paragloboside, but it failed to react with other gangliosides, including N-acetylneuraminosyl(alpha 2-3)lactoneotetraosylceramide (sialyl (alpha 2-3)paragloboside) and "Ii"-type gangliosides. MSG-15 was found to recognize NeuAc alpha 2-6Gal beta structure of the ganglioside. Gangliosides obtained from human hepatomas were analyzed by immunostaining on high-performance thin-layer chromatography plates using the monoclonal antibody MSG-15. All primary hepatoma samples used in this study (nine samples) were found to contain sialyl(alpha 2-6)paragloboside, which accounted for 13-31% of the monosialoganglioside fractions in the hepatomas. Furthermore, MSG-15 recognized several monosialogangliosides in addition to sialyl(alpha 2-6)paragloboside. These gangliosides apparently also contain a terminal NeuAc alpha 2-6Gal beta structure. Other ganglioside fractions obtained from hepatoma and meconium were immunostained on thin layer chromatography plates with MSG-15. Additionally, another monoclonal antibody (H-11), which recognizes terminal lactosamine structure, was used to immunostain these fractions after sialidase treatment. Bands stained with both monoclonal antibodies showed similar mobilities to each other in the di- and trisialoganglioside fractions as well as monosialoganglioside fraction. In control liver, GM3 ganglioside accounted for 92% of monosialoganglioside fraction, and sialyl(alpha 2-6)paragloboside accounted for less than 1% of the fraction. Immunohistochemical study by using MSG-15 in tissue sections from hepatocellular carcinoma and normal liver tissues demonstrated that only hepatocellular carcinoma cells gave a positive reaction. These results suggest that the biosynthetic pathway of gangliosides containing NeuAc alpha 2-6Gal beta 1-4GlcNAc beta structure is activated in hepatoma cells.

Animals↗
PubMed1998

Inhibition of hemopoiesis in vitro by neuroblastoma-derived gangliosides.

Hemopoiesis is disturbed in bone marrow-involving cancers like leukemia and neuroblastoma. Shedding of gangliosides by tumor cells may contribute to this tumor-induced bone marrow suppression. We studied in vitro the inhibitory effects of murine neuroblastoma cells (Neuro-2a and C1300) and their gangliosides on hemopoiesis using normal murine hemopoietic progenitor colony-forming assays. Transwell cultured neuroblastoma cells showed a dose-dependent inhibition on hemopoiesis, indicating that a soluble factor was responsible for this effect. Furthermore, the supernatant of Neuro-2a cultured cells inhibited hemopoietic proliferation and differentiation. To determine whether the inhibitory effect was indeed due to shed gangliosides and not, for instance, caused by cytokines, the effect of DL-threo-1 -phenyl-2-decanoylamino-3-morpholino-1-propanol (DL-PDMP) on Neuro-2a cells was studied. DL-PDMP is a potent inhibitor of glucosylceramide synthase, resulting in inhibition of the synthesis and shedding of gangliosides. The initially observed inhibitory effect of supernatant of Neuro-2a cells was abrogated by culturing these cells for 3 days in the presence of 10 microM DL-PDMP. Moreover, gangliosides isolated from Neuro-2a cell membranes inhibited hemopoietic growth. To determine whether the described phenomena in vitro are a reflection of bone marrow suppression occurring in vivo, gangliosides isolated from plasma of neuroblastoma patients were tested for their effects on human hemopoietic progenitor colony-forming assays. These human neuroblastoma-derived gangliosides inhibited normal erythropoiesis (colony-forming unit-erythroid/burst-forming unit-erythroid) and myelopoiesis (colony-forming unit-granulocyte/macrophage) to a higher extent compared with gangliosides isolated from control plasma. Altogether these results suggest that gangliosides shed by neuroblastoma cells inhibit hemopoiesis and may contribute to the observed bone marrow depression in neuroblastoma patients.

Animals↗
PubMed2002

Interaction of the extracellular domain of the epidermal growth factor receptor with gangliosides.

Ganglioside GM3 inhibits epidermal growth factor (EGF)-dependent cell proliferation in a variety of cell lines. Both in vitro and in vivo, this glycosphingolipid inhibits the kinase activity of the EGF receptor (EGFR). Furthermore, membrane preparations containing EGFR can bind to GM3-coated surfaces. These data suggest that GM3 may interact directly with the EGFR. In this study, the interaction of gangliosides with the extracellular domain (ECD) of the EGFR was investigated. The purified human recombinant ECD from insect cells bound directly to ganglioside GM3. The ganglioside interaction site appears to be distinct from the EGF-binding site. In agreement with previous reports on the effects of specific gangliosides on EGFR kinase activity, the ECD preferentially interacted with GM3. The order of relative binding of other gangliosides investigated was as follows: GM3 GM2, GD3, GM4 > GM1, GD1a, GD1b, GT1b, GD2, GQ1b > lactosylceramide. These data suggest that NeuAc-lactose is essential for binding and that any sugar substitution reduces binding. In agreement with the specificity of soluble ECD binding to gangliosides, GM3 specifically inhibited EGFR autophosphorylation. Identification of a ganglioside interaction site on the ECD of the EGFR is consistent with the hypothesis that endogenous GM3 may function as a direct modulator of EGFR activity.

Animals↗
PubMed1999

Alterations in ganglioside expression during the differentiation of human mast cells.

Gangliosides are physiological components of the outer cell membrane. In the present study, the role of ganglioside expression during differentiation of human mast cells was evaluated. After 11 days of culture in medium known to induce mast cell differentiation, 70% of peripheral blood mononuclear cells (PBMC) showed positive staining for the high affinity IgE receptor and tryptase on immunocytochemistry and an associated 20-fold increase of ganglioside GM3 expression. Furthermore, exogenous addition of GM3 during cultivation of PBMC in medium containing low levels of growth factors induced an increase of mast cell specific tryptase. The association of ganglioside expression with mast cell differentiation was confirmed by experiments with the human mast cell line HMC-1. FcepsilonRI-positive cultured cells enriched with immunobeads exhibited a 3-fold higher expression of GM3, compared to FcepsilonRI negative HMC-1 cells. Furthermore, measurable amounts of the gangliosides GM2, GM1 and GD1a were found only in the FcepsilonRI positive cells. A corresponding transient increase of mRNA for GalNAcT, the key enzyme in the production of these latter gangliosides, could be detected preceding the expression of these gangliosides and the FcepsilonRI by RT-PCR. Taken together, these data point to a functional role of gangliosides in the differentiation of human mast cells.

Basophils↗
PubMed1991

Humoral immune response in disease-free advanced melanoma patients after vaccination with melanoma-associated gangliosides. EORTC Cooperative Melanoma Group.

Several studies have shown that melanoma-associated gangliosides are immunogenic in melanoma patients and that antibodies against them have a favorable prognostic effect. Our study aims at characterizing the humoral immune response in disease-free, advanced melanoma patients vaccinated with a total ganglioside fraction extracted from pooled metastases of human melanoma, containing as major gangliosides GM3 and GD3, and as minor ones GM2 and GD2. Prior to vaccination, all patients were made disease-free by surgical removal of skin, lymph-node or other distant metastases. Repeated vaccinations were carried out intradermally with gangliosides either in the native form in buffered solution, or in the form of liposomes. Serum samples were collected at regular intervals and assayed by ELISA for the presence of specific IgG and IgM antiganglioside antibodies. Selected samples were tested by immunostaining on thin-layer plates to specify the ganglioside species involved in the reactivity. Out of 32 evaluable patients, 17 presented a significant increase in antibody titer, mostly of the IgG isotype, which was maximal between 2 and 4 months after starting injections of gangliosides, and gradually disappeared within 1 year. No significant difference could be seen between the group of 20 patients treated with native gangliosides and the group of 12 vaccinated with the gangliosides in liposomes. All gangliosides seemed to be immunogenic, but GM2 and GD2 were somewhat more reactive. The disease-free intervals for the patients who showed an antibody response to the treatment were significantly higher (p less than 0001) than those of the non-responding group, as compared by the Kaplan-Meier method.

Antibody Formation↗
PubMed1994

Generation of monoclonal antibodies specific for ganglioside lactones: evidence of the expression of lactone on human melanoma cells.

We generated 3 murine monoclonal antibodies (MAbs) specific for ganglioside lactones by immunizing C3H/HeN mice with purified lactones adsorbed to Salmonella minnesota followed by fusion with mouse myeloma cells. The use of a wide variety of glycolipids, including ganglioside lactones, enabled the precise structures recognized by these MAbs to be elucidated through an ELISA and by immunostaining on thin-layer chromatography. MAb AMR38, which was generated with GM1 lactone, showed restricted specificity, detecting only the GM1 lactone used for immunization. None of the other ganglioside lactones, intact gangliosides (including GM1) or neutral glycolipids tested were recognized. In contrast, MAbs AMR40 and AMR19, which were generated with GD1a lactone and GD3 lactone, respectively, showed broader specificities, recognizing several ganglioside lactones. However, the precise epitopes were different. MAb AMR40 reacted intensely with ganglioside lactones having an external NeuAc alpha 2-->3Gal-sequence (GD1a, GM3, GM1b, GT1b, and IV3NeuAc alpha-nLc4Cer), but not with those having a NeuAc alpha 2-->8NeuAc alpha 2-->3Gal- sequence. On the other hand, MAb AMR19 reacted with ganglioside lactones having a NeuAc alpha 2-->8NeuAc alpha 2-->3Gal- sequence (GD3, O-Ac-GD3, GD2, GDlb, GTlb, GQlb and GTla), but not with those having a NeuAc alpha 2-->3Gal- sequence. None of the intact gangliosides or neutral glycolipids tested were recognized by the MAbs. We also determined the expression of ganglioside lactones on human melanoma cells grown in athymic nude mice by means of an immunofluorescence technique.

Animals↗
PubMed2002

Ethanol-induced increase of sphingosine recycling for ganglioside biosynthesis: a study performed on cerebellar granule cells in culture.

The effect of ethanol on ganglioside metabolism was assessed in cultured rat cerebellar granule cells. Cells were incubated in the presence of tritiated serine or galactose, and the synthesis of radioactive gangliosides was followed. The rate of de novo biosynthesis of gangliosides labeled in the oligosaccharide moiety (deriving from tritiated galactose) was not affected by the presence of ethanol. On the contrary, the biosynthesis of gangliosides labeled in the ceramide long chain base moiety (deriving from tritiated serine), dramatically decreased in the presence of alcohol. These results suggest that the gap between the extent of the biosynthesis of lipid and polar portions observed in the presence of ethanol, is filled by an increased recycling of sphingosine produced from ganglioside degradation. This hypothesis was confirmed by pulse-chase experiments with GM1 ganglioside, tritiated in the sphingosine moiety, and following radiolabeled gangliosides deriving from its metabolic processing. In fact, the radioactivity carried by gangliosides whose labeling could derive exclusively (GD1b + GT1b) or partially (GD1a) from the recycling of catabolic radiolabeled sphingosine, dramatically increased in ethanol-treated cells during the chase period. Taken together, these results suggest that ethanol increases ceramide sphingosine recycling for ganglioside biosynthesis.

Animals↗
PubMed1984

Metabolism and function of gangliosides in developing neurons.

Previous experiments have shown that the addition of a mixture of gangliosides to the growth medium induced morphological changes in primary neuronal cultures, producing especially a trophic effect and a sprouting of neurites (neuritogenesis). The study reported here examined the changes of some biochemical parameters that paralleled the morphological modifications of cultured neurons from chick brain hemispheres treated with gangliosides. Neurons cultured from 3 to 7 days in the presence of various concentrations of a purified mixture or of single-species of gangliosides (GM1, GD1a, GT1b) revealed that these glycolipids were easily incorporated into the cells as a function of their exogenous concentrations. Incubation of neurons with N-acetyl-D-[U-14C]mannosamine showed a final labeling of all endogenous cellular and exogenous incorporated gangliosides; however, the radioactivity recovered decreased as a function of the number of sialic acid units of the exogenously added gangliosides. The treatment of neuronal cells from 3 to 7 days in culture with a mixture of 10(-8) M and 10(-5) M gangliosides led to the following observations on some neurochemical parameters: no effect on the influx of choline and dopamine; no effect on the spontaneous choline efflux, whereas the K+-provoked one is abolished; decrease of the spontaneous and K+-stimulated release of dopamine; no effect on the spontaneous release of GABA for 10(-8) M gangliosides but an increase of both spontaneous and K+-provoked release for 10(-5) M gangliosides. The data suggest that the possible insertion of gangliosides into the neuronal membranes may imply structural modifications that may influence enzymatic activities, neurotransmitter transport, and finally, some nerve cell mechanisms.

Animals↗
PubMed1993

Rat pancreatic ganglioside expression: differences between a model of autoimmune islet B cell destruction and a normal strain.

Islet cell antibodies (ICA) bind antigens expressed in both human and rat pancreatic islets. Biochemical studies have shown that an ICA-autoantigen has the properties of a monosialo-ganglioside migrating between GM2 and GM1 standards (GM2-1). We therefore aimed to isolate and characterize gangliosides from whole pancreas and isolated islets of bio breeding diabetes-prone (BB-DP), bio breeding diabetes-resistant (BB-DR), and Wistar Furth (WF) rat strains. Gangliosides were characterized by TLC, HPLC, diode array analysis, and ganglioside-specific staining. ICA binding was studied by indirect immunostaining. The GM2-1 fraction was present in BB-DP, BB-DR, and WF rat pancreases (11, 17, and 9.5%, respectively, of total ganglioside content). Substantial differences were found in other fractions: in BB-DP pancreas, in addition to GM2-1, the main fractions were GM3 (49%), GD1a (12%), GT1b (5%), and a ganglioside migrating between GM1 and GD3 standards (23%), while in BB-DR pancreas the above components were 71, 5.5, 2, and 4.5%, respectively; in WF pancreas, the main fractions were GM3, GD3, GD1a, GT1b and a trisialoganglioside (GT*) migrating above the GT1b standard (42.7, 7, 20.2, 13.8, and 6.8, respectively). A different pattern of ganglioside expression was found in isolated islets of BB-DP, BB-DR, and WF rats: the GM2-1 fraction represented, respectively, 29.1, 30.4, and 31.6% of total ganglioside content; GM3 51.1, 66, and 68.4%. A fraction migrating between GM1 and GD3 standards was present only in BB-DP and BB-DR islets (19.8 and 3.6%, respectively). ICA-positive human sera reacted with pancreas of all rat strains studied, with similar end-point titers. In conclusion, (1) the GM2-1 ganglioside, in the same way as a putative target antigen of ICA, is equally expressed in BB-DP, BB-DR, and WF rat pancreata; and (2) the GM1-GD3 is expressed in higher amounts in BB-DP than in BB-DR pancreas and islets and is absent in WF.

Animals↗
PubMed1995

Ganglioside GT1b induces keratinocyte differentiation without activating protein kinase C.

The altered patterns of expression of gangliosides during density-dependent growth inhibition, oncogenic transformation, and embryogenesis suggest that gangliosides, sialylated membrane glycolipids, may affect cellular proliferation and differentiation. Gangliosides of the "b" pathway of ganglioside synthesis, including GM3, GD3, and GD1b, inhibit the proliferation of cultured keratinocytes without increasing differentiation. We have examined the effect on keratinocyte proliferation and differentiation of supplemental ganglioside GT1b, a more highly sialylated ganglioside of the "b" synthetic pathway that is also present in cultured keratinocytes. In contrast to the lack of effect on differentiation of these other gangliosides, we noted significant induction of keratinocyte differentiation by GT1b, as evidenced by early desmosome formation, and increased cornified envelope formation and expression of involucrin and of the differentiation-specific keratin K1. The addition of GT1b did not cause a shift in intracellular free calcium or alter protein kinase C activity. Alterations in the membrane concentration of ganglioside GT1b, a minor ganglioside component of the keratinocyte membrane, may participate in regulating keratinocyte differentiation.

Calcium↗