Mixture Of Bovine Brain Gangliosides Research Articles

Animal model of axonal Guillain-Barré syndrome induced by sensitization with GM1 ganglioside

Some humans develop the axonal form of Guillain-Barré syndrome after receiving bovine brain ganglioside. On sensitization with the ganglioside mixture, all of a group of rabbits injected developed high anti-GM1 IgG antibody titers, flaccid limb weakness of acute onset, and a monophasic illness course. Pathological findings for the peripheral nerves showed predominant Wallerian-like degeneration, with neither lymphocytic infiltration nor demyelination. IgG was deposited on the axons of the anterior roots, and GM1 was proved to be present on the axons of peripheral nerves. Sensitization with purified GM1 also induced axonal neuropathy, indicating that GM1 was the immunogen in the mixture. A model of human axonal Guillain-Barré syndrome has been established that uses inoculation with a bovine brain ganglioside mixture or isolated GM1. This model may help to clarify the molecular pathogenesis of the syndrome and to develop new treatments for it.

Animal model of axonal Guillain‐Barré syndrome induced by sensitization with GM1 ganglioside

Some humans develop the axonal form of Guillain-Barré syndrome after receiving bovine brain ganglioside. On sensitization with the ganglioside mixture, all of a group of rabbits injected developed high anti-GM1 IgG antibody titers, flaccid limb weakness of acute onset, and a monophasic illness course. Pathological findings for the peripheral nerves showed predominant Wallerian-like degeneration, with neither lymphocytic infiltration nor demyelination. IgG was deposited on the axons of the anterior roots, and GM1 was proved to be present on the axons of peripheral nerves. Sensitization with purified GM1 also induced axonal neuropathy, indicating that GM1 was the immunogen in the mixture. A model of human axonal Guillain-Barré syndrome has been established that uses inoculation with a bovine brain ganglioside mixture or isolated GM1. This model may help to clarify the molecular pathogenesis of the syndrome and to develop new treatments for it.

Dietary gangliosides positively modulate the percentages of Th1 and Th2 lymphocyte subsets in small intestine of mice at weaning.

We studied the influence of dietary gangliosides on the number of spontaneous cytokine-secreting cells from two intestinal lymphocyte populations: lamina propria lymphocytes and Peyer's patches lymphocytes in Balb/c mice for 28 days after weaning. Weanling mice were separated into two groups, designated as Control and BG. The Control group was fed with a semipurified diet without gangliosides and the BG group was fed with the semipurified diet supplemented with 47 mg/kg of a mixture of bovine brain gangliosides. Intestinal lymphocytes were isolated from mice killed at 3, 7, 14 and 28 days after weaning, and the percentages of spontaneous Th1 as well as Th2 cytokine-secreting lymphocytes were determined using the ELISPOT assay. The BG group animals showed an earlier development in the number of cytokine-secreting cells, which appeared one week later in Control animals. In addition, mice fed with the ganglioside-supplemented diet showed a significantly higher number of Th1 and Th2 cytokine-secreting lymphocytes than Control mice in lamina propria and Peyer's patches lymphocytes at the end of the experimental period (28 days). Our results suggest that dietary gangliosides influence the maturation process of the intestinal immune system that take place during weaning.

GANGLIOSIDE-MEDIATED METABOLIC SYNCHRONIZATION OF PROTEIN SYNTHESIS ACTIVITY IN CULTURED HEPATOCYTES

An ultradian oscillation of protein synthesis was detected by synchronization of metabolic activity in rat hepatocyte cultures. This oscillation occurs in dense cultures in fresh medium, but not in sparse ones. Metabolic synchronization of sparse cultures, however, was initiated by conditioned medium or addition of 0.3–0.5μm of a mixture of bovine brain gangliosides to fresh culture medium along with either 0.06–0.2μm GM1 or 0.1–0.2μm GDIa. GTIb and GDIb did not produce oscillations, nor did human liver ganglioside GM3. High expression of GM1 ganglioside determinants in hepatocytes maintained in the conditioned medium purified polyclonal antibodies to GM1 was coupled with protein synthetic oscillatory activity, i.e. metabolic synchronization. Incubation of dense cultures with GM1-antibodies for 24h decreased the amplitude of these oscillations. In sparse cultures maintained in fresh medium where protein synthesis showed no oscillatory pattern, GM1 expression was low.

Cholesterol-induced alterations of the packing properties of gangliosides: an EPR study

The effect of cholesterol (Chol) on two kinds of glycolipid assemblies, one composed of monosialogangliosides (G M1a) and the other formed by a natural mixture of bovine brain gangliosides (TBG), has been analysed. The experimental approach involves spin label electron paramagnetic resonance (EPR) in aqueous lipid dispersions. The employment of a hydrosoluble spin label and a ‘quencher’ of the EPR signal that is not able to permeate lipid interfaces, allowed us to conclude that G M1a/Chol mixtures give rise to vesicles at Chol proportions for which TBG/Chol mixtures form micelles. The use of different liposoluble spin labels reveals that cholesterol produces a straightening of the hydrocarbon chains in both lipid systems. In G M1a/Chol mixtures, this feature is more pronounced and it is coupled with a decrease in polarity at the chain ends.

Isolation of three novel cholinergic neuron-specific gangliosides from bovine brain and their in vitro syntheses.

In the present study, three extremely minor but novel Chol-1 antigens, termed X1, X2, and X3 have been isolated from bovine brain gangliosides. Based on the results of sialidase degradation, TLC-immunostaining with anti-Chol-1 antibody and fast atom bombardment mass spectrometry, their chemical structures were identified as: III6NeuAc-GgOse4Cer (X1: GM1 alpha) III6NeuAc,II3NeuAc-GgOse4Cer (X2: GD1a alpha) III6NeuAc,II3NeuAc-NeuGc-GgOse4Cer (X3: GT1b alpha) The yields of GM1 alpha, GD1a alpha, and GT1b alpha, were approximately 150, 20, and 10 micrograms, respectively, from 10 g of the bovine brain ganglioside mixture. In conjunction with our previous observations, all gangliosides with anti-Chol-1 reactivity were found to contain a common sialyl alpha 2-6 N-acetylgalactosamine residue, indicating that this unique sialyl linkage is the specific antigenic determinant. We subsequently examined the biosynthesis of the three novel Chol-1 gangliosides using rat liver Golgi fraction as an enzyme source. The results showed that GM1 alpha, GD1a alpha, and GT1b alpha were synthesized from asialo-GM1, GM1a, and GD1b, respectively, by the action of a GalNAc alpha 2-6sialyltransferase.

Complex gangliosides affect GD 3 accessibility to antibody in developing neuronal cells

Ganglioside expression of embryonic chick retina cells developed in vitro was analyzed by indirect immunofluorescence. Immature neurons were GD 3 positive cells and the labeling was chiefly distributed all over their cell membrane. Mature neurons became GD 3 negative and expressed complex gangliosides of the a- and b-pathways; nevertheless, the content of GD 3 accounted for approximately 40% of the total gangliosides in these cells. Neuraminidase hydrolysis pointed out that GD 3 was located in membrane of differentiated cells. The frequency of cells with the GD 3 immunostain localized in restricted areas of membrane of undifferentiated neurons increased significantly after adding a mixture of bovine brain gangliosides (largely complex gangliosides). Antibody binding to immobilized GD 3 showed a dose-dependent inhibition by adding a mixture of bovine brain gangliosides, GM 1, GD 1a or asialo-GM 1. Glycosphingolipids with shorter oligosaccharide chains, as cerebrosides or sulfatides, did not affect this binding. These results suggest that, concomitant with the accretion of content of complex gangliosides, a rearrangement in the membrane would occur, which progressively masks GD 3 to its antibody. This rearrangement might affect putative ganglioside functions involved in neuronal differentiation.

Isolation and structural characterization of N-acetyl- and N-glycolylneuraminic-acid-containing GalNAc-GD1a isomers, IV4GalNAcIV3Neu5AcII3Neu5GcGgOse4Cer and IV4GalNAcIV3Neu5GcII3Neu5AcGgOse4Cer, from bovine brain.

A ganglioside preparation containing two structurally related minor gangliosides (Gg 1 + 2) was isolated from bovine brain ganglioside mixture and characterized. Treatment of 50 g ganglioside mixture with Clostridium perfrigens sialidase, followed by chromatography on DEAE-Sepharose and silica gel columns, yielded 20 mg Gg 1 + 2. By chemical analyses, 1H- and 13C-NMR spectroscopy, enzymic hydrolyses using human beta-hexosaminidase A and clostridial sialidase, and TLC overlay with the conjugated cholera toxin B subunit, the two novel gangliosides Gg 1 and Gg 2 were identified to be: Gg 1, GalNAc-GD1a(Neu5Ac/Neu5Gc), beta-GalNAc-(1-4)-[alpha-Neu5Ac-(2-3)]-beta- Gal-(1-3)-beta-GalNAc-(1-4)-[alpha-Neu5Gc-(2-3)]-beta-Gal-(1-4)-be ta- Glc-(1-1)-Cer; Gg 2, GalNAc-GD1a(Neu5Gc/Neu5Ac), beta-GalNAc-(1-4)-[alpha-Neu5Gc-(2-3)]- beta-Gal-(1-3)-beta-GalNAc-(1-4)-[alpha-Neu5Ac-(2-3)]-beta-Gal-(1- 4)-beta- Glc-(1-1)-Cer. These two gangliosides contain the identical pentasaccharide backbone except that the substitution of the two sialic acids, Neu5Ac and Neu5Gc, are in the reversed position of the external and the internal Gal residues. Our analyses showed that the content of Gg 1 and Gg 2 were approximately 0.12% and 0.08%, respectively, of the total brain ganglioside mixture.

In vitro changes of ganglioside immunostain localization in developing neural cells.

The immunostains of GD3, c-pathway polysialogangliosides and gangliotetraosylgangliosides belonging to the a- and b-pathways were analyzed in embryo optic lobe cells. Cells were cultured in serum free media with, and without, bovine brain ganglioside mixture (largely gangliotetraosylgangliosides). Control cells differentiated in vitro, whereas ganglioside treated cells emitted almost no neurites. In immature cells, GD3 and c-gangliosides were extensively expressed and their immunostains were observed all over the cell (general localization), whereas gangliotetraosylgangliosides were scanty and their stain was preferentially restricted to discrete areas (in clusters). In control differentiated cells, the GD3 expression was strikingly reduced and its immunostain appeared in clusters; c-gangliosides were abundant and their stain was found with general localization; the expression of gangliotetraosylgangliosides became important and their stain was observed with general localization in most of the positive cells. In ganglioside treated cells, in spite of their undifferentiated morphology, the gangliotetraosylganglioside stain appeared with general localization. These results suggest that gangliosides involved in neural ontogenesis are preferentially located in clusters when they are scarce and have general localization when they are abundant in membranes.

Novel lacto-ganglio type gangliosides with GM2-epitope in bovine brain which react with IgM from a patient of the amyotrophic lateral sclerosis-like disorder.

A motor neuron disorder resembling that of amyotrophic lateral sclerosis was found in a patient who had received the intramuscular administration of a mixture of bovine brain gangliosides (Yuki, N., Sato, S., Miyatake, T., Sugiyama, K., Katagiri, T., and Sasaki, H. (1991) Lancet 337, 1109-1110). A very high titer of anti-GM2 IgM was detected in the patient's serum and the patient quickly recovered after plasmapheresis. The clinical course of the patient appeared to be different from amyotrophic lateral sclerosis and the anti-GM2 IgM was thought to be the culprit. The IgM reacted with GM2, GM1b-GalNAc, SPG(alpha 2-3)-GalNAc, and GD1a-GalNAc, but not with GA2 or GD2, meaning that the epitope recognized by the IgM was the GM2-like terminal structure, GalNAc beta 1-4(Neu-Ac alpha 2-3)Gal beta 1-. In this study, we found two novel GM2-epitope containing gangliosides, X1 and X2, in bovine brain gangliosides by TLC immunostaining using the patient's IgM. They were characterized as unique lacto-ganglio type gangliosides containing the following branching structures. [formula: see text] Their unusual structures may be immunogenic to humans to induce anti-GM2 antibody.

Effects of ganglioside administration on experimental autoimmune neuritis induced by peripheral nerve myelin or P 2-specific T cell lines

We studied the effects of ganglioside administration in two animal models of inflammatory demyelinating polyneuropathy. We administered a mixture of bovine brain gangliosides intraperitoneally to Lewis rats with myelin-induced or T cell line-mediated experimental autoimmune neuritis (EAN). Under the experimental conditions we had chosen, we only detected marginal but not statistically significant effects on disease course and severity, as evidenced by motor function, electrophysiological findings, and morphological signs of inflammation and demyelination. There was no significant induction of antibody production against gangliosides, and we did not detect signs of increased cellular reactivity towards gangliosides. We conclude that the administration of gangliosides modulates EAN at best marginally, and does not induce a cellular or humoral immune reaction.

Gangliosides protect from TNFα-induced apoptosis

The modulation of tumor necrosis factor α-mediated cytotoxicity by gangliosides was analyzed. When cells of the TNFα-sensitive fibrosarcoma cell line L929 were incubated for 16 h with recombinant TNFα (156 or 312 pg/ml), they were lysed to 65.2% or 78.0%, respectively. The presence of a bovine brain ganglioside mixture (BBG, Cronassial) inhibited the TNFα-mediated lysis in a dose-dependent manner (IC50 ≈ 1 mg/ml). Maximum inhibition was achieved with 2 mg/ml BBG (82.6% inhibition for 156 pg/ml TNFα and 88.5% inhibition for 312 pg/ml TNFα). In situ nick translation revealed that BBG (2 mg/ml) significantly reduces the number of cells with TNFα-induced DNA-strand breaks from 71.2% to 6.6% (P < 0.0001), indicating a protection against TNFα-mediated apoptosis. We conclude that BBG prevents the cytotoxic action of TNFα on tumor cells. The inhibitory effect may be due to an interference of gangliosides with intracellular signal transduction pathways, resulting in an inhibition of the activation of DNA-cleaving endonucleases.

Effects of postnatal ganglioside administration and hypoxia-exposure on the dopamine release from striatal slices, the behaviour and the ganglioside pattern of 2-3 months old rats.

Neonatal rats were injected with a mixture of bovine brain gangliosides (30 mg/kg body weight, s.c.) immediately before the exposure to hypoxia (pO2 = 10 kPa, 10 hrs daily) from the 2nd to the 11th day of life. At the age of 2-3 months the potential protective or restitutive effect of gangliosides on the radiolabelled dopamine release from striatal slices and on the conditioned avoidance learning was studied. No change in the content and the pattern of gangliosides of the rat striata was found after exposure to hypoxia and ganglioside administration, respectively. Both hypoxia exposure and ganglioside treatment of controls increased the dopamine release whereas hypoxic animals treated with gangliosides showed a diminished release. Hypoxia-induced impaired conditioned avoidance learning was improved by ganglioside treatment. The changed release of dopamine and the altered behavioural performance after ganglioside treatment alone indicate the necessity of a very cautious application of gangliosides to the developing brain.

Structural characterization of a novel cholinergic neuron-specific ganglioside in bovine brain.

A new ganglioside antigen, termed Chol-1 alpha-b, recognized by cholinergic neuron-specific antibody, Chol-1 alpha, was isolated from bovine brain ganglioside mixture using Q-Sepharose. The yield was approximately 1.3 mg from 5 g of the total ganglioside. The chemical structure was characterized as a novel ganglioside by means of gas-liquid chromatography, a permethylation study, mild acid hydrolysis, thin layer chromatography-enzyme immunostaining, fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectroscopy. The ganglioside has the following unique structure. [formula: see text] When examined by thin layer chromatography immunostaining and enzyme-linked immunosorbant assays, this ganglioside has the most intense immunoreactivity with Chol-1 alpha antibody among bovine brain gangliosides. As combined with our previous results (Hirabayashi, Y., Hyogo, A., Nakao, T., Tsuchiya, K., Suzuki, Y., Matsumoto, M., Kon, K., and Ando, S. (1990) J. Biol. Chem. 265, 8144-8151; Ando, S., Hirabayashi, Y., Kon, K., Inagaki, F., Tate, S., and Whittaker, X. (1992) J. Biochem. (Tokyo), 111, 287-290), the present study indicates the occurrence of a new series of gangliosides containing N-acetylneuraminic acid residue attaching to N-acetylgalactosamine as cholinergic specific antigens.

Calmodulin, a ganglioside-binding protein. Binding of gangliosides to calmodulin in the presence of calcium.

Ca(2+)-dependent ganglioside-binding protein was isolated from a soluble cytosol fraction of mouse brains using a ganglioside affinity column prepared with a mixture of bovine brain gangliosides. It was identified as calmodulin based on the following features identical with those of calmodulin: molecular weight, pI, chromatographic profile and amino acid sequences of lysyl-endopeptidase digests, and ability to activate cyclic nucleotide phosphodiesterase. Bovine brain calmodulin derivatized with 5-dimethylaminonaphthalene-1-sulfonyl (dansyl-calmodulin), tetramethylrhodamine isothiocyanate, or biotin was also shown to bind to the ganglioside affinity column Ca2+ dependently and elute with gangliosides GD1a, GD1b, GT1b, GQ1b, GM1, and GM2, melittin, and trifluoperazine but not with GgOse4Cer and oligosaccharides of GM1, GD1a, and GT1b. Modification of the Lys94 residue of calmodulin by biotinylation drastically reduced the capacity for ganglioside binding. Ganglioside GD1b caused a blue shift and increase in intensity of the fluorescence emission spectrum of dansyl-calmodulin in the presence of Ca2+. The increment in fluorescence was proportional to the amount of GD1b added and was maximal at the molar ratio of GD1b to calmodulin, approximately 7.8. Gangliosides are thus shown to specifically bind to calmodulin, and this binding may be a general mechanism for regulating calmodulin-dependent enzymes with consequent cellular response, such as cell differentiation.

Lasting effects of postnatal hypoxia and saline injection on the striatal dopamine transport and their modification by gangliosides

Hypobaric hypoxia (10 h daily, pO 2 10 kPa) and saline administration (2.5 μl/g body wt) from the 2nd till the 11th day of life both induced a long-lasting increase of the low-affinity dopamine (DA) uptake capacity in SI-fractions of the rat striatum. Additionally, the potassium-stimulated DA release was enhanced in adult control rats postnatally injected with saline. The administration of a mixture of bovine brain gangliosides (30 μg/g body wt) was found to prevent these effects. However, the kinetic constants of the DA uptake of hypoxic rats treated with gangliosides were reduced in comparison to untreated controls. Thus, the effects of gangliosides appear to differ between hypoxic and control conditions. The modification of the dopaminergic activity during brain development is discussed as a possible mechanism of the preventive effects of gangliosides against long-term cerebral dysfunctions following hypoxia or stress.

Protection of neuro‐2a cells against calcium ionophore cytotoxicity by gangliosides

Gangliosides are known to assert both neuritogenic and neuroprotective effects when applied to a variety of neuroblastoma and primary neuronal cultures. We have developed a model employing Neuro-2a neuroblastoma cells with Ca2+ ionophore A23187 as neurotoxic agent causing neurite retraction and eventual cell death. Gangliosides attenuated the toxicity of this substance, increasing both cell survival and neurite stability. In one series of experiments, cells were exposed to A23187 for 24 hr and then incubated in fresh medium (washout) for 18 hr; gangliosides were present at varying times. The paradigm in which cells were only preincubated (2 hr) with ganglioside provided no benefit, nor did incubation of the cells in both ionophore and ganglioside during the 24-hr exposure period. Significant protection was achieved by exposing the cells to ganglioside after washout of A23187, or continuously throughout the whole period. Bovine brain ganglioside mixture and the four major components (GM1, GD1a, GD1b, GT1b) applied individually were all effective. By contrast, GM3 and GM1-alcohol, a neutral derivative of GM1, provided little or no protection. Dichlorobenzamil, an inhibitor of the Na(+)-Ca2+ exchanger, tended to block the neurite stabilizing effect of gangliosides, suggesting that the mechanism might involve potentiation of this antiporter.

Rapid isolation of monosialogangliosides from bovine brain gangliosides by selective-overload chromatography.

A procedure for rapid isolation of monosialogangliosides from purified bovine brain gangliosides has been developed. It utilizes the selective difference in association between monosialogangliosides and polysialogangliosides for the ion-exchange resin Q-Sepharose. When the ion-exchange column is overloaded with a bovine brain ganglioside mixture in the proper ganglioside to column bed-volume ratio, the polysialogangliosides are selectively retained by the column while the monosialogangliosides emerge with the void volume without the use of salt for elution. With the critical ganglioside to bed-volume ratio (1 g:8.32 ml), and an appropriate column bed-height to column radius ratio of 6.9, monosialogangliosides are reproducibly obtained in high purity with greater than 90% yield. The method has been used at both the analytical and preparative scale. We call this separation technique selective-overload chromatography.

Exogenous Gangliosides Stimulate Breakdown of Neuro‐2A Phosphoinositides in a Manner Unrelated to Neurite Outgrowth

Gangliosides administered exogenously are well-known effectors of differentiation in many neuroblastoma lines and primary neuronal cultures. Previous studies suggested the phosphoinositide signaling mechanism could be a contributing factor. We have found that treatment of Neuro-2A cells with bovine brain ganglioside mixture (BBG) causes breakdown of phosphoinositides, as measured by increased levels of inositol phosphates. The effect was optimal at 60 min and required a minimal BBG concentration of 25 microM. However, addition of neomycin, which blocked phosphoinositide breakdown, had no observable effect on ganglioside-stimulated neurite outgrowth. A similar result was obtained with psi-tectorigenin, which also inhibited phosphoinositide hydrolysis. When cells were treated with maitotoxin, an agent that promotes phosphoinositide breakdown, there was no enhancement of neurite outgrowth. These findings indicate that although exogenous gangliosides elevate inositol phosphate formation over a prolonged period in neuro-2A cells, this reaction is not integral to the differentiation of these cells. The possibility of secondary effects influencing neurite type and structure cannot be excluded.

Gangliosides offer partial protection in experimental allergic neuritis

The effect of gangliosides on the clinical course of experimental allergic neuritis was tested in Lewis rats sensitized with bovine intradural root myelin in complete Freund's adjuvant. A mixture of bovine brain gangliosides (GM1, GD1a, GD1b, GT1b) was injected intramuscularly at a daily dose of 20 mg per kilogram of body weight, beginning 6 days after inoculation. The results from seven different experiments show that in most cases, the administered gangliosides were partially protective. Particularly striking was the reduction in mortality rate to half or less of saline-injected controls. Cumulative clinical index scores were also significantly lower with ganglioside treatment in five of the seven experiments. The cause of the wide variability is not known, but it was noted that better results were obtained when the animals were sensitized with freshly isolated myelin.