Effect of serum on ganglioside uptake and choleragen responsiveness of transformed mouse fibroblasts.

NCTC 2071 cells, transformed mouse fibroblasts, did not respond to choleragen when grown in chemically defined medium. When grown in medium containing 10 percent fetal calf serum, however, the cells accumulated cyclic AMP upon exposure to the toxin. Gangliosides isolated from the fetal calf serum were as effective as whole serum in inducing choleragen responsiveness in the cells. The putative choleragen receptor, the monosialo-ganglioside GM1, could not be detected by chemical analysis in cells exposed to serum. 3H-Labeled GM1 was detected in these cells, however, following sequential exposure to galactose oxidase and sodium borotritide. Thus, uptake of minute amounts of GM1 from serum by these cells sensitized them to choleragen.     

Loss of choleragen receptors and ganglioside upon differentiation of 3T3-L1 preadipocytes

3T3-L1 preadipocytes differentiate in culture into cells having the enzymatic and morphological characteristics of adipocytes. Differentiation is accompanied by a decrease in total cellular ganglioside content; the ganglioside level is 1.8 to 2.5-fold higher in undifferentiated than in differentiated cells. Gangliosides GM3 and GD1a constitute a majority of total cell gangliosides in both cell types, while ganglioside GM1, the putative choleragen receptor, constitutes less than 5%. Differentiation results in a 75 to 85% decrease in ganglioside GM1. An inverse correlation exists between the percentage of adipocytes in the cell population and: 1) total ganglioside and ganglioside GM1 content, and 2) surface ganglioside GM1 as estimated by choleragen binding or fluorescent staining of bound choleragen. Nondifferentiating 3T3-C2 control cells do not exhibit changes in total ganglioside, ganglioside GM1, or choleragen binding that are observed with 3T3-L1 cells.     

Incorporation and metabolism of ganglioside GM2 in skin fibroblasts from normal and GM2 gangliosidosis subjects

Ganglioside GM2, 3H-labeled in the sphingoid base, was added to the culture medium of normal and GM2 gangliosidosis fibroblasts. Ganglioside was found to adsorb rapidly to the cell surface, most of it could however be removed by trypsination. The trypsin-resistant incorporation was about 10 nmol/mg cell protein, after 48 h. The rates of adsorption and incorporation depended strongly on the concentration of fetal calf serum in the medium, higher serum concentrations being inhibitory. After various incubation times, the lipids were extracted, separated by thin-layer chromatography and visualized by fluorography. In normal cells a variety of degradation products as well as sphingomyelin was found whereas in GM2 gangliosidosis cells, only trace amounts of such products (mainly GA2) were found. In contrast, the higher gangliosides GM1 and GD1a were formed in comparable amounts (2.2-3.6% of total radioactivity after 92 h) in normal and pathologic cell lines. Supplementation of cells from GM2 gangliosidosis, variant AB, with purified GM2-activator protein restored ganglioside GM2 degradation to almost normal rates but had no effect on its glycosylation to gangliosides GM1 and GD1a. From these results we conclude that the synthesis of higher gangliosides from incorporated GM2 can occur by direct glycosylation and not only via lysosomal degradation and resynthesis from [3H]sphinganine-containing degradation products. Preliminary studies with subcellular fractionation after various times of [3H]ganglioside incorporation indicated biphasic kinetics for the net transport of membrane-inserted ganglioside to lysosomes, compatible with the notion that a portion of the glycolipids can also escape from secondary lysosomes and migrate to Golgi compartment or cell surface.     

Efflux of sphingolipids metabolically labeled with [1-3H]sphingosine, L-[3-3H]serine and [9,10-3H]palmitic acid from normal cells in culture

The membrane complex lipids of human fibroblasts and differentiated rat cerebellar granule cells in culture were metabolically radiolabeled with [1-³H]sphingosine, L-[3-³H]serine and [9,10-³H]palmitic acid. A relevant efflux of radioactive sphingolipids and phosphatidylcholine was observed from cells to the culture medium in the presence of fetal calf serum. This event was independent of the concentration and structure of the metabolic precursor administered to cells, and it was linearly time-dependent. The radioactive lipid patterns present in the medium were different from those present in the cells. Radioactive sphingomyelin and ganglioside GM3 containing short acyl chains were the main species present in the medium from human fibroblasts, while sphingomyelin and GD3 ganglioside in that from neuronal cells. In the absence of proteins in the culture medium, the efflux of complex lipids was much lower than in the presence of serum, and the patterns of released molecules were again different from those of cells. This work was supported by COFIN-PRIN, Consiglio Nazionale delle Ricerche (PF Biotechnology), Italy.     

Studies on the turnover and subcellular localization of membrane gangliosides in cultured neuroblastoma cells

To compare the subcellular distribution of endogenously synthesized and exogenous gangliosides, cultured murine neuroblastoma cells (N1E-115) were incubated in suspension for 22h in the presence ofd-[1-³H]galactose or [³H]GM1 ganglioside, transferred to culture medium containing no radioisotope for periods of up to 72 hr, and then subjected to subcellular fractionation and analysis of lipidsialic acid and radiolabeled ganglioside levels. The results indicated that GM2 and GM3 were the principal gangliosides in the cells with only traces of GM1 and small amounts of disialogangliosides present. About 50% of the endogenously synthesized radiolabelled ganglioside in the four major subcellular membrane fractions studied was recovered from plasma membrane and only 10–15% from the crude mitochondrial membrane fraction. In contrast, 45% of the exogenous [³H]GM1 taken up into the same subcellular membrane fractions was recovered from the crude mitochondrial fraction; less than 15% was localized in the plasma membrane fraction. The results are similar to those obtained from previously reported studies on membrane phospholipid turnover. They suggest that exogenous GM1 ganglioside, like exogenous phosphatidylcholine, does not intermix freely with any quantitatively major pool of endogenous membrane lipid.     

The role of gangliosides in the interaction of human chorionic gonadotropin and cholera toxin with murine Leydig tumor cells

A clonal line of murine Leydig tumor cells (MLTC-1) bound both human chorionic gonadotropin (hCG) and cholera toxin (CT) with high affinity and accumulated cyclic AMP in response to either effector. The major cellular ganglioside was GM3 with small amounts of GM2, GM1, and GD1a. The gangliosides became labeled when the cells were grown in medium containing [3H] galactose or were exposed to galactose oxidase or NaIO4 followed by NaB3H4. CT specifically protected GM1 from surface labeling whereas hCG did not protect any gangliosides from being labeled. When the cells were exposed to sialidase, surface GD1a was eliminated, and GM1 increased with a corresponding increase in CT binding. When sialidase-treated cells were first incubated with the B component of CT, binding and action of CT was blocked. The cells, however, retained their ability to bind and respond to hCG. Addition of purified gangliosides to the medium effectively inhibited the binding and action of CT but not hCG. The cells incorporated the exogenous gangliosides and exhibited increased binding of and responsiveness to CT but not hCG. Both hCG- and CT-receptor complexes were extracted from the cells with nonionic detergent and analyzed by sucrose gradient centrifugation. The hCG-receptor complex had an apparent molecular weight of 190,000 whereas the CT-receptor complex sedimented only slightly faster than CT itself. MLTC-1 gangliosides were separated on thin layer chromatograms which were overlayed with either iodinated CT or hCG. The toxin bound to a ganglioside corresponding to GM1 whereas the hormone did not bind to any of the gangliosides. When the cells were incubated overnight with hCG, they lost their hCG receptors but exhibited an increase in CT binding and gangliosides. Our results indicate that GM1 is the specific receptor for CT whereas gangliosides are not involved in the binding and action of hCG.     

The role of the ganglioside lipid moiety in the process of ganglioside-cell interactions.

The role of the ceramide moiety of gangliosides, together with the deriving aggregative properties of ganglioside in solution, in the process of ganglioside-cell interactions was studied. The natural GM1(stearoyl) and the synthetic GM1(acetyl), containing the stearoyl and acetyl groups as the acyl moiety, respectively, were used in binding experiments to rat cerebellar granule cells. Regardless of the cell culture conditions, such as the presence of absence of fetal calf serum, the association of GM1(acetyl) to the cells was much greater than that of GM1(stearoyl). GM1(acetyl) was present in the incubation medium as monomers. After incubation, a large part of the total GM1(acetyl) associated to cells, 76-93% depending on the experimental conditions, was removed by washing with protein solutions. The remaining associated ganglioside was not removed by repeating washing with protein solutions or trypsin treatments and was considered as a component of the membrane. The cell association of GM1(stearoyl), present in solution as monomers as well as micelles, could be classified as serum-labile, trypsin-labile and trypsin-stable. The trypsin-stable form of association, corresponding to the molecules stably inserted into the membrane, was proportionally higher, the proportions varying with increasing incubation time and decreasing ganglioside concentration. This form of association was particularly high when incubation was performed in the presence of fetal calf serum. Incubation experiments performed with a mixture of GM1(stearoyl) and GM1(acetyl) in a molar ratio which allowed their presence in the medium as monomers as well as mixed micelles, led to a ganglioside association suggesting that besides the aggregative properties of the molecule other ganglioside properties are involved in the ganglioside-cell interaction process.     

Incorporation of exogenous ganglioside GM1 into neuroblastoma membranes: Inhibition by calcium ion and dependence upon membrane protein

Since exogenous gangliosides are known to promote neuritogenesis, the incorporation of exogenous GM1 into neuroblastoma membranes was examined. Neuro-2A cells, synchronized in the G1/G0 phase, were suspended in HEPES buffered saline containing 10^–4 M [³H]GM1, and membrane incorporation was measured as radioactivity remaining with the cell pellet following incubation with serum-containing medium and trypsin. Calcium ion (0.01 to 10 mM) reduced incorporation of exogenous GM1, due to its interaction with GM1 micelles in solution. When cells were treated with proteases prior to incubation with GM1, the inhibitory effect of Ca²⁺ was lost and total incorporation into membranes was lowered by approximately one order of magnitude. Pretreatment of cells with 0.05% trypsin resulted in an inhibition of GM1 incorporation within 5 minutes. When trypsinized cells were resuspended in complete growth medium, the cells recovered the ability to incorporate GM1 with time, and this paralleled labeling of cellular protein with [³H]leucine. The role of membrane protein in the incorporation of exogenous GM1 could not be explained by the lytic release of cytosolic transfer proteins nor the artifactual coating of the cell surface by serum proteins. These results suggest that the incorporation of exogenous gangliosides into cellular membrane lipid bilayers cannot be fully explained by considerations of lipophilicity alone, and leads us to propose that initial recognition by membrane protein(s) is necessary.Abbreviations used GM1 H³NeuAc-GgOse₄Cer - HBS HEPES buffered saline - DMEM Dulbecco's modified Eagle's medium - FCS fetal calf serum     

Rapid Internalization and Intracellular Metabolic Processing of Exogenous Ganglioside by Cerebellar Granule Cells Differentiated in Culture

Ganglioside GM1, tritiated at the level of the long chain base (sphingosine) [( Sph-3H]GM1), sialic acid (N-acetylneuraminic acid) [( NeuAc-3H]GM1), or terminal galactose [( Gal-3H]GM1) was supplied to cerebellar granule cells differentiated in vitro, and its metabolic processing was followed with pulse time. Using [Sph-3H]GM1 and [NeuAc-3H]GM1 the formation of radioactive compounds of catabolic origin (GM2, GM3, lactosylceramide, glucosylceramide, and ceramide) started being detectable at 10-15 min of pulse, whereas compounds of biosynthetic origin (GD1a, GD1b, GT1b, O-acetylated GT1b, spingomyelin, and sialoglycoprotein) appeared after 15-30 min of pulse. Using [Gal-3H]GM1 two radioactive substances were formed, GD1a and GT1b, with the former (produced by direct sialosylation of GM1) appearing after 30 min of pulse and the latter (formed by biosynthetic recycling of released galactose) appearing after 2 h. The radioactivity linked to all metabolites increased with increasing pulse time until 4 h. The percentage of GM1 taken up and subjected to metabolic processing was found to increase from 1.8% after 10 min of pulse to 12.5% after 4 h. Cerebellar granule cells were able to release enzymes of lysosomal origin, beta-D-N-acetylhexosaminidase and beta-D-galactosidase, into the culture medium, with the release being markedly decreased by the absence in the medium of fetal calf serum, a condition that was used for studying exogenous GM1 uptake and metabolization. However, these enzymes exerted no activity at the pH of the culture medium, and no radioactive gangliosides, besides GM1, were detected in the culture medium during pulse.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preparation of GM1 ganglioside with sialidase-producing marine bacteria as a microbial biocatalyst.

This paper describes the preparation of monosialoganglioside GM1 with sialidase-producing marine bacteria as a microbial biocatalyst. A new sialidase-producing bacterium, identified tentatively as Pseudomonas sp. strain YF-2, was isolated from seawater by enrichment culture with ganglioside as the sole source of carbon. When YF-2 was cultured in a synthetic medium containing crude bovine brain gangliosides at 25 degrees C for 3 days, 80 to 90% of the gangliosides were converted to GM1. GM1 was then purified from the supernatant of YF-2 culture by C18 reverse-phased chromatography, followed by DEAE-Sephadex A25 anion-exchange chromatography. In a typical experiment, 178 mg of highly purified GM1 was obtained from 500 mg of the crude ganglioside fraction. The GM1 induced neurite outgrowth of neuroblastoma Neuro2a cells at a concentration of 33 to 100 microM in the presence of fetal calf serum. Sialidase was purified 33-fold with 13.3% recovery from the culture supernatant of YF-2. The purified enzyme hydrolyzed polysialogangliosides to produce GM1 but did not act on GM1. It was therefore concluded that polysialogangliosides in the culture of strain YF-2 were converted to GM1 by this sialidase.     

Binding of choleragen and anti-ganglioside antibodies to gangliosides incorporated into preformed liposomes

Exogenously added gangliosides were taken up and incorporated into liposomes just as they are incorporated into cells. Ganglioside GM1 was rapidly taken up by liposomes containing dimyristoyl- or dipalmitoylphosphatidylcholine, cholesterol and dicetyl phosphate. When incubated with a wide range of GM1 concentrations for 18 h, the liposomes incorporated about 10% of the added ganglioside. The rate of GM1 uptake by preformed liposomes was both time- and temperature-dependent. The liposomes also incorporated other gangliosides to a similar extent. The GM1 taken up by preformed liposomes was predominantly located on the outer surface of the liposomes and did not appear to be internalized into the inner half of the lipid bilayer. Liposomes containing GM1 added after liposome formation bound as many anti-GM1 antibodies and as much choleragen as liposomes having GM1 added during the formation of the lipid bilayers. Thus, preformed liposomes sensitized by incubation with GM1 are a good model system for studying the interactions of antibodies and toxins with membrane-associated gangliosides.     

The plasma membrane-associated sialidase MmNEU3 modifies the ganglioside pattern of adjacent cells supporting its involvement in cell-to-cell interactions

We describe herein the enzyme behavior of MmNEU3, the plasma membrane-associated sialidase from mouse (Mus musculus). MmNEU3 is localized at the plasma membrane as demonstrated directly by confocal microscopy analysis. In addition, administration of the radiolabeled ganglioside GD1a to MmNEU3-transfected cells, under conditions that prevent lysosomal activity, led to its hydrolysis into ganglioside GM1, further indicating the plasma membrane topology of MmNEU3. Metabolic labeling with [1-(3)H]sphingosine allowed the characterization of the ganglioside patterns of COS-7 cells. MmNEU3 expression in COS-7 cells led to an extensive modification of the cell ganglioside pattern, i.e. GM3 and GD1a content was decreased to about one-third compared with mock-transfected cells. At the same time, a 35% increase in ganglioside GM1 content was observed. Mixed culture of MmNEU3-transfected cells with [1-(3)H]sphingosine-labeled cells demonstrates that the enzyme present at the cell surface is able to recognize gangliosides exposed on the membrane of nearby cells. Under these experimental conditions, the extent of ganglioside pattern changes was a function of MmNEU3 transient expression. Overall, the variations in GM3, GD1a, and GM1 content were very similar to those observed in the case of [1-(3)H]sphingosine-labeled MmNEU3-transfected cells, indicating that the enzyme mainly exerted its activity toward ganglioside substrates present at the surface of neighboring cells. These results indicate that the plasma membrane-associated sialidase MmNEU3 is able to hydrolyze ganglioside substrates in intact living cells at a neutral pH, mainly through cell-to-cell interactions.     

GM1 Ganglioside Treatment of PC 12 Cells Stimulates Ganglioside, Glycolipid, and Lipid, but Not Glycoprotein Synthesis Independently from the Effects of Nerve Growth Factor

The incorporation of radioactive precursors into gangliosides and other glycolipids, glycoproteins, and total lipids has been studied in rat pheochromocytoma PC12 cells. Starting with the same PC12 cell pool, cultures displaying different degrees of neuritic expression in response to nerve growth factor (NGF) and combinations of serum ganglioside GM1 were produced. Attempts were then made to correlate neuritic regulation with biochemical performances of these cells. NGF stimulates the incorporation of [3H]galactose into gangliosides and other glycolipids and glycoproteins and [14C]acetate into total lipids, regardless of the serum concentration. NGF both increased their initial labeling rates and promoted additional and more extensive labeling from culture day 4 onward. Unexpectedly, exogenous GM1 also elicited an increase in ganglioside labeling as well as that of the other lipid classes, but not of glycoproteins. The GM1-induced increase was evident at higher serum concentrations (1%) regardless of the presence or absence of NGF, but not apparent in low (0.15%) serum. Serum levels themselves did not affect labeling patterns in the absence of NGF and GM1. GM1-induced stimulation of labeling reflects an increase in the synthetic activities of the cells, and not increased precursor uptake or reduced product degradation. For all constituents stimulated by GM1, concurrent treatment with NGF produces cumulative effects, suggesting independent mechanisms of action by the two molecules.     

Ionic responses and growth stimulation in rat astroglial cells: differential mechanisms of gangliosides and serum

Rat astroglial cells respond to fetal calf serum (FCS) and gangliosides, including GM1, by undergoing proliferation. Here, we show that addition of FCS but not GM1 causes an increase in Na+, K+-pump activity, as measured by ouabain-sensitive 86Rb+ influx. The increase of Na+, K+-pump activity by FCS was due to increased Na+ influx (measured with 22Na+). This increased Na+ influx was sensitive to amiloride, an inhibitor of Na+/H+ exchange. Amiloride also blocked the FCS-stimulated incorporation of [3H]thymidine into DNA. Two defined polypeptide growth factors, epidermal growth factor and fibroblast growth factor were also able to elicit an amiloride-sensitive Na+ influx and an ouabain-sensitive K+ uptake in these astroglial cells, in the presence of FCS or insulin. Thus, GM1 differs from serum and growth factors in the mechanisms by which these agents stimulate the proliferation of the astroglial cells used here.     

Gangliosides released by the perfused rat liver are associated to high density lipoprotein.

We examine here the delivery of gangliosides from the perfused rat liver into the perfusate. One hour after the administration of [3H]GM1 to recirculating perfused livers, almost 80% of the perfusate radioactive gangliosides were recovered associated to the HDL fraction. This fraction was relatively enriched in radioactive GD1a. The pattern of endogenous gangliosides from perfused livers, rat serum and perfusates were very different: GM3 was the main liver ganglioside, GM1 and GD1a were the most abundant in perfusates being GM3 almost absent; GM3, GM1 and GD1a were present in rat serum in similar proportions. Using a non-recirculating perfusion protocol, radioactive gangliosides were found in the HDL fraction since 15 minutes after the administration of [3H]GM1. These results suggest that rat liver supplies the perfusates with some gangliosides and that they are associated to HDL. These facts arise the possibility that the liver is one of the source of serum gangliosides.     

Induction of cholera toxin receptors in cultured cells by butyric acid.

Exposure of HeLa cells to sodium butyrate caused an increase in choleragen (cholera toxin) receptors as measured by increased binding of 125I-choleragen to the intact cells. The process was dependent on time and butyrate concentration; maximal increases (over 40-fold) were observed at 48 h and 5 mM sodium butyrate. Other short chain fatty acids were less effective in elevating choleragen receptors in the order: butyrate greater than pentanoate greater than hexanoate greater than propionate. Acetate and isobutyrate had no effect. The increase in toxin receptors caused by butyrate was reversible and occurred in serum-free medium. The affinity of choleragen for control and butyrate-treated HeLa cells appeared to be similar. Butyrate also induced an elevation in choleragen receptors in rat C6 glial and Friend erythroleukemic cells but not in a butyrate-resistant HeLa mutant. The increase observed in Friend cells paralleled the increase in ganglioside GM1 (galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide), the reported choleragen receptor. Although no GM1 could be detected in untreated Hela cells, small amounts were found in cells exposed to butyrate.     

Enzymatic and chemical oxidation of gangliosides in cultured cells: effects of choleragen.

Cell surface glycolipids of normal human fibroblasts and NCTC2071 cells (transformed mouse fibroblasts) were labeled by incubating the intact cells with either galactose oxidase or sodium periodate, followed by reduction of the oxidized sugar residues with NaB3H4. In intact human fibroblasts, incorporation of 3H was increased with increasing time of exposure to galactose oxidase prior to treatment with NaB3H4. Following limited exposure to galactose oxidase, more label was incorporated into the larger glycolipids. Although labeling of the monosialoganglioside GM1 was maximal by 16 h, not all of the GM1 in the intact cells appeared to be accessible to galactose oxidase, since 10 to 12 times more GM1 was labeled when cells were disrupted before incubation with the enzyme. The human fibroblasts contained approximately 8 X 10(6) molecules of GM1 per cell. Maximal binding of choleragen (5 X 10(5) molecules of [125I]choleragen per cell) completely prevented cholevented oxidation of GM1 in intact fibroblasts by galactose oxidase but only partially protected the sialic acid moiety of GM1 from oxidation by periodate. Choleragen had little effect on the enzymatic or chemical oxidation of other glycolipids. NCTC 2071 cells do not contain endogenous GM1 but incorporate exogenous GM1 from the culture medium. When bound to NCTC 2071 cells, exogenous GM1 was protected by choleragen from oxidation by galactose oxidase or whether endogenous or taken up from the incubation medium, are, after interaction with choleragen, less accessible to oxidation by periodate or galactose oxidase.     

Promotion of Neuritogenesis in Mouse Neuroblastoma Cells by Exogenous Gangliosides. Relationship Between the Effect and the Cell Association of Ganglioside GM1

Ganglioside GM1 promoted neuritogenesis of neuroblastoma cells, neuro-2a clone, in monolayer culture. GM1 bound to neuro-2a cells in three distinct forms, one removable by treatment with serum-containing solutions, one serum-resistant and labile to trypsin treatment, and one resistant to serum and trypsin treatments. The proportions among the three forms of cell-associated GM1 varied in relation to duration of exposure to ganglioside, ganglioside concentration in the medium, and number of cells in culture. The form removable by serum was predominant at the initial stages of association and at the highest ganglioside concentrations (over 10(-6)M); the trypsin-labile and -stable forms tended to increase with increasing cell number and decreasing ganglioside concentration. The neuritogenic effect of GM1 was higher when neuro-2a cells were incubated for 24 h in the presence of GM1 and fetal calf serum. Under this condition the percentage of neurite-bearing cells increased from 11% of control to 62% at the optimal ganglioside concentration of 10-4M. The effect was still present, although to a lower extent (from 11% to 28% of neurite-bearing cells), when cells were first exposed for only 2 h to GM1, then washed and incubated for 24 h in the presence of fetal calf serum. The trypsin-labile and -stable forms of cell-associated GM1 had a fundamental role in the effect, whereas the form removable by serum was not involved. The preparation of GM1 used was extremely pure (99%) and, in particular, had a peptide contamination, if any, less than 1:20,000-1:50,000.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth- and development-dependent expression of gangliosides in rat hepatocytes and liver tissues.

Expression of gangliosides in the liver was examined in primary cultures of hepatocytes from adult rats and liver tissues from rats of different ages. Hepatocytes were isolated from 7-week-old rat liver and cultured in L-15 medium containing insulin, dexamethasone and 10% fetal bovine serum. Hepatocytes proliferated only on the first day, and then ceased proliferation. The content of GD3 and GD1a increased during the period of active proliferation and reached a nearly constant level, whereas GM1, GD1b, GT1b, and GQ1b gradually increased throughout culture. Addition of EGF to the culture medium caused significant increases in the content of GD3, and to a lesser degree of GM3, but exhibited little effect on the expression of other ganglioside species. The specific induction of GD3 and GM3 expression by EGF was reproduced under serum-free conditions, despite the lack of hepatocyte proliferation. Expression of gangliosides in cultured hepatocytes was also modulated by cell density; higher cell density brought about increased content of GM1, GD1a, GD1b, GT1b, and GQ1b with concomitant reduction of GM3 in cells. The composition of gangliosides in liver tissues demonstrated a unique developmental pattern. GD3 and GD1a were strongly expressed in E-16 embryonic tissue and rapidly decreased with increasing age. GD1b, GT1b, and GQ1b were found only in postnatal liver tissues. These findings suggest that the expression of gangliosides in rat hepatocytes and liver tissues are regulated by growth- and development-dependent factors.     

Ganglioside composition of astrocytes

Short-term and long-term (greater than 7 months) cultured astrocytes from 14-day-old rat brain were analyzed for ganglioside content. Analysis of the extracted gangliosides by HPTLC revealed that ganglioside GM1 was absent in 35 days and 235 days cultured astrocytes, and that the predominant ganglioside was GM3, showing a double band in both cases. A small amount of the disialogangliosides (GD3, GD1a) was also detected. More than 70% of radioactivities into ganglioside fractions by cultured astrocytes, in the presence of N-[3H]-acetylmannosamine, appeared in ganglioside GM3. The upper band component of GM3 increased 60% in long-term astrocyte cultures compared to 35-day-old cultures. Also, an increased GD3 content in long-term astrocyte cultures was detected. These results suggest that the increase of GD3 and upper band GM3 in long-term cultured astrocytes might be related to the appearance of small processes showing strong reactivity against GFAP and vimentin during astrocyte-subculture.