1,721,067 research outputs found
BIOSYNTHESIS OF GANGLIOSIDES CONTAINING C18/1 AND C20/1 [3-C-14]SPHINGOSINE AFTER ADMINISTRATING [1-C-14]PALMITIC ACID AND [1-C-14]STEARIC ACID TO RAT CEREBELLAR GRANULE CELLS IN CULTURE
No full textThe biosynthesis of ganglioside molecular species containing sphingosine of different structure was investigated by administrating rat cerebellar granule cells in culture with [1-C-14]palmitic and [1-C-14]stearic acids which are the precursors for sphingosine biosynthesis. The incorporation of radioactivity into the sphingosine of the ganglioside species containing C20:1 sphingosine after administrating [1-C-14]stearic acid was low in comparison with the incorporation of radioactivity into the sphingosine of ganglioside species containing C18:1 sphingosine after administration of [1-C-14]palmitic acid, but the ratio between the radioactivity incorporated in the C20:1 and the C18:1 sphingosine of C20 and C18 ganglioside species progressively increased when the cell culture was prolonged. Ceramide-containing radioactive sphingosine was found after palmitic or stearic acid administration. Ceramide-containing C20:1 sphingosine found after adding stearic acid was about 5% of that synthesized starting from palmitic acid and containing C18:1 sphingosine. Free radioactive C18:1 and C20:1 sphingosine were found after adding radioactive palmitic or stearic acid. This is representative of a catabolic process occurring after biosynthesis of the complex sphingolipid starting from the radioactive precursor. In fact it has been proved that only saturated sphingosines are used for the synthesis of complex sphingolipids, the introduction of the double bond at position four of the sphingoid chain occurring at the level of ceramide [Rother, J., van Echten, G., Schwarzmann, G. and Sandhoff, K. (1992) Biochem. Biophys. Res. Commun. 189, 14-20]. Saturated sphingosines were not present. The lack of free C20:0 sphingosine confirms the hypothesis that the C20:0 sphingosine synthesis and the process (C20:0 sphingosine-->C20:0 ceramide-->C20:1 ceramide) occur in the correct quantity for the synthesis of C20:1 gangliosides. Moreover, we found only traces of free C20:1 sphingosine, at days 8 and 15 of cell culture when the biosynthesis of complex C20:1 gangliosides and the related catabolic processes occur to a higher extent, thus excluding the idea that a large amount of C20:0 sphingosine can be acylated to C20:0 ceramide and dehydrogenated to C20:1 ceramide which, being not used for ganglioside biosynthesis, is immediately catabolised to C20:1 sphingosine
Metabolic processing of gangliosides by normal and Salla human fibroblasts in culture - A study performed by administering radioactive G(M3) ganglioside
No full textCultured fibroblasts from normal subjects and from subjects affected by Salla disease, characterized by the lack or misfunction of the membrane carrier responsible for the egress of sialic acid from lysosomes, were fed with ganglioside G(M3) labeled at the sialic acid acetyl group, [Neu5Ac-H-3]G(M3), or at C-3 of sphingosine (Sph), [Sph-H-3]G(M3), or at C-1 of stearoyl chain, [stearoyl-C-14]G(M3). After a 15-h pulse the total amount of cell-bound G(M3) corresponded to about 2% of the endogenous ganglioside content. Cells were then subjected to a 72-h chase, and the radioactive products from both ganglioside catabolism and salvage processes of catabolic fragments were measured. These data indicated that about 50% of the cell-bound ganglioside underwent metabolic processing, suggesting a ganglioside half-life of 2-3 days. [Neu5Ac-H-3] formed from [Neu5Ac-H-3]G(M3) degradation was mostly re-cycled for the biosynthesis of gangliosides and sialoglycoproteins, only a minor part being degraded to [H-3]water, which constituted only 1.6% of total metabolite linked radioactivity. [Sph-H-3] from the [Sph-H-3]G(M3) degradation was partly re-cycled for the biosynthesis of gangliosides, neutral glycosphingolipids and sphingomyelin, and partly (about 20% of the total metabolite linked radioactivity) degraded to [H-3]water. In Salla fibroblasts metabolic processing of [Neu5Ac-H-3]G(M3) produced large amounts of free [H-3]Neu5Ac, and a reduced incorporation of radioactivity into glycoconjugates (as compared to normal cells). However, the accumulation of free Neu5Ac was not accompanied by an increase of tritiated water. LacCer and Cer from [stearoyl-C-14]G(M3) catabolism were found to accumulate in Salla fibroblasts, an indication that the enzymes of glycosphingolipid metabolism were affected by the impairment of Neu5Ac egress from lysosomes. Particularly relevant was the accumulation of ceramide which was hardly detectable in control cells
Human fibroblasts in culture metabolize differently exogenous G(M3) ganglioside species containing C18 and C20 sphingosine
No full textPreparation of radioactive G(M3) Species containing isotopically labeled C18 sphingosine or C20 sphingosine is reported and their use for studying some aspects of the sphingolipid biosynthesis in cells is discussed. Human fibroblasts in culture that have only C18 sphingolipids and G(M3) as the major gangliosides, were fed with the two radioactive G(M3) Species. The radioactive gangliosides were taken up by the cells and metabolized. The analyses of the radioactivity metabolic fate, in this model provides the following information. i - About 70-80% of the total catabolic sphingosine is re-cycled for biosynthesis of complex sphingolipids. ii - A small amount of the catabolic C20 sphingosine was re-cycled for biosynthesis of C20 sphingolipids, thus yielding complex lipids that are not naturally present in fibroblast cells. iii - A regulatory step in the biosynthesis of sphingolipid species differring long chain base content, C18 or C20 sphingosine, is in some way involved in the first steps of sphingolipid biosynthesis, and thus plays a decisive role in the availability of the long chain bases
Ganglioside molecular species containing C18-and C20-sphingosine in mammalian nervous tissues and neuronal cell cultures
No full textGangliosides exist as a very complex mixture of species differing in both the hydrophilic and hydrophobic moieties. They are particularly abundant in the central nervous system (CNS), where they have been associated with development and maturation of the brain, neuritogenesis, synaptic transmission, memory formation and synaptic aging. Today, many data suggest that some of the effects exerted by gangliosides are due to interactions with proteins that participate in the transduction of signals through the membrane in membrane microdomains. A specific characteristic of CNS gangliosides is the structure of their long-chain base (LCB). In fact, considering all the mammalian cell sphingolipids,,gangliosides, sulphatides, neutral glycosphingolipids, sphingomyelin and ceramides, it would seem that while the LCB with 18 carbons is the main component of all sphingolipids, only CNS gangliosides contain significant amounts of LCB with 20 carbons. C18-Sphingosine is always present in cell gangliosides; the individual ganglioside species containing C18-sphingosine increase during cell differentiation then remain constant during cell aging. Gangliosides containing C20-sphingosine are absent, or present only in traces, in undifferentiated cells but with the onset of cell differentiation they appear, their content slowly but continuously increasing throughout the life span. In this review we discuss the chemistry, physico-chemistry and metabolism of ganglioside species differing in LCB length and introduce the hypothesis that the varying ratio between C18- and C20-gangliosides during CNS development and aging can be instrumental in modulating membrane domain organisation and cell properties. (C) 2000 Elsevier Science B.V. All rights reserved
CHANGES IN THE GANGLIOSIDE LONG-CHAIN BASE COMPOSITION OF RAT CEREBELLAR GRANULE CELLS DURING DIFFERENTIATION AND AGING IN CULTURE
No full textChanges in the ganglioside long-chain base (LCB) composition in rat cerebellar granule cells in culture were studied during differentiation and aging. The total native ganglioside mixtures, extracted from the cells maintained in culture up to 22 days, were fractionated by reversed-phase HPLC, each ganglioside homogeneous in the oligosaccharide chain as well as in the LCB being quantified. Two main LCBs were components of the ganglioside species of cultured cells, the C18:1 LCB and the C20:1 LCB. The content of C20:1 ganglioside molecular species was low and quite constant during differentiation, comprising approximately 8% of the total ganglioside species content, the C20:1 LCB appearing to be represented more in the ganglioside of the ''b series'' (GD1b, GT1b, and GQ1b) than in the ''a series'' (GM1 and GD1a). During aging in culture, for 8-22 days, the content of the C20:1 species of all gangliosides increased, being more pronounced for GM1 and GD1a
Efflux of sphingolipids metabolically labeled with [1-3H]sphingosine, L-[3-3H]serine and [9,10-3H]palmitic acid from normal cells in culture
No full textThe 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 obsd. from cells to the culture medium in the presence of fetal calf serum. This event was independent of the concn. 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 contg. 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 mols. were again different from those of cells. [on SciFinder (R)
THE SIALIC-ACID RESIDUE OF EXOGENOUS GM1 GANGLIOSIDE IS RECYCLED FOR BIOSYNTHESIS OF SIALOGLYCOCONJUGATES IN RAT-LIVER
No full textIn order to assess metabolic recycling of sialic acid, GM1 ganglioside [nomenclature of Svennerholm (1964) J. Lipid. Res. 5, 145-155; IUPAC-IUB Recommendations (1977) Lipids 12, 455-468], 14C-radiolabelled at the acetyl group of sialic acid, was intravenously injected into Wistar rats, and the presence of radioactive sialic acid in liver sialoglycolipids (gangliosides) and sialoglycoproteins was ascertained. A time-course study (20 min-72 h) showed that the radioactivity present in the liver distributed in the following fractions, with reciprocal proportion varying with time: the protein (glycoprotein) fraction, the ganglioside fraction and the diffusible fraction, which contained low-Mr compounds, including sialic acid. Ganglioside-linked radioactivity gradually decreased with time; protein-linked radioactivity appeared soon after injection (20 min), reached a maximum around 20 h, then slowly diminished; diffusible radioactivity provided a sharp peak at 4 h, then rapidly decreased till disappearing after 40 h. The behaviour of bound radioactivity in the individual liver gangliosides was as follows: (a) rapid diminution with time in GM1, although with a lower rate at the longer times after injection; (b) early appearance (20 min) with a peak at 1 h, followed by continuous diminution, in GM2; (c) early appearance (20 min), peak at 1 h, diminution till 4 h, followed by a plateau, in GM3; (d) appearance at 60 min, maximum around 40 h and slow diminution thereafter, in GD1a, GD1b and GT1b. A detailed study, accomplished at 40 h after injection, demonstrated that almost all radioactivity present in the protein fraction was released by mild acid treatment and recovered in purified sialic acid; most of radioactive glycoprotein-bound sialic acid was releasable by sialidase action. In addition, the radioactivity present in the different gangliosides was exclusively carried by sialic acid and present in both sialidase-resistant and sialidase-labile residues. Only in the case of GD1a was the specific radioactivity of sialidase-resistant sialic acid superior to that of sialidase-releasable sialic acid. The results obtained lead to the following conclusions: (a) radioactive GM3 and GM2 were produced by degradation of GM1 taken up; GM3 originated partly by a process of neosynthesis; (b) radioactive GM1 consisted in part of residual exogenous GM1 and in part of a neosynthetized product; (c) radioactive GD1a originated in part by direct sialylation of GM1 taken up and in part by a neosynthetic process; (d) radioactive GD1b and GT1b resulted only from neosynthesis.(ABSTRACT TRUNCATED AT 400 WORDS
Recognition by two-dimensional thin-layer chromatography and densitometric quantification of alkali-labile gangliosides from the brain of different animals
No full textA simple method for recognition and quantification of alkali-labile gangliosides is described. The method was worked out using authentic alkali-labile gangliosides in pure form (9-O-Ac-GT1b; 9-O-Ac-GQ1b; lactone form of GD1b) and applied to ganglioside mixtures from the brain of mouse, rat, rabbit, pig, and pigeon. The method consists of two-dimensional thin-layer chromatography on silica gel high-performance thin-layer chromatography plates employing the same solvent, chloroform/methanol/0.2% aqueous CaCl2, 50/40/10, for both runs. Prior to the second run the plate is exposed at room temperature for 5 h to ammonia vapors in order to split alkali-labile linkages. At the end of chromatography alkali-stable gangliosides appear lined along a diagonal starting from the origin; the spots corresponding to alkali-labile gangliosides lie out of the diagonal and can be individually detected and quantified on the basis of their sialic acid content. Up to 15 different spots, corresponding to as many alkali-labile gangliosides, can be recognized by this procedure
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