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1.
Neurochem Res ; 27(7-8): 793-800, 2002 Aug.
Article in English | MEDLINE | ID: mdl-12374215

ABSTRACT

The therapeutic potential of bone marrow-derived stromal cells for the therapy of Tay-Sachs disease is primarily related to the restoration of their own GM2 ganglioside storage. With this aim, we produced bone marrow-derived stromal cells from the adult Tay-Sachs animal model and transduced them with a retroviral vector encoding for the alpha-subunit of the lysosomal enzyme beta-hexosaminidase A (E.C. 3.2.1.52). Our results demonstrate that transduced Tay-Sachs bone marrow-derived stromal cells have beta-hexosaminidase A comparable to that of bone marrow-derived stromal cells from wild-type mice. Moreover, beta-hexosaminidase A in transduced Tay-Sachs bone marrow-derived stromal cells was able to hydrolyze the GM2 ganglioside in a feeding experiment, thus demonstrating the correction of the altered phenotype.


Subject(s)
Bone Marrow Cells/metabolism , G(M2) Ganglioside/metabolism , Models, Animal , Stromal Cells/metabolism , Tay-Sachs Disease/metabolism , Animals , Chromatography, Thin Layer , Genetic Vectors , Mice , Retroviridae/genetics
2.
J Neurochem ; 78(5): 1162-7, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11553690

ABSTRACT

Rat cerebellar granule cells differentiated in culture were fed [1-(3)H]sphingosine, allowing the metabolic radiolabelling of all cell sphingolipids and phosphatidylethanolamine. A detergent-insoluble sphingolipid-enriched membrane fraction, containing about 60% of cell sphingolipids, but only trace amounts of phosphatidylethanolamine, was prepared from [1-(3)H]sphingosine-fed cells by sucrose gradient centrifugation. This fraction was enriched in the Src family protein tyrosine kinases c-Src, Lyn and Fyn and in the GPI-anchored neuronal adhesion molecule TAG-1. The cell lysate and the sphingolipid-enriched membrane fraction were subjected to immunoprecipitation with anti-GD3 ganglioside monoclonal antibody R24, under experimental conditions designed to preserve the integrity of the domain. The radioactive lipid composition of the immunoprecipitates obtained from the cell lysate and from the sphingolipid-enriched fraction were very similar, and closely resembled the sphingolipid composition of the whole sphingolipid-enriched membrane fraction. In fact, the immunoprecipitates contained, together with GD3 ganglioside, all cell glycosphingolipids and sphingomyelin, whereas they did not contain phosphatidylethanolamine. Moreover, cholesterol and phosphatidylcholine were detected in the immunoprecipitates by qualitative TLC analysis followed by colourimetric visualization. c-Src, Lyn, Fyn and TAG-1 were associated with the anti-GD3 antibody immunoprecipitate. These proteins were not detected in the immunoprecipitates obtained under experimental conditions different from those designed to preserve the integrity of the domain. These data suggest that a membrane domain containing cholesterol, phosphatidylcholine, sphingolipids and proteins can be separated from the total cell membranes by anti-GD3 antibody immunoprecipitation, and that the association of c-Src, Fyn, Lyn, and TAG-1 with the sphingolipid-enriched domain is mediated by the interaction with a complex lipid environment, rather than by specific interactions with a single sphingolipid species.


Subject(s)
Cell Adhesion Molecules, Neuronal , Gangliosides/isolation & purification , Membrane Glycoproteins/isolation & purification , Membrane Microdomains/enzymology , Neurons/enzymology , Precipitin Tests/methods , src-Family Kinases/isolation & purification , Animals , Antibodies, Monoclonal , CSK Tyrosine-Protein Kinase , Cell Fractionation/methods , Cells, Cultured , Cerebellum/cytology , Contactin 2 , Gangliosides/immunology , Neurons/cytology , Protein-Tyrosine Kinases/isolation & purification , Proto-Oncogene Proteins/isolation & purification , Proto-Oncogene Proteins c-fyn , Rats , Rats, Sprague-Dawley , Sphingosine/isolation & purification , Tritium
3.
J Biol Chem ; 276(24): 21136-45, 2001 Jun 15.
Article in English | MEDLINE | ID: mdl-11264283

ABSTRACT

In the present paper, we report on the properties of sphingolipid-enriched domains of rat cerebellar granule cells in culture at different stages of neuronal development. The major lipid components of these domains were glycerophospholipids and cholesterol. Glycerophospholipids were 45-75% and cholesterol 15-45% of total lipids of the domains. This corresponded to 5-17% of total cell glycerophospholipids and 15-45% of total cell cholesterol. Phosphatidylcholine, mainly dipalmitoylphosphatidylcholine, was 66-85% of all the glycerophospholipids associated with these domains. Consequently, the palmitoyl residue was significantly enriched in the domains. The surface occupied by these structures increased during development. 40-70% of cell sphingolipids segregated in sphingolipid-enriched membrane domains, with the maximum ganglioside density in fully differentiated neurons. A high content of ceramide was found in the domains of aging neurons. Then, the sphingolipid/glycerophospholipid molar ratio was more than doubled during the initial stage of development, whereas the cholesterol/glycerophospholipid molar ratio gradually decreased during in vitro differentiation. Phosphorylated phosphoinositides, which were scant in the domains of undifferentiated cells, dramatically increased during differentiation and aging in culture. Proteins were minor components of the domains (0.1-2.8% of all domain components). Phosphotyrosine-containing proteins were selectively recovered in the sphingolipid-enriched domain. Among these, Src family protein-tyrosine kinases, known to participate to the process of neuronal differentiation, were associated with the sphingolipid-enriched domains in a way specific for the type of kinase and for the developmental stage of the cell. Proteins belonging to other signaling pathways, such as phosphoinositide 3-kinase and its downstream target, Akt, were not associated with the domains.


Subject(s)
Cerebellum/metabolism , Lipid Metabolism , Neurons/metabolism , Sphingolipids/metabolism , Animals , Animals, Newborn , Cell Membrane/metabolism , Cells, Cultured , Ceramides/metabolism , Cerebellum/cytology , Cholesterol/metabolism , Gangliosides/metabolism , Glycerides/metabolism , Kinetics , Membrane Lipids/metabolism , Methionine/metabolism , Nerve Tissue Proteins/isolation & purification , Nerve Tissue Proteins/metabolism , Neurons/cytology , Phosphates/metabolism , Phosphorus Radioisotopes , Rats , Rats, Sprague-Dawley , Sphingomyelins/metabolism , Sphingosine/metabolism , Sulfur Radioisotopes , Tritium
4.
Biochim Biophys Acta ; 1469(2): 63-77, 2000 Sep 18.
Article in English | MEDLINE | ID: mdl-10998569

ABSTRACT

Gangliosides 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.


Subject(s)
Central Nervous System/chemistry , Gangliosides/chemistry , Sphingosine/chemistry , Acyl Coenzyme A , Acyltransferases , Aging , Animals , Brain Chemistry , Carbohydrate Sequence , Cells, Cultured , Central Nervous System/metabolism , Gangliosides/biosynthesis , Gangliosides/metabolism , Humans , Isotope Labeling , Mass Spectrometry , Molecular Sequence Data , Molecular Structure , Neurons/chemistry , Radiochemistry , Serine C-Palmitoyltransferase , Sphingosine/analogs & derivatives , Sphingosine/metabolism
5.
Immunity ; 13(2): 255-64, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10981968

ABSTRACT

The structural basis for the T cell recognition of lipoglycans remains to be elucidated. We have described autoreactive T cells responsive to GM1 ganglioside presented by CD1b. We show that glycosphingolipids bind to CD1b on the cell surface at neutral pH and are recognized without internalization or processing. Furthermore, soluble GM-CD1b complexes stimulate specific T cells. Oligosaccharide groups containing five or more sugars are required to build a minimal epitope for TCR recognition. This suggests a mechanism for T cell recognition of glycosphingolipids in which much of the CD1b-bound ligand is exposed. Binding to CD1b is a highly reversible process and other ceramide-containing glycosphingolipids displace GM1. These nonantigenic compounds act as blockers and may prevent harmful autoreactivity in vivo.


Subject(s)
Antigen Presentation , Antigens, CD1/immunology , Epitopes, T-Lymphocyte/immunology , G(M1) Ganglioside/immunology , T-Lymphocytes/immunology , Ligands
6.
Eur J Biochem ; 267(13): 4187-97, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10866823

ABSTRACT

Cultures of MDCK II and human fibroblast cells were fed radioactive sphingosine and a radioactive GM3 ganglioside derivative containing a photoactivable group. The derived cell homogenates were treated with Triton X-100 and fractionated by sucrose-gradient centrifugation to prepare a detergent-insoluble membrane fraction known to be enriched in sphingolipid and caveolin-1, i.e. of caveolae. The detergent-insoluble membrane fraction prepared after feeding [1-3H]sphingosine to cells, was found to be highly enriched, with respect to protein content, in metabolically radiolabeled sphingomyelin and glycosphingolipids (about 18-fold). By feeding cells photoactivable radioactive GM3, after 2 h-chase, caveolin-1, CAV1, and proteins of high molecular mass became cross-linked to GM3, the cross-linking complexes being highly concentrated in the detergent-insoluble membrane fraction. The interaction between the ganglioside derivative and CAV1 was a time-dependent, transient process so that CAV1 cross-linking to GM3 was hardly detectable after a 24-h chase followed the pulse time. After a 24-h chase, only the high molecular mass proteins cross-linked to GM3 could be clearly observed. These results suggest that a portion of the GM3 administered to cells enters caveolae and moves to the glycosphingolipid domains, or enters caveolae that are then rapidly catabolized. Electron microscopy of cells in a culture immunostained with a monoclonal antibody to GM3 and a secondary gold-conjugated antibody detected several clusters of gangliosides on the plasma membranes separate from caveolae; gangliosides located inside the caveolae could not be detected. Scanning confocal microscopy of cells immunostained with anti-GM3 and anti-CAV1 Ig showed only a very small overlap with the CAV1 and GM3 signals. Thus, the biochemical and microscopic studies suggest that caveolae contain at most a low level of gangliosides and are separate from the GM3 ganglioside enriched domains.


Subject(s)
Cell Membrane/chemistry , Gangliosides/analysis , Animals , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Fibroblasts/chemistry , Humans , Molecular Weight , Sphingosine/metabolism
7.
J Biol Chem ; 275(16): 11658-65, 2000 Apr 21.
Article in English | MEDLINE | ID: mdl-10766784

ABSTRACT

Sphingolipid-enriched membrane domains, characterized by a particular protein and lipid composition, have been detected in a variety of cells. However, limited data are available concerning these domains in neuronal cells. We analyzed the lipid and protein composition of a sphingolipid-enriched membrane fraction prepared from primary rat cerebellar granule cells differentiated in culture. Although the protein content of this fraction was only 1.4% of total cellular protein, 60% of the gangliosides, 67% of the sphingomyelin, 50% of the ceramide, and 40% of the cholesterol were located in this fraction. The protein pattern of the sphingolipid-enriched domain fraction was dramatically different from that associated with the cell homogenate. This fraction contained 25% of the tyrosine-phosphorylated proteins and was enriched in two proteins with apparent molecular masses of 135 and 15 kDa. 12% of cellular glycerophospholipids were located in the fraction, with phosphatidylcholine having the highest enrichment. The molar ratio between proteins, glycerophospholipids, cholesterol, sphingomyelin, ceramide and gangliosides in cerebellar granule cells was 1.6:41.6:6. 1:1.3:0.3:1 in the cell homogenate and 0.04:8.3:4.0:1.4:0.2:1 in the sphingolipid-enriched membrane fraction. These data indicate that selected proteins segregate with sphingolipids in specialized domains in the membrane of cultured neurons.


Subject(s)
Cell Membrane/chemistry , Cerebellum/cytology , Sphingolipids/analysis , Animals , Cell Differentiation , Cell Membrane/metabolism , Cells, Cultured , Chromatography, Thin Layer , Cytoplasmic Granules , Electrophoresis, Polyacrylamide Gel , Methionine/analysis , Phosphates/analysis , Rats , Rats, Sprague-Dawley , Sphingosine/analysis
8.
Glycoconj J ; 17(3 -4): 223-32, 2000.
Article in English | MEDLINE | ID: mdl-11201794

ABSTRACT

Src family kinases play a relevant role in the development and differentiation of neuronal cells. They are abundant in sphingolipid-enriched membrane domains of many cell types, and these domains are hypothesized to function in bringing together molecules important to signal transduction. We studied the association of Src family tyrosine kinases and their negative regulatory kinase, Csk, with sphingolipids in sphingolipid-enriched domains of rat cerebellar granule cells differentiated in culture. We find that c-Src, Lyn and Csk are enriched in the sphingolipid-enriched fraction prepared from these cells. Coimmunoprecipitation experiments show that these and sphingolipids are part of the same domain. Cross-linking experiments with a photoactivable, radioactive GD1b derivative show that c-Src and Lyn, which are anchored to the membrane via a myristoyl chain, associate directly with GD1b. Csk, which is not inserted in the hydrophobic core of the membrane, is not photolabeled by this ganglioside. These results suggest that lipid-lipid, lipid-protein, and protein-protein interactions cooperate to maintain domain structure. We hypothesize that such interactions might play a role in the process of neuronal differentiation.


Subject(s)
Cerebellum/metabolism , Sphingolipids/metabolism , src-Family Kinases/metabolism , Animals , CSK Tyrosine-Protein Kinase , Carbohydrate Sequence , Cell Differentiation , Cell Membrane/metabolism , Cells, Cultured , Cerebellum/cytology , Gangliosides , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Molecular Sequence Data , Precipitin Tests , Protein-Tyrosine Kinases/metabolism , Rats , Rats, Sprague-Dawley
9.
Glycoconj J ; 17(3 -4): 261-8, 2000.
Article in English | MEDLINE | ID: mdl-11201799

ABSTRACT

This paper is the first report on the use of the electron microscopy autoradiography technique to detect metabolically tritium labeled sphingolipids in intact cells in culture. To label cell sphingolipids, human fibroblasts in culture were fed by a 24 hours pulse, repeated 5 times, of 3 x 10(-7) M [1-(3)H]sphingosine. [1-(3)H]sphingosine was efficently taken up by the cells and very rapidly used for the biosynthesis of complex sphingolipids, including neutral glycolipids, gangliosides, ceramide and sphingomyelin. The treatment with [1-(3)H]sphingosine did not induce any morphological alteration of cell structures, and well preserved cells, plasma membranes, and intracellular organelles could be observed by microscopy. Ultrathin sections from metabolic radiolabeled cells were coated with autoradiographic emulsion. One to four weeks of exposition resulted in pictures where the location of radioactive sphingolipids was evidenced by the characteristic appearance of silver grains as irregular coiled ribbons of metallic silver. Radioactive sphingolipids were found at the level of the plasma membranes, on the endoplasmic reticulum and inside of cytoplasmic vesicles. Thus, electron microscopy autoradiography is a very useful technique to study sphingolipid-enriched membrane domain organization and biosynthesis.


Subject(s)
Autoradiography/methods , Membrane Lipids/metabolism , Microscopy, Electron/methods , Sphingolipids/metabolism , Carbohydrate Sequence , Cells, Cultured , Fibroblasts , Humans , Isotope Labeling , Membrane Lipids/chemistry , Molecular Sequence Data , Phosphatidylethanolamines/chemistry , Phosphatidylethanolamines/metabolism , Sphingolipids/chemistry , Sphingosine/chemistry , Sphingosine/metabolism , Tritium
11.
Acta Biochim Pol ; 45(2): 385-92, 1998.
Article in English | MEDLINE | ID: mdl-9821869

ABSTRACT

Preparation of radioactive GM3 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 GM3 as the major gangliosides, were fed with the two radioactive GM3 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.


Subject(s)
Fibroblasts/metabolism , G(M3) Ganglioside/metabolism , Cells, Cultured , G(M3) Ganglioside/chemistry , Humans , Sphingosine/chemistry
12.
J Lipid Res ; 38(6): 1163-9, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9215544

ABSTRACT

Changes in the enzyme 3-ketosphinganine synthase activity in rat cerebellar granule cells in culture were studied during differentiation and aging. The enzyme activity with palmitoyl-CoA and stearoyl-CoA, precursors of, respectively, C18-sphinganine and C20-sphinganine, was studied on the total cell homogenate using radioactive serine. The enzyme assay was performed by thin-layer chromatography (TLC) separation of the enzyme reaction mixture, and the resultant radioactive 3-ketosphinganine was identified by chromatographic comparison with a chemically synthesized 3-ketosphinganine, and quantified by determination of the TLC radioactivity distribution on the basis of the radioactivity content of cell lipid extract that was determined by scintillation counting. Using palmitoyl-CoA, the enzyme activity progressively increased from 40 to 54 pmol of 3-ketosphinganine/mg cell DNA per min in the first 8 days and then progressively decreased, and was 39 pmol of C18-(3-ketosphinganine)/mg cell DNA per min at day 22 in culture. For stearoyl-CoA the enzyme activity was very low at day one and then increased to a constant value of about 15 pmol of C20-(3-ketosphinganine)/mg cell DNA per min. These results are in good agreement with the finding that the ganglioside species that contain C18-sphingosine increase during cell differentiation and remain constant during cell aging, while the ganglioside species that contain C20-sphingosine continuously increase during both cell differentiation and aging.


Subject(s)
Carbon-Carbon Ligases , Cerebellum/enzymology , Ligases/metabolism , Neurons/enzymology , Acyl Coenzyme A/metabolism , Acyl Coenzyme A/pharmacology , Animals , Cells, Cultured , Cerebellum/cytology , Cerebellum/metabolism , Chromatography, Thin Layer , Dose-Response Relationship, Drug , Ligases/analysis , Magnetic Resonance Spectroscopy , Neurons/cytology , Neurons/metabolism , Osmolar Concentration , Palmitoyl Coenzyme A/metabolism , Proteins/analysis , Proteins/drug effects , Rats , Rats, Sprague-Dawley , Serine/pharmacology , Sphingosine/analogs & derivatives , Sphingosine/chemical synthesis , Sphingosine/chemistry , Sphingosine/metabolism , Time Factors
13.
Indian J Biochem Biophys ; 34(1-2): 150-6, 1997.
Article in English | MEDLINE | ID: mdl-9343943

ABSTRACT

We report the preparation of radioactive GM3 ganglioside and its use in the study of sialic acid storage disorders. For the first time GM3 was isotopically radiolabeled in three positions of the molecule: at the sialic acid acetyl group, [3H-Neu5Ac]GM3, at the C1 of the fatty acid moiety, [14C-Stearoyl]GM3, and at C3 of sphingosine, [3H-Sph]GM3. The radioactive GM3 administered to cultured human fibroblasts from a patient suffering from Salla disease was taken up by the cells and metabolized. An analysis of the distribution of radioactivity within the ganglioside metabolic derivatives showed an accumulation of free sialic acid and ceramide in the pathological cells.


Subject(s)
G(M3) Ganglioside/metabolism , Lysosomal Storage Diseases/metabolism , Sialic Acids/metabolism , Animals , Carbohydrate Sequence , Carbon Radioisotopes , Cattle , Cells, Cultured , G(M3) Ganglioside/chemistry , Humans , Molecular Sequence Data , Molecular Structure , Tritium
14.
Eur J Biochem ; 250(3): 661-9, 1997 Dec 15.
Article in English | MEDLINE | ID: mdl-9461288

ABSTRACT

Human cultured fibroblasts were fed with GM3 ganglioside species isotopically labeled at C3 of C18-sphingosine, or at C3 of C18-sphinganine, or at the sialic acid acetyl group, and with C18-sphingosine and C18-sphinganine, both labeled at C1. After a lipid pulse the cells were subjected until 7-day chase; measurements were then made of the radioactive products resulting from the administered long-chain base and ganglioside species catabolism and the salvage processes of catabolic fragments. From the data we drew the following conclusions. The GM3 species differing in the long-chain base structure were taken up by the cells and metabolized. About 80% of the total catabolic C18-sphingosine and C18-sphinganine were recycled for the biosynthesis of complex sphingolipids, the rest being degraded. Results obtained by administering ganglioside species of GM3 containing radioactive sphingosine or the free radioactive sphingosine to fibroblasts suggested the existence in the cells of two quite separate pools of sphingosine. One pool was the direct result of either the catabolism of radioactive GM3 high-density microdomains or the diffusion of exogenous sphingosine into the cell; this pool was mainly used for the biosynthesis of the GD3 species that contain palmitic and stearic acids. The other pool of sphingosine, the cell basal pool, came from the catabolism of radioactive sphingolipids in the recycling of sphingosine, and was used for the biosynthesis of the GD3 species that mainly contain very long fatty acid chains, the main fibroblast endogenous species of GD3. Administration of the ganglioside species of GM3 containing sphinganine or free sphinganine to fibroblasts yielded the GD3 species containing mainly very long-chain fatty acids and sphingosine. These results show the possible existence of a pool of ganglioside-derived sphingosine, quite separate from the cell basal pool of sphingosine, suggesting that sphingosine derived from sphingolipid catabolism is reduced to sphinganine before entering the sphingolipid biosynthetic pathway.


Subject(s)
G(M3) Ganglioside/metabolism , Sphingosine/metabolism , Autoradiography , Cell Line , Cells, Cultured , Ceramides/analysis , Chromatography, High Pressure Liquid , Chromatography, Thin Layer , Fatty Acids/biosynthesis , Fatty Acids/metabolism , Fibroblasts , G(M3) Ganglioside/biosynthesis , Gangliosides/analysis , Gangliosides/biosynthesis , Gangliosides/chemistry , Humans , Phospholipids/analysis , Sphingolipids/biosynthesis , Sphingomyelin Phosphodiesterase/metabolism , Sphingomyelins/analysis , Sphingosine/analogs & derivatives
15.
J Neurochem ; 67(5): 1866-71, 1996 Nov.
Article in English | MEDLINE | ID: mdl-8863491

ABSTRACT

Changes in the free long-chain base (LCB) composition in rat cerebellar granule cells in culture were studied during differentiation and aging. The total LCB mixtures, extracted from the cells maintained in culture up to 22 days, were derivatized with o-phthalaldehyde and fractionated by reversed-phase HPLC, and each LCB species was quantified. Four main LCBs were components of the total LCB mixtures of cultured cells: C18-sphingosine, C18-sphinganine, C20-sphingosine, and C20-sphinganine. They were found in all the cells analyzed, from 0 to 22 days of culture, with their contents being in the sequence C18-sphingosine > C18-sphinganine > C20-sphingosine > C20-sphinganine and varying from 0.02 +/- 0.015 pmol/mg of cell protein for C20-sphinganine at day 0 to 223 +/- 22 pmol/mg of cell protein for C18-sphingosine at day 8. Sphinganines were found to be minor components of the total LCB mixture, with C20-sphinganine being particularly scarce in nondifferentiated cells. The cell content of C20-sphinganine progressively increased from day 0 to 22 of culture; that of C18-sphinganine increased up to day 8, when cells are differentiated, and then remained quite constant. The changes of C18- and C20-sphingosine levels during cell culture were qualitatively similar to those of C18- and C20-sphinganine, but the content of the sphingosines was much higher than that of the sphinganines.


Subject(s)
Neurons/cytology , Neurons/metabolism , Sphingosine/analogs & derivatives , Sphingosine/metabolism , Animals , Cell Differentiation , Cells, Cultured , Cellular Senescence , Cerebellum/cytology , Cerebellum/metabolism , Chromatography, High Pressure Liquid , Chromatography, Thin Layer , Kinetics , Rats , Rats, Sprague-Dawley , Sphingosine/chemistry , Sphingosine/isolation & purification , Time Factors
16.
J Biol Chem ; 271(36): 21738-44, 1996 Sep 06.
Article in English | MEDLINE | ID: mdl-8702969

ABSTRACT

Cultured 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 GM3 labeled at the sialic acid acetyl group, [Neu5Ac-3H]GM3, or at C-3 of sphingosine (Sph), [Sph-3H]GM3, or at C-1 of stearoyl chain, [stearoyl-14C]GM3. After a 15-h pulse the total amount of cell-bound GM3 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-3H] formed from [Neu5Ac-3H]GM3 degradation was mostly re-cycled for the biosynthesis of gangliosides and sialoglycoproteins, only a minor part being degraded to [3H]water, which constituted only 1.6% of total metabolite linked radioactivity. [Sph-3H] from the [Sph-3H]GM3 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 [3H]water. In Salla fibroblasts metabolic processing of [Neu5Ac-3H]GM3 produced large amounts of free [3H]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-14C]GM3 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.


Subject(s)
G(M3) Ganglioside/metabolism , Lysosomal Storage Diseases/metabolism , Adolescent , Animals , Cattle , Cells, Cultured , Child , DNA/metabolism , Fibroblasts/metabolism , Gangliosides/metabolism , Humans , Male , N-Acetylneuraminic Acid , Proteins/metabolism , Sialic Acids/metabolism , Sialoglycoproteins/metabolism , Sphingomyelins/metabolism
18.
Glycoconj J ; 13(3): 347-52, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8781964

ABSTRACT

An efficiency assessment of a ganglioside assay procedure was carried out on human serum gangliosides from healthy subjects of different sex and age. The analysis of the gangliosides, extracted with chloroform/methanol and purified by lipid partitioning, ion exchange column chromatographic separation and desalting procedures as described by Senn et al. (1989) Eur J Biochem 181: 657-62, was performed by HPTLC followed by densitometric quantification. The yield of the procedure, expressed as radioactivity recovery, was determined by adding GM3 ganglioside, tritium labelled at the sialic acid acetyl group and at the C3 position of sphingosine, to the lyophilized serum or by associating it with the serum lipoproteins. In spite of the fact that the extraction and purification procedures were performed exactly as described we found the radioactivity recovery to be variable (25-50%) and much lower than that proposed. Much of the radioactivity was found in the organic phase after lipid partitioning, whilst all the ganglioside purification steps led to some further loss. After the introduction of some modifications to the procedure the recovery improved, reaching 67-79%. The analyses on 33 samples of 5 ml showed a human serum ganglioside content of about 10 nmol ml-1 (as corrected for the recovery), and confirmed that GM3 ganglioside is the main component of the total serum ganglioside mixture.


Subject(s)
Gangliosides/blood , Adult , Age Factors , Carbohydrate Conformation , Carbohydrate Sequence , Chromatography, High Pressure Liquid/methods , Chromatography, Thin Layer/methods , Densitometry/methods , Female , G(M3) Ganglioside/blood , Gangliosides/chemistry , Gangliosides/isolation & purification , Humans , Male , Middle Aged , Molecular Sequence Data , Reference Values , Reproducibility of Results , Sex Characteristics
19.
Clin Chim Acta ; 247(1-2): 143-57, 1996 Mar 29.
Article in English | MEDLINE | ID: mdl-8920233

ABSTRACT

The metabolic processing of sialoglycolipids (gangliosides) was investigated in cultures of skin fibroblasts obtained from two patients affected with Salla disease. Cultured fibroblasts were fed with GM1 ganglioside [3H]-radiolabelled at the sialic acid ([NeuAc-3H]GM1) or sphingosine ([Sph-3H]GM1) moiety. Formation of metabolites was followed in pulse-chase experiments. It was observed that: (a) Salla fibroblasts, fed with [NeuAc-3H]GM1 accumulate radioactive free sialic acid in the lysosomal compartment and show a much lower sialic acid re-cycling for biosynthetic purposes than control fibroblasts, as demonstrated by decreased incorporation of the label into glycolipids and glycoproteins; (b) Salla fibroblasts, fed with [NeuAc-3H]GM1 or [Sph-3H]GM1, tend to accumulate gangliosides GM2 and GM3, and to reduce the breakdown products following the desialosylation step, presumably as a consequence of the inhibition of sialidase by free sialic acid; (c) owing to (b) the basal production of the bioregulators of sphingoid nature, ceramide and sphingosine, is reduced, as well as re-cycling of these substances for biosynthetic purposes, with further reduction of the turnover rate of sphingolipids. The decreased turnover rate of sialoglycoconjugates and sphingolipids, together with the diminished formation of bioregulators of sphingoid nature, may play a relevant role in the pathogenesis of the disease.


Subject(s)
Fibroblasts/metabolism , G(M1) Ganglioside/metabolism , Lysosomal Storage Diseases/metabolism , N-Acetylneuraminic Acid/metabolism , Cells, Cultured , Humans
20.
Neurochem Int ; 28(2): 183-7, 1996 Feb.
Article in English | MEDLINE | ID: mdl-8719707

ABSTRACT

The ganglioside mixture from the cerebellum of young, 6 month old and two years old rats, was fractionated by reversed phase high performance liquid chromatography, each ganglioside homogeneous in the oligosaccharide chain as well as in the long-chain base being subsequently quantified. Two long-chain bases, LCB, were components of the five major gangliosides GM1, GD1a, GD1b, GT1b and GQ1b, these being the C18:1 LCB and C20:1 LCB. The content of C20:1 ganglioside molecular species was lower than that of the C18:1 one. In very young animals, day 8, the C20:1 ganglioside species represented about 8% of the total ganglioside content, then they progressively increased and reached, at 2 years, about 42% of the total. C18:1 GD1a and C18:1 GT1b, were the major species in young animals and reached their highest content at day 29, being 1.45 and 1.28 nmol/mg protein, respectively. The content of these two species decreased in adult and old animals and at two years it was 0.71 and 0.82 nmol/mg protein, respectively.


Subject(s)
Aging/metabolism , Cerebellum/growth & development , Cerebellum/metabolism , Gangliosides/metabolism , Animals , Chromatography, High Pressure Liquid , Gangliosides/chemistry , Gas Chromatography-Mass Spectrometry , Rats , Rats, Sprague-Dawley , Spectrophotometry, Ultraviolet
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