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Biomedical subjects

Jacques Portoukalian

Publications and source records attributed to Jacques Portoukalian.

11 recordsLinked to original sources
PubMed2004

Gangliosides of organ-confined versus metastatic androgen-receptor-negative prostate cancer.

Prior development of a unique androgen-receptor (AR)-negative cell line (HH870) from organ-confined (T2b) human prostate cancer (CaP) enabled comparison of the gangliosides associated with normal and neoplastic prostate epithelial cells, organ-confined versus metastatic (DU 145, PC-3), and AR-negative versus AR-positive CaP cell lines. Resorcinol-HCl and specific monoclonal antibodies were used to characterize gangliosides on 2D-chromatograms, and to visualize them on the cell surface with confocal-fluorescence microscopy. AR-negative cells expressed GM1b, GM2, GD2, GD1a, and GM3. GM1a, GD1b, and GT1b were undetectable. GM1b and GD1a were more prominent in AR-negative than in AR-positive cells. PC-3 and HH870 cells were unique in the expression of O-acetylGD2 (O-AcGD2) and two alpha2,3-sialidase-resistant, alkali-susceptible GMR17-reactive gangliosides. Expression of GD1a, GM1b, doublets of GD3, GD2, and O-AcGD2, and the presence of an additional alkali-labile-14.G2a-reactive ganglioside, two alkali-susceptible, and three alkali-resistant GMR17-reactive gangliosides makes HH870 a potential component of a polyvalent-vaccine for active-specific immunotherapy of CaP.

Animals↗
PubMed2004

Subcellular compartmentalization of ceramide metabolism: MAM (mitochondria-associated membrane) and/or mitochondria?

Recent studies by our group and others have disclosed the presence of ceramides in mitochondria, and the activities of ceramide synthase and reverse ceramidase in mitochondria have also been reported. Since a possible contamination with the ER (endoplasmic reticulum)-related compartment MAM (mitochondria-associated membrane) could not be ruled out in previous studies, we have re-investigated the presence of the enzymes of ceramide metabolism in mitochondria and MAM highly purified from rat liver. In the present paper, we show that purified mitochondria as well as MAM are indeed able to generate ceramide in vitro through both ceramide synthase or reverse ceramidase, whereas the latter enzyme activity is barely detectable in microsomes. Moreover, ceramide synthase activities were recovered in outer mitochondrial membranes as well as in inner mitochondrial membranes. Using radiolabelled sphingosine as a substrate, mitochondria could generate ceramide and phytoceramide. However, the in vitro sensitivity of ceramide synthase toward FB1 (fumonisin B1) in mitochondria as well as in MAM was found to depend upon the sphingoid base: whereas dihydrosphingosine N-acyltransferase was inhibited by FB1 in a concentration-dependent manner, FB1 actually activated the ceramide synthase when using sphingosine as a substrate. Acylation of sphingosine 1-phosphate and dihydrosphingosine 1-phosphate, generating ceramide 1-phosphate, was also shown with both subcellular fractions. Moreover, the same difference in sensitivity towards FB1 for the ceramide synthase activities was seen between the two phosphorylated sphingoid bases, raising the possibility that distinct base-specific enzymes may be involved as ceramide synthases. Collectively, these results demonstrate the involvement of mitochondria in the metabolism of ceramides through different pathways, thereby supporting the hypothesis that topology of ceramide formation could determine its function.

Animals↗
PubMed2004

Menaquinone-4 concentration is correlated with sphingolipid concentrations in rat brain.

Studies with animals support a role for vitamin K (VK) in the biosynthesis of sphingolipids, a class of complex lipids present in high concentrations in the brain. In mice and rats, VK deficiency decreases levels of brain sulfatides and causes behavioral alterations. In light of its heterogeneity and to better understand the role of VK in the brain, we characterized the distribution of the two main VK vitamers, phylloquinone (K1) and menaquinone-4 (MK-4), in nine distinct brain regions. Weaning female Sprague-Dawley rats (n=5/dietary group) were fed diets containing either low (L, 80 microg/kg diet), adequate (A, 500 microg/kg diet) or high (H, 2000 microg/kg diet) levels of K1 for 6 mo. The main form of VK in the brain was MK-4, and it was present in significantly higher concentrations in myelinated regions (the pons medulla and midbrain) than in nonmyelinated regions. Both regional K1 and MK-4 increased with K1 intake (P<0.05). Sphingolipid distribution varied across brain regions (P<0.001) but was not affected by K1 intake. In the L and A groups but not the H group, brain MK-4 concentration was positively correlated with the concentrations of sulfatides (L, r=0.518; A, r=0.479) and sphingomyelin (L, r=0.515; A, r=0.426), and negatively correlated with ganglioside concentration (L, r=-0.398); A, r=-0.353). Sphingolipids are involved in major cellular events such as cell proliferation, differentiation and survival. The strong associations reported here between brain MK-4 and sphingomyelin, sulfatides and gangliosides suggest that this vitamer may play an important role in the brain.

Animals↗
PubMed2003

Characterization of the ceramide moieties of sphingoglycolipids from mouse brain by ESI-MS/MS: identification of ceramides containing sphingadienine.

Sphingoglycolipids (SGLs) are cell membrane constituents. As the ceramide structure influences the biological properties of the SGL, we characterized by electrospray ionization tandem mass spectrometry the molecular species of ceramides present in SGL of mouse brain. We report here for the first time the presence in mammalian brain of sphingadienine (d18:2). Sphingenine (d18:1) is present in all SGL species, in contrast to eicosasphingenine (d20:1), which is a constituent of only gangliosides. Sphingadienine is present in galactosylceramide and sulfatides. Free ceramides contain the three types of bases. Thus, there could be two separate pools of free ceramides (d18:1, d18:2 and d20:1, d18:1) as precursors of complex SGL.

Animals↗
PubMed2003

The mitochondria-associated endoplasmic-reticulum subcompartment (MAM fraction) of rat liver contains highly active sphingolipid-specific glycosyltransferases.

Although most glycosphingolipids (GSLs) are thought to be located in the outer leaflet of the plasma membrane, recent evidence indicates that GSLs and their precursor, ceramide, are also associated with intracellular organelles and, particularly, mitochondria. GSL biosynthesis starts with the formation of ceramide in the endoplasmic reticulum (ER), which is transported by controversial mechanisms to the Golgi apparatus, where stepwise addition of monosaccharides on to ceramides takes place. We now report the presence of GSL-biosynthetic enzymes in a subcompartment of the ER previously characterized and termed 'mitochondria-associated membrane' (MAM). MAM is a membrane bridge between the ER and mitochondria that is involved in the biosynthesis and trafficking of phospholipids between the two organelles. Using exogenous acceptors coated on silica gel, we demonstrate the presence of ceramide glucosyltransferase (Cer-Glc-T), glucosylceramide galactosyltransferase and sialyltransferase (SAT) activities in the MAM. Estimation of the marker-enzyme activities showed that glycosyltransferase activities could not be ascribed to cross-contamination of MAM by Golgi membranes. Cer-Glc-T was found to have a marked preference for ceramide bearing phytosphingosine as sphingoid base. SAT activities in MAM led to the synthesis of G(M3) ganglioside and small amounts of G(D3). G(M1) was also synthesized along with G(M3) upon incubation of the fraction with exogenous unlabelled G(M3), underlying the presence of other sphingolipid-specific glycosyltransferases in MAM. On the basis of our results, we propose MAM as a privileged compartment in providing GSLs for mitochondria.

Animals↗
PubMed2003

Gangliosides from human melanoma tumors impair dendritic cell differentiation from monocytes and induce their apoptosis.

Gangliosides are ubiquitous membrane-associated glycosphingolipids, which are involved in cell growth and differentiation. Most tumor cells synthesize and shed large amounts of gangliosides into their microenvironment, and many studies have unraveled their immunosuppressive properties. In the present study we analyzed the effects of GM3 and GD3 gangliosides, purified from human melanoma tumors, on the differentiation of monocyte-derived dendritic cells (MoDC). At concentrations close to those detected in the sera from melanoma patients, both gangliosides dose-dependently inhibit the phenotypic and functional differentiation of MoDC, as assessed by a strong down-regulation of CD1a, CD54, CD80, and CD40 Ags and impaired allostimulatory function on day 6 of culture. Furthermore, GM3 and GD3 gangliosides decreased the viable cell yield and induced significant DC apoptosis. Finally, addition of GD3 to differentiating DC impaired their subsequent maturation induced by CD154. The resulting DC produced low amounts of IL-12 and large amounts of IL-10, a cytokine pattern that might hamper an efficient antitumor immune response. In conclusion, the results demonstrate that gangliosides impair the phenotypic and functional differentiation of MoDC and induce their apoptosis, which may be an additional mechanism of human melanoma escape.

Antigens, Neoplasm↗
PubMed2003

Sulfatide and GM1 ganglioside modulate the high-affinity dopamine uptake in rat striatal synaptosomes: evidence for the involvement of their ionic charges.

The present study was undertaken to examine the effects of the anionic glycolipids GM1 ganglioside and sulfatide on the high-affinity dopamine (DA) uptake in rat striatal synaptosomes. After 1h of incubation, GM1 stably bound to synaptosomes and modified the activity of the neuronal dopamine transporter (DAT). With 1.2 and 12 microM GM1, V(max) decreased by 13 and 23%, respectively, reflecting a slight reduction of the number of functional uptake sites and K(m) was lowered by 21 and 33%, thus showing an increase of the affinity. Treatment of synaptosomes with 1.2 microM of sulfatide, which possesses an anionic sulfated group, led to a similar decrease of V(max) (19%) than GM1, but to a significantly higher reduction of K(m) (35%). In fact, sulfatide associated to synaptosomes in a 3.5-fold higher extent than GM1. Conversely, when GM1 and sulfatide were replaced by GM1 alcohol and galactosylceramide, respectively, no modification of the DA uptake occurred, although these neutral glycolipids incorporated into the synaptosomes to the same extent as the related anionic compounds.Altogether, these results demonstrate the key role of negative charges linked to the oligosaccharide chains of glycolipids in the modulation of DA transport across the synaptosomal membrane.

Animals↗
PubMed2003

[Cellular lipid dynamics].

During the past years, the notion of microdomains at the surface of cellular membranes has been developed. These are constituted by lipid rafts which involve sphingoglycolipids and cholesterol. To these rafts are associated proteins which have a lipid anchor or are transmembrane proteins. These lipid rafts target specific proteins at the plasma membrane surface and can remain associated with them. They are present in surface receptors and endocytosis occurs upon binding of the specific ligands. Thus these rafts participate to major aspects of cellular dynamics. These rafts are complex structures, insoluble in non-ionic detergents. According to the detergent used, many types of rafts can be isolated. Any alteration of cholesterol, sphingoglycolipids, or abnormalities of the proteins themselves, can lead to abnormal targeting at the membrane surface. It is possible that specific sphingoglycolipids are necessary to target specific proteins at the membrane surface. This may explain the complexity of the sphingoglycolipid molecules, both in relation to their oligosaccharide and to their ceramide structures. There is both a cellular and a tissue specificity of these constituents. Complex sphingoglycolipids are involved in cellular differentiation, cellular polarization, and modified in relation to cancer. Virus and bacteria can be linked to the sphingoglycolipids of these microdomains and alter cellular signaling and function. Sphingoglycolipids are involved in autoimmune diseases as antibody targets and in neurolipidoses which are genetic diseases involving their catabolism. The dynamics of the lipid rafts, in relation to cholesterol, can be altered in Niemann-Pick's disease type C and in Alzheimer's disease. Thus these microdomains are involved in many aspects related to normal and pathological cellular dynamics.

Animals↗
PubMed2002

A sensitive flow cytometry method for anti-GM1 antibodies detection.

High-affinity anti-GM1 antibodies are frequently described in several nervous system diseases, mainly in multifocal motor neuropathy (MMN) and some acute neuropathies. These antibodies are currently detected using enzyme-linked immunosorbent assay (ELISA) and immuno-thin-layer-chromatography (immuno-TLC) methods. We report in this article a new method based on the incorporation of exogenous GM1 in a selected cell line to detect anti-GM1 antibodies using flow cytometry (FC). This method is evaluated on 80 sera from normal blood donors (NBD) and patients suffering various nervous system diseases. It appears to be as sensitive as our method ELISA for the diagnosis of some motor neuron syndromes.

Autoantibodies↗
PubMed2002

Recovery of gangliosides from aqueous solutions on styrene-divinylbenzene copolymer columns.

An investigation was made using a styrene-divinylbenzene copolymer as a solid phase sorbent to recover gangliosides from aqueous solutions. A comparison with octadecyl-bonded (C18) silica gel showed that the general procedure used to purify gangliosides on C18 silica gel could be used with the copolymer. The yield of gangliosides depended on various parameters such as the composition of the conditioning solution, the salt concentration of the loading solution, and the amount of applied gangliosides per gram of copolymer. In optimal conditions, the recovery of gangliosides and other lipids present in the upper phases of partition was higher than 95%. Using radiolabeled gangliosides, it was found that gangliosides present in serum-containing medium could also be quantitatively recovered on copolymer, provided the medium was diluted with an equal volume of methanol prior to its application onto the column. The major advantage of the copolymer is its high stability in acidic or alkaline conditions that allows multiple cycles of cleaning and reconditioning of the sorbent without alteration of its chromatographic properties.

Animals↗