Production of Multiple Brain-Like Ganglioside Species Is Dispensable for Fas-Induced Apoptosis of Lymphoid Cells

Activation of an acid sphingomyelinase (aSMase) leading to a biosynthesis of GD3 disialoganglioside has been associated with Fas-induced apoptosis of lymphoid cells. The present study was undertaken to clarify the role of this enzyme in the generation of gangliosides during apoptosis triggered by Fas ligation. The issue was addressed by using aSMase-deficient and aSMase-corrected cell lines derived from Niemann-Pick disease (NPD) patients. Fas cross-linking elicited a rapid production of large amounts of complex a- and b-series species of gangliosides with a pattern and a chromatographic behavior as single bands reminiscent of brain gangliosides. The gangliosides were synthesized within the first ten minutes and completely disappeared within thirty minutes after stimulation. Noteworthy is the observation that GD3 was not the only ganglioside produced. The production of gangliosides and the onset of apoptotic hallmarks occurred similarly in both aSMase-deficient and aSMase-corrected NPD lymphoid cells, indicating that aSMase activation is not accountable for ganglioside generation. Hampering ganglioside production by inhibiting the key enzyme glucosylceramide synthase did not abrogate the apoptotic process. In addition, GM3 synthase-deficient lymphoid cells underwent Fas-induced apoptosis, suggesting that gangliosides are unlikely to play an indispensable role in transducing Fas-induced apoptosis of lymphoid cells

Iminosugar-Based Inhibitors of Glucosylceramide Synthase Increase Brain Glycosphingolipids and Survival in a Mouse Model of Sandhoff Disease

The neuropathic glycosphingolipidoses are a subgroup of lysosomal storage disorders for which there are no effective therapies. A potential approach is substrate reduction therapy using inhibitors of glucosylceramide synthase (GCS) to decrease the synthesis of glucosylceramide and related glycosphingolipids that accumulate in the lysosomes. Genz-529468, a blood-brain barrier-permeant iminosugar-based GCS inhibitor, was used to evaluate this concept in a mouse model of Sandhoff disease, which accumulates the glycosphingolipid GM2 in the visceral organs and CNS. As expected, oral administration of the drug inhibited hepatic GM2 accumulation. Paradoxically, in the brain, treatment resulted in a slight increase in GM2 levels and a 20-fold increase in glucosylceramide levels. The increase in brain glucosylceramide levels might be due to concurrent inhibition of the non-lysosomal glucosylceramidase, Gba2. Similar results were observed with NB-DNJ, another iminosugar-based GCS inhibitor. Despite these unanticipated increases in glycosphingolipids in the CNS, treatment nevertheless delayed the loss of motor function and coordination and extended the lifespan of the Sandhoff mice. These results suggest that the CNS benefits observed in the Sandhoff mice might not necessarily be due to substrate reduction therapy but rather to off-target effects

Exercice interlaboratoire sur les dosages des hydrocarbures dans l'eau et les sédiments Interlaboratory Exercise on Hydrocarbon Measurement in Water and Sediments

Cet exercice d'intercalibration organisé au niveau national sous l'égide du Réseau National d'Observation portait sur le dosage des hydrocarbures présents dans J'eau et les sédiments. Les résultats sont comparés et discutés en fonction des techniques analytiques employées. Vingt-cinq ,laboratoires ont participé à l'exercice pour le substrat eau et vingt-deux pour le substrat sédiment en suivant dans la majorité des cas un protocole analytique commun à tous les participants. Pour chaque substrat deux niveaux de concentrations en hydrocarbures ont été proposés pouvant correspondre à des cas réels de pollution. La spectrométrie infrarouge (IR) et la spectrofluorimétrie ultraviolette (SFUV) ont été les techniques les plus utilisées. Les plus faibles dispersions dans les résultats sont relevées pour le dosage des hydrocarbures dans l'eau par SFUV et pour celui des sédiments par IR. Bien que chacune de ces techniques présente une spécificité vis-à-vis de certaines classes d'hydrocarbures, les résultats montrent dans leur ensemble un bon degré d'homogénéité lorsque l'étalon de référence est une coupe pétrolière. La microgravimétrie est la méthode qui présente les plus faibles variabilités dans les résultats, mais semble limitée dans le dosage de teneurs très faibles en hydrocarbures (< 100 ppm). Les limites d'utilisation des différentes techniques sont discutées. L'analyse individuelle des hydrocarbures aliphatiques et aromatiques a été effectuée par quelques laboratoires en utilisant la chromatographie en phase gazeuse (CPG) et/ou la chromatographie en phase liquide (CLHP). En l'absence d'un protocole imposé, on observe pour ces deux techniques une dispersion plus marquée des résultats, ce qui rend leur exploitation plus délicate et peu utilisable dans l'hypothèse d'une quantification globale des hydrocarbures. Cet exercice correspond à une première tentative d'harmonisation, au niveau national, des méthodes de dosage des hydrocarbures totaux dans l'eau et les sédiments marins dans le but d'améliorer la comparabilité des résultats entre des laboratoires susceptibles d'être engagés dans des programmes de surveillance de pollution par hydrocarbures. <br> This intercomparison exercice organized among French laboratories, under the aegis of RNO (Réseau National d'Observation), concerned the measurement of hydrocarbons present in water and sediments. The results are compared and discussed according to the analytical techniques used. Twenty-five laboratories took part in the project involving water substrate and twenty-two for the part involving sediment substrate. In most cases, the same analytical procedure was followed by all the participants. For each substrate two levels of hydrocarbon concentrations were proposed that might correspond to possible cases of pollution in environmental studies. Infrared spectrometry (IR) and ultraviolet spectrofluorometry (UVSF) were the most widely used techniques. The smallest dispersions in the results were found for hydrocarbon measurement in water by UVSF and in sediments by IR. Although each of these techniques has its own specificity with regard to various classes of hydrocarbons, the results on the whole show a good degree of homogeneity when the oil is used as a reference standard. Microgravimetry is the method showing the smallest variability in the results, but it seems to be limited for measuring very low hydrocarbon contents (< 100 ppm). The limits of use of the different techniques are discussed. Aliphatic and aromatic hydrocarbons were analyzed individually by several laboratories, using gas chromatography (GC) and/or high performance liquid chromatography (HPLC). In the absence of a defined analytical procedure, we observed a quater dispersion of results, so these two techniques, are difficult to use for the evaluation of total hydrocarbon contents. This intercomparison exercice corresponds to an initial attempt to achieve national harmonization of methods for measuring total hydrocarbons in water and marine sediments with the aim of improving the comparability of results among laboratories liable to be involved in monitoring pollution by hydrocarbons

Distribution and Characterization of Tar Ball Pollution on Beaches in Brittany (France) and Indonesia

Stranded tar along the Brittany coast and four test sites in Indonesia (Jakarta Bay, Malacca and Makassar Straits, Central South Java) were surveyed in 1982 in order to quantify and identify the possible origin of oil pollutants. The estimation of tar pollution was determined by a statistical methodology based on a stratified random sampling. Western Brittany and Jakarta Bay must be regarded among the areas most vulnerable to chronic oil pollution with respectively gross weight values of 76.9 ±115.6g/m and 812.7±219g/m, comparable to those of other coasts along major tanker routes such as Kuwait or Bermuda. The levels on the other sampled sites were relatively low in spite of such dense maritime traffic as in Malacca straits (15.4 ±5.4 g/m) or near the mouth of La Loire (12.2±15.9 g/m and less than 0.5 g/m in the Bay of Saint-Brieuc). Hydrocarbons in tar samples were examined by liquid and gas chromatography for a classification in crude oil residues, sludge residues and fuel oil. A multimethod approach combining GC, IR, SFUV and sulfur content was used for a characterization of oil extracts and their tentative matching with the physico-chemical parameters of some reference oils

Glucosylceramide and glucosylsphingosine modulate calcium mobilization from brain microsomes via different mechanisms

We recently demonstrated that elevation of intracellular glucosylceramide (GlcCer) levels results in increased functional Ca2+ stores in cultured neurons, and suggested that this may be due to modulation of ryanodine receptors (RyaRs) by GlcCer (Korkotian, E., Schwarz, A., Pelled, D., Schwarzmann, G., Segal, M. and Futerman, A. H. (1999) J. Biol. Chem. 274, 21673–21678). We now systematically examine the effects of exogenously added GlcCer, other glycosphingolipids (GSLs) and their lyso-derivatives on Ca2+ release from rat brain microsomes. GlcCer had no direct effect on Ca2+ release, but rather augmented agonist-stimulated Ca2+ release via RyaRs, through a mechanism that may involve the redox sensor of the RyaR, but had no effect on Ca2+ release via inositol 1,4,5-trisphosphate receptors. Other GSLs and sphingolipids, including galactosylceramide, lactosylceramide, ceramide, sphingomyelin, sphingosine 1-phosphate, sphinganine 1-phosphate, and sphingosylphosphorylcholine had no effect on Ca2+ mobilization from rat brain microsomes, but both galactosylsphingosine (psychosine) and glucosylsphingosine stimulated Ca2+ release, although only galactosylsphingosine mediated Ca2+ release via the RyaR. Finally, we demonstrated that GlcCer levels were ∼10-fold higher in microsomes prepared from the temporal lobe of a type 2 Gaucher disease patient compared with a control, and Ca2+ release via the RyaR was significantly elevated, which may be of relevance for explaining the pathophysiology of neuronopathic forms of Gaucher disease

Increasing fat content from 20 to 45 wt% in a complex diet induces lower endotoxemia in parallel with an increased number of intestinal goblet cells in mice

International audienceThe impacts of high-fat diets (HFDs) on the onset of metabolic endotoxemia and low-grade inflammation are well established in rodent models. However, the dose-effect of dietary lipid intakes on these parameters is not known. We hypothesized that increasing dietary lipid amounts could be linked to parallel increases of endotoxemia, low-grade inflammation, and metabolic and intestinal alterations. Six-week-old male C57BL/6J mice were fed a low-fat diet (LFD, 2.6 wt% of lipids), a moderate HFD (mHFD, 22 wt% of lipids), or a very HFD (vHFD, 45 wt% of lipids) formulated mainly using chow ingredients and milk fat. After 12 weeks, white adipose tissues, liver, intestine, distal colon contents, and plasma were collected. Only vHFD mice significantly increased body weight and fat mass vs LFD mice. This was associated with increases of plasma concentrations of triglycerides, leptin and adiponectin, and liver lipids. No such differences were observed between LFD and mHFD mice. However, mHFD developed metabolic endotoxemia and inflammation, unlike vHFD mice. In turn, vHFD mice showed more goblet cells in all intestine segments vs both other groups and a decrease of Bacteroides-Prevotella in their microbiota vs LFD mice. Finally, mHFD mice colon exhibited a decrease in lactobacilli and in the levels of occludin phosphorylation. Altogether, using complex HFD, no associations were observed between dietary lipid amounts and the magnitude of endotoxemia, inflammation, and physiological alterations developed. These results reveal the impact of the diet composition on intestinal goblet cells and mucus coat, bringing new insights about further consequences on HFD-induced metabolic disorders