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H Dreyfus

Publications and source records attributed to H Dreyfus.

At least 73 records · Page 4Linked to original sources
PubMed1986

Effect of neuraminidase treatment on the topological distribution of phospholipids in chick brain microsomes.

The desialylation of chick brain microsomal membranes affects the transbilayer distribution of phospholipids. When intact microsomes were treated with neuraminidase, less phosphatidylcholine and sphingomyelin could be hydrolysed with phospholipase C under experimental conditions which allowed the hydrolysis of the phospholipids of the external leaflet only. In contrast, the accessibility of phosphatidylethanolamine and phosphatidylserine to the external probes (trinitrobenzene sulfonic acid or phospholipase C) was not affected. After neuraminidase treatment of a microsomal fraction, less phosphatidylcholine, newly synthesized through the cytidine pathway, could be hydrolysed by phospholipase C, whereas the reaction of newly synthesized phosphatidylethanolamine molecules with trinitrobenzene sulfonic acid was not affected. The results suggest that in biological membranes some choline phospholipid molecules may interact with the sialyl residue of sialocompounds. This interaction may contribute to the maintenance of phospholipid asymmetry in brain membranes.

Animals↗
PubMed1986

Uptake of ethanolamine in neuronal and glial cell cultures.

The uptake of radioactive ethanolamine has been studied in exclusively neuronal and glial cell cultures from dissociated cerebral hemispheres of chick embryos. Both cell types show saturable kinetics; neurons have an apparent Km of 6.7 microM, Vmax 41.4 pmol mg prot.-1 min-1 and glial cells a Km of 119.6 microM, Vmax 3,917 pmol mg prot-1 min-1. The lower affinity of the transport and the 100 fold increase in Vmax observed in glial cells correlated with a more important accumulation of free ethanolamine found in glial cells and with a higher degree of phosphorylation of ethanolamine. The uptake appeared to be temperature and Na+ ions dependent but was not affected by CN- or ouabain. Monomethyl-, dimethylethanolamine and choline were effective in inhibiting the uptake. Little or no effect was observed with serine, methionine, carnitine, alanine or glutamate.

Animals↗
PubMed1986

Effect of CDP-choline on hypocapnic neurons in culture.

Neuronal cultures from chick embryo cerebral hemispheres were protected against a hypocapnic injury by adding to their growth medium 10(-6)M CDP-choline before or after the injury. The protection obtained with CDP-choline was analyzed by a morphometric analysis and showed that pretreatment of neuronal cultures with CDP-choline maintained the number of cell aggregates and of primary neuronal processes at control values after hypocapnic shock. Various experiments showed that the intact molecule was responsible for the protective action, since pretreatment with different concentrations of various nucleosides and nucleotides (up to 10(-5) M), choline, and phosphorylcholine was without protective effect. The addition of CDP-choline after the hypocapnic injury resulted in a protection of the cultures as shown by morphological observation. Incubation of neurons with radioactive choline showed that hypocapnia increased the incorporation of the label into phospholipids whereas the presence of CDP-choline reduced it. The de novo synthesis of choline was affected by neither hypocapnia nor CDP-choline treatment. The results indicate that CDP-choline may have the capacity to protect neurons under conditions of basic pH and that cellular proliferation may be stimulated by the compound.

Animals↗
PubMed1986

Surface glycosyltransferase activities during development of neuronal cell cultures.

Neurons in culture obtained from dissociated cerebral hemispheres of 8-day-old chick embryos showed measurable activities of galactosyl-, fucosyl-, and sialyl-transferases at the external surface of their plasma membrane. Important changes in these activities were observed during cell proliferation and maturation, in particular the surface fucosyltransferase activity, and/or the amount of intracellular fucosylated acceptors increased during synaptogenesis, between 3 and 5 days in culture (d.i.c.). A sodium dodecyl sulfate radioelectrophoretic analysis of the fucosylated neuronal acceptors labelled with [14C]fucose showed, during synaptogenesis, the high labelling of two protein bands of 116 and 50 X 10(3) daltons. The fucosylation of glycoconjugates occurred preferentially, in neurons, upon glycoproteins whereas in glial cell cultures glycolipids were more fucosylated. The reasons for such a difference are not yet understood but the results suggest that the surface fucosyltransferase activity and fucosylated proteins in particular may play a role during the synaptogenesis of neurons in culture.

Animals↗
PubMed1986

Evidence for sialidase activity in K 562 cells: inhibition by adriamycin treatment.

Sialidase activity has been studied in the human erythroleukemia K 562 cell line grown in vitro. The total sialidase activity was determined using disialoganglioside GD1a and fetuin as exogenous substrates. The enzymatic activity was stimulated by 0.08% Triton X-100 and reached the highest level at pH 4.0. Results obtained showed that gangliosides are hydrolysed more extensively than glycoproteins by K 562 sialidases. This finding could suggest that endogenous gangliosides may be the main source of metabolically available sialic acid in K 562 cell line. After treatment of K 562 cells by Adriamycin (40 nM), a potent anticancer drug, sialidase activity decreased by 40% as compared to control cells. This decrease occurs early during the first day of incubation with Adriamycin. This inhibition of sialidase activity could explain previous results obtained in our laboratory which show an enhanced sialylation of the membrane glycoconjugates after Adriamycin treatment.

Cell Line↗
PubMed1985

Topological biosynthesis of phosphatidylcholine in brain microsomes.

The sidedness of the biosynthesis of phosphatidylcholine and its transbilayer movement in brain microsomes were investigated. Microsomes were labelled in vitro or in vivo either through Kennedy's pathway or by the base-exchange reaction. The vesicles were treated with phospholipase C under conditions where only the phospholipids present in the external leaflet were hydrolyzed. The incubation of microsomes with CDP-[14C]choline or [14C]choline showed that most of the newly synthesized phosphatidylcholine molecules were localized in the external leaflet. With time a few molecules were transferred into the inner leaflet. When phosphatidylcholine was labelled in vivo by intraventricular injection of [3H]choline the specific activities of the phosphatidylcholine in the outer leaflet were higher than those in the inner leaflet after short times of labelling but became similar after long times of labelling. The results suggest that in brain microsomes the synthesis of phosphatidylcholine through Kennedy's pathway or by the base-exchange reaction takes place on the external leaflet which corresponds to the cytoplasmic one in situ. The transfer of these molecules from the outer leaflet to the inner one is a slow process and the mechanisms that control the transbilayer movement of the phosphatidylcholine seem to be independent of those that control their biosynthesis.

Animals↗
PubMed1984

Metabolism and function of gangliosides in developing neurons.

Previous experiments have shown that the addition of a mixture of gangliosides to the growth medium induced morphological changes in primary neuronal cultures, producing especially a trophic effect and a sprouting of neurites (neuritogenesis). The study reported here examined the changes of some biochemical parameters that paralleled the morphological modifications of cultured neurons from chick brain hemispheres treated with gangliosides. Neurons cultured from 3 to 7 days in the presence of various concentrations of a purified mixture or of single-species of gangliosides (GM1, GD1a, GT1b) revealed that these glycolipids were easily incorporated into the cells as a function of their exogenous concentrations. Incubation of neurons with N-acetyl-D-[U-14C]mannosamine showed a final labeling of all endogenous cellular and exogenous incorporated gangliosides; however, the radioactivity recovered decreased as a function of the number of sialic acid units of the exogenously added gangliosides. The treatment of neuronal cells from 3 to 7 days in culture with a mixture of 10(-8) M and 10(-5) M gangliosides led to the following observations on some neurochemical parameters: no effect on the influx of choline and dopamine; no effect on the spontaneous choline efflux, whereas the K+-provoked one is abolished; decrease of the spontaneous and K+-stimulated release of dopamine; no effect on the spontaneous release of GABA for 10(-8) M gangliosides but an increase of both spontaneous and K+-provoked release for 10(-5) M gangliosides. The data suggest that the possible insertion of gangliosides into the neuronal membranes may imply structural modifications that may influence enzymatic activities, neurotransmitter transport, and finally, some nerve cell mechanisms.

Animals↗
PubMed1983

A study on the topological distribution of phospholipids in microsomal membranes of chick brain using phospholipase C and trinitrobenzenesulfonic acid.

The transbilayer distribution of phospholipids in chicken brain microsomal membranes has been investigated using trinitrobenzenesulfonic acid and phospholipase C from Clostridium welchii. The exposure of intact microsomes to trinitrobenzenesulfonic acid showed that the labelling of aminophospholipids followed biphasic kinetics, indicating that these membranes contain a fast- and a slow-reacting pool of aminophospholipids. Use of microsomes radioiodinated on their surface led to the conclusion that the fast-reacting pool may be located on the outer leaflet of the microsomal vesicles. It contains about 35% of the phosphatidylethanolamine, 29% of the ethanolamine plasmalogens and 18% of the phosphatidylserine. The treatment of intact microsomes with the phospholipase C Cl. welchii produced the hydrolysis of 50% of the phospholipids without any loss of their permeability properties, indicating that they are not permeable to the hydrolase. Phospholipids extracted from the microsomes were hydrolyzed rapidly by the phospholipase C with the exception of phosphatidylserine and phosphatidylinositol. In intact microsomes about 90% of phosphatidylcholine, 32% of ethanolamine phospholipids and 60% of sphingomyelin were accessible to the phospholipase. These results suggest that the phospholipids have an asymmetric distribution in chicken brain microsomes, the external leaflet containing about 75% of the choline phospholipids and 25% of the aminophospholipids, whereas an opposite distribution is observed in the inner leaflet.

Animals↗
PubMed1983

Ectoglycosyltransferase activities at the surface of cultured neurons.

Glycosyltransferase activities (ectogalactosyl, ectofucosyl and ectosialyl) were studied at the external surface of exclusively neuronal cultures. An appropriate methodology gave the possibility to eliminate sources of errors due to the hydrolysis of nucleotide sugar substrates or due to cellular uptake of free sugars. Ovomucoid and asialofetuin coupled to Sepharose and Ultrogel beads were used as exogenous substrate to circumvent possible substrates pinocytosis. Ectoglycosyltransferase activities were studied as function of protein concentration, incubation time and amount of bead coupled exogenous acceptors. The data show that these enzymes are present at the external surface of the neuronal membrane; their possible role in cell - cell interactions is suggested.

Animals↗
PubMed1983

Neuraminidase in calf retinal outer segment membranes.

An enzyme catalyzing the hydrolysis of sialic acid (N-acetylneuraminic acid: NeuNAc)-containing glycoconjugates has been found in bovine retinal rod outer segment (ROS) membranes. The enzymatic activity is optimal at pH 4.0 and is stimulated by 0.15% Triton X-100. Total activity was determined by the release of NeuNac from endogenous and exogenous substrates (GD1a). The ROS enzyme preferentially hydrolyses the ROS gangliosides, possibly because they are more accessible than the glycoproteins as substrates for the neuraminidase. Release of NeuNAc from gangliosides leads to important changes in the ganglioside patterns; whereas the amounts of GM1 increased throughout the incubation, the levels of polysialogangliosides GT1b and GD3 diminished owing to their rapid hydrolysis. The finding that gangliosides are hydrolysed more extensively than glycoproteins suggests that endogenous ROS gangliosides may be the principal source of metabolically available sialic acid in ROS. It was also observed that the activity of ROS neuraminidase is not affected by illumination of the membranes.

Animals↗
PubMed1983

[Existence of UDP galactose: galactosyltransferase activity at the external surface of cultured neurons].

The existence of UDP-galactose:galactosyltransferase activities at the external surface of cultured neurons from chick embryo hemispheres, was demonstrated. [14C]-galactose was transferred upon cellular substrates present on the neuronal surface and upon exogenous substrates (ovomucoïd) bound to Sepharose beads. The activity of ectogalactosyltransferase was not due to a leakage of intracellular enzymes into the incubation medium nor to an intracellular enzymatic activity utilizing [14C]-galactose obtained from the hydrolysis of UDP-[14C]-galactose. Kinetics of these ectogalactosyltransferases were analysed. The results support the hypothesis of a communication and/or interaction among the nerve cell surfaces via glycoconjugates or their biosynthetic enzymes.

Animals↗
PubMed1982

Efflux of choline from neurons and glia in culture.

The efflux of radioactive choline from exclusively neuronal or glial cell cultures was dependent upon the concentrations of choline present in the cells and in the incubation medium, suggesting the possible presence of a homoexchange phenomenon between influx and efflux. The ionic dependence of the outward movement of choline from these cells showed that is could be stimulated by high K+ concentrations and by the absence of Ca2+. In glial cells, however, the efflux of choline was increased with a much lower concentration of K+ compared to neurons. The result may suggest that during nerve stimulation the release of K+ from neurons could stimulate, from glia, the efflux of choline which would then be taken up in neurons.

Animals↗
PubMed1982

Gangliosides in various brain areas of three inbred strains of mice.

The ganglioside patterns of cerebellum, cortex, pons-medulla, hypothalamus, hippocampus and caudate nucleus of three inbred strains of mice (C57BL/6J, DBA/2J and BALB/cJ) have been analysed. All brain areas contained both the simple and complex species of gangliosides. GD1a was the major ganglioside in cortex, hippocampus and caudate nucleus whereas GT1b was the major species in cerebellum, hypothalamus and pons-medulla. In hippocampus, the percentages of GT1b and GD1a were quite similar. Pons and medulla exhibited the highest levels of GM1 (which approaches the value of GT1b) and the lowest values of GD1a. A ganglioside, which was present in highest amounts in cerebellum disappeared after alkali treatment. Highly significant differences were observed in the amounts and patterns of gangliosides among brain areas of the three strains. Highly significant differences (p less than 0.001) were also found in the ganglioside distribution of various brain areas among the strains, especially for tri- and tetrasialogangliosides between Balb and DBA. A significant difference of GM1 was observed in the cerebellum when comparing DBA with the two other strains. It is likely that the differences might be related to their relative abundances in certain cell types and for defining synaptic circuits in brain areas of some strains.

Animals↗
PubMed1982

Possible role of sialocompounds in the uptake of choline into synaptosomes and nerve cell cultures.

Incubation of primary nerve cell cultures and of crude synaptosomal preparations with neuraminidase released sialic acid from both gangliosides and sialoglycoproteins. After this treatment, the pattern of ganglioside distribution was severely modified with a decrease of polysialogangliosides (GD1b, GT1b, Gt1L, GQ1) and a dramatic increase in monosialoganglioside GM1. The choline influx into neuraminidase treated cells and organelles was reduced by 30--50% but the efflux was unmodified. In particular the high affinity mechanism of choline uptake disappeared and the low affinity mechanism was modified in both cases. The disappearance of the high affinity uptake mechanism was not followed by a decreased acetylcholine synthesis as it should be if the current theories on choline uptake and acetylcholine synthesis are correct. Our present data thus confirm our previous hypothesis that choline metabolism regulates choline uptake rather than the other way round as is suggested by the theories most widely accepted at present. Choline uptake was unaffected by pretreatment of cells and organelles with tetanus toxin suggesting that the effect of neuraminidase on the choline uptake were either mediated through glycoproteins or through gangliosides other than those which bind to tetanus toxin (GD1b and GT1b). Several speculative models for explaining the effect of neuraminidase on choline uptake are proposed.

Animals↗