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Biomedical subjects

P Louisot

Publications and source records attributed to P Louisot.

At least 109 records · Page 6Linked to original sources
PubMed1990

Effect of desipramine on a glycoprotein sialyltransferase activity in C6 cultured glioma cells.

The tricyclic antidepressant desipramine, when added to culture medium, gave rise in C6 rat glioma cells to a decrease of the activity of the enzyme asialofetuin sialyltransferase. The inhibition was dose and time dependent and was observed in both multiplying cells and cells blocked with 2 mM thymidine or depletion of amino acids. This inhibition was rather specific to the sialyltransferase, as under the conditions where this enzyme was inhibited up to 70%, other enzymes such as dolichol phosphate mannose synthetase, glutamine synthetase, and glycerol phosphate dehydrogenase remained unaffected. This inhibition was not reversed after removal of desipramine from the medium and was not observed by direct addition of desipramine to the sialyltransferase incubation assay. Under the same conditions, W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], which is known to be a potent calmodulin antagonist and an inhibitor of calmodulin-dependent kinases, gave the same concentration-dependent inhibition profile of sialyltransferase as desipramine, whereas H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine], which is an inhibitor of protein kinase C and cyclic nucleotide-dependent kinases, had no effect. So, it is suggested that desipramine inhibits the sialyltransferase activity in C6 glioma cells through a calmodulin-dependent system.

Animals↗
PubMed1990

Glycosyltransferase activities in chondrocytes from osteoarthritic and normal human articular cartilage.

Six glycosyltransferases (mannosyl-, glucosyl-, N-acetyl-glucosaminyl-, galactosyl-, sialyl- and fucosyltransferases) are studied and characterized for their optimal conditions and their relations with interfering reactions (glycosyl-nucleotide pyrophosphatases, glycosidases and proteinases) in chondrocytes from osteoarthritic and normal human articular cartilage. Osteoarthritis induces increased activities for five glycosyl-transferases. The observed modifications are not explained by alterations in physico-chemical parameters of the enzymes or by intervention of glycosyl-nucleotide pyrophosphatases, glycosidases or proteolytic enzymes.

Cartilage, Articular↗
PubMed1990

Intestinal glycosyl-transferase activities. Nutritional regulation by a chemically-modified protein: methionyl-casein.

In order to estimate the effect of a chemically modified casein, glycosylation processes were studied in rats fed a diet containing this protein. Two groups of rats were fed either a methionyl-casein diet or a normal casein diet. The methionyl-casein was enriched in methionine by covalent linkage of this amino-acid. The nutritional data (growth, protein intake...) were not modified by the diet. The microsomal N-acetylgalactosaminyl- and fucosyl-transferase activities were unaffected by the diet. On the contrary, the soluble fucosyl-transferase activity was enhanced and the activation of fucose transfer in cytosol from methionyl-casein-diet fed rats disappeared after enzyme purification by DEAE-cellulose chromatography. The activation was not explained by changes in some interfering reactions (glycosyl-nucleotide pyrophosphatase, oxidase, protease activities).

Animals↗
PubMed1990

Histaminergic H1, serotoninergic, beta adrenergic and dopaminergic receptors in human osteoarthritic cartilage.

Using radioligand binding assays, histaminergic H1, serotoninergic, dopaminergic and Beta adrenergic receptors were studied in human normal and osteoarthritic cartilage. The four studied receptors were present in normal cartilage; serotoninergic, dopaminergic and Beta adrenergic receptors were significantly increased in osteoarthritic cartilage while histaminergic H1 receptors were significantly increased only in osteophytic cartilage. The results are consistent with a non specific activation of osteoarthritic chondrocytes.

Cartilage, Articular↗
PubMed1990

Biosynthesis of glycoconjugates in mitochondrial outer membranes. Preliminary characterization of the oligosaccharide moiety of a N-glycoprotein.

Outer mitochondrial membranes synthesize a N-glycoprotein by a direct incorporation of sugars from their nucleotide-donors into an endogenous protein acceptor. To characterize the oligosaccharide moiety of this N-glycoprotein, we sequentially incorporated [14C]-sugars into the protein acceptor. After pronase digest, the released [14C]-glycopeptides were fractionated on a QAE-Sephadex column which gave rise to 9% of neutral glycopeptides and 91% of charged glycopeptides. These latter were identified as bearing phosphate residues in the form of monoester (28%) and acid-stable diester (63%). The oligosaccharide moiety of this mitochondrial glycoprotein has been characterized as incomplete biantennary complex-type chains.

Chromatography, Affinity↗
PubMed1990

Lipid accumulation in prosthetic vascular grafts. Experimental study.

The present study demonstrates that the endoprosthetic tissue, developed at the contact of Dacron and Gore-Tex vascular prostheses replacing the infrarenal aortae of healthy dogs, presents a particular lipidic pattern as compared with the adjacent intimal arterial layer. The modified lipidic pattern is characterized by a significant increase in the total amounts of cholesterol, phospholipids, and triglycerides, despite a normal lipidic plasma profile. Histochemical studies showed that lipid droplets are accumulated in the cytoplasm of deeply situated cells and in the extracellular matrix. These findings support the idea that lipids may be trapped within the pseudo-intima of synthetic vascular grafts, even in the absence of a major plasma lipid disorder, and contribute to the prosthesis failure.

Animals↗
PubMed1989

Characterization of the submitochondrial compartments: study of the site of synthesis of dolichol and dolichol-linked sugars.

The fractionation of mitochondrial membranes on discontinuous sucrose gradient leads to the obtaining of free outer membranes, free inner membranes and two distinct membrane contact site populations characterized as follows. Only outer membrane contact sites and inner membrane contact sites bind hexokinase. Outer membranes and outer membrane contact sites are cholesterol-rich fractions. The endogenous dolichol content is twice fold higher in outer membranes and outer membrane contact sites than in inner membranes and inner membrane contact sites, only the biosynthesis of dolichol in inner membrane contact sites is not stimulated by addition of exogenous [14C]-IPP and FPP. The glycosylation of endogenous dolichol from labeled nucleotide-sugars (UDP-GlcNAc, GDP-Man and UDP-Glc) leads to the synthesis of dolichol-pyrophosphoryl-sugars and dolichol-monophosphoryl-sugars with the rate of synthesis proportional to the dolichol content of each submitochondrial fraction.

Animals↗
PubMed1989

Glycosyltransferase activities in normal and leukaemic monocytic cells.

Glycosyltransferase activities were measured in normal monocytes and in leukaemic monoblasts. Biosynthesis of glycosylated derivatives of dolichyl-monophosphate, which act as intermediates in glycosylation, was measured. Transfer of mannose from GDP-mannose was greatly increased in leukaemic monoblasts. Galactosyltransferase activities, using endogenous protein acceptors, were increased in leukaemic cells of the monocytic lineage compared to normal cells. No significant difference was observed on specific exogenous glycoprotein acceptors. These selective increases of some glycosyltransferase activities in normal and leukaemic monocytic cells can be correlated either with different expression of specific carbohydrate structures or with changes in glycosylation regulation.

Biomarkers, Tumor↗
PubMed1989

Evidence for an O-glycan sialylation system in brain. Characterization of a beta-galactoside alpha 2,3-sialyltransferase from rat brain regulating the expression of an alpha-N-acetylgalactosaminide alpha 2,6-sialyltransferase activity.

We present evidence for the existence in rat brain of several sialyltransferases able to sialylate sequentially asialofetuin. [14C]Sialylated glycans of asialofetuin were analyzed by gel filtration. Three types of [14C]sialylated glycans were synthesized: N-glycans and monosialylated and disialylated O-glycans. The varying effects of N-ethylmaleimide, lysophosphatidylcholine (lysoPtdCho) and trypsin, were helpful in the identification of these different sialyltransferases. One of them, selectively inhibited by N-ethylmaleimide, was identified as the Neu5Ac alpha 2----3Gal beta 1----3GalNAc-R:alpha 2----6 sialyltransferase previously described [Baubichon-Cortay, H., Serres-Guillaumond, M., Louisot, P. and Broquet, P. (1986) Carbohydr. Res. 149, 209-223]. This enzyme was responsible for the synthesis of disialylated O-glycans. LysoPtdCho and trypsin selectively inhibited the enzyme responsible for the synthesis of monosialylated O-glycan. N-ethylmaleimide, lysoPtdCho and trypsin did not inhibit Neu5Ac transfer onto N-glycans, giving evidence for three different molecular species. To identify the enzyme responsible for monosialylated O-glycan synthesis, we used another substrate: Gal beta 1----3GalNAc--protein obtained after galactosylation of desialylated ovine mucin by a GalNAc-R:beta 1----3 galactosyltransferase from porcine submaxillary gland. This acceptor was devoid of N-glycans and of NeuAc in alpha 2----3 linkages on the galactose residue. When using N-ethylmaleimide we obtained the synthesis of only one product, a monosialylated structure. After structural analysis by HPLC on SAX and SiNH2 columns, we identified this product as Neu5Ac alpha 2----3Gal beta 1----3GalNAc. The enzyme leading to synthesis of this monosialylated O-glycan was identified as a Gal beta 1----3GalNAc-R:alpha 2----3 sialyltransferase. When using lysoPtdCho and trypsin, sialylation was completely abolished, although the Neu5Ac alpha 2----3Gal beta 1----3GalNAc-R:alpha 2----6 sialyltransferase was not inhibited. We provided thus evidence for the interpendence between the two enzymes, the alpha 2----3 sialyltransferase regulates the alpha 2----6 sialyltransferase activity since it synthesizes the alpha 2----6 sialyltransferase substrate.

Animals↗
PubMed1989

Topological orientation of mitochondrial GDPmannose: dolichyl-monophosphate mannosyltransferase in the outer membrane.

Previous studies have shown the existence of an autonomous mitochondrial GDPmannose:dolichylmonophosphate mannosyltransferase, located in mitochondrial outer membrane of liver cells. As nothing is known about glycosylation sites in mitochondria, we have investigated the topological orientation of this enzyme in intact mitochondria, using controlled proteolysis with trypsin. Mitochondria were purified sequentially by mild ultrasonic treatment and sucrose density gradient. Purity and homogeneity of mitochondrial fraction were assessed by electron microscopy and specific marker enzymes measures. Our data provide evidence for a mitochondrial GDPmannose:dolichylmonophosphate mannosyltransferase facing the cytoplasmic side of the outer membrane. However, the exposure of this enzyme to the water phase has been shown to be dependent on the ionic strength of the environment.

Animals↗
PubMed1989

Different reactivity of two brain sialyltransferases towards sulfhydryl reagents. Evidence for a thiol group involved in the nucleotide-sugar binding site of the NeuAc alpha 2-3Gal beta 1-3GalNAc alpha(2-6)sialyltransferase.

We have studied the amino-acid residues involved in the catalytic activity of two distinct brain sialyltransferases acting on fetuin and asialofetuin. These two enzymes were strongly inhibited by N-bromosuccinimide, a specific blocking reagent for tryptophan residues. This result suggests the involvement of such residues in the catalytic process of the two sialyltransferases. Furthermore, chemical modifications by various sulfhydryl reagents led to a strong inhibition of the fetuin sialyltransferase while the asialofetuin sialyltransferase was only slightly inhibited. For a more thorough understanding of the thiol inactivation mechanism of the fetuin sialyltransferase, we studied in more detail the reactivity of this enzyme with NEM (N-ethylmaleimide), an irreversible reagent. The time-dependent inactivation followed first-order kinetics and these kinetic data afforded presumptive evidence for the binding of 1 mol NEM per mol of enzyme. Only CMP-NeuAc protected the enzyme against NEM inactivation effectively. MnCl2 did not enhance the protective effect of CMP-NeuAc. The modifications of the fetuin sialyltransferase kinetic parameters by NEM showed a competitive mechanism between NEM and CMP-NeuAc. The results suggest the involvement of a sulfhydryl residue in or near the nucleotide-sugar binding site of the fetuin sialyltransferase (but we could not excluded that CMP-NeuAc binding may induce a change in conformation of the protein, leading to a decreased accessibility of this thiol group located near the nucleotide-sugar binding site). This SH group is essential to the enzyme activity, which is not the case for the asialofetuin sialyltransferase.

Animals↗
PubMed1989

Organization of mitochondrial UDP-glucose:dolichylmonophosphate glucosyltransferase in the outer membrane.

Previous studies have shown the existence of an autonomous mitochondrial UDP-glucose: dolichylmonophosphate glucosyltransferase, located in mitochondrial outer membrane of liver cells. To improve our knowledge about the topographical aspects of glycosylation in mitochondria, we have investigated the organization of this enzyme in intact mitochondria, using controlled proteolysis with trypsin and sensitivity towards amino-acid specific reagents. Our data provides evidence: --for a mitochondrial glucosyltransferase facing the cytoplasmic side of the outer membrane --and for the involvement of histidine and tryptophan residues as well as sulfhydryl groups in the catalytic activity of the enzyme.

Amino Acids↗
PubMed1989

Galactosyltransferase activities in mitochondria outer membrane: biosynthesis of galactosylated proteins.

1. Mitochondria outer membranes prepared from mouse livers were purified on a discontinuous sucrose gradient. Control in electron microscopy and marker enzymes assays confirmed purity and homogeneity of this fraction. 2. Purified mitochondria outer membranes exhibited significant UDP-galactose: glycoprotein galactosyltransferase activities when incubated with endogenous or exogenous glycoprotein acceptors in presence of detergent (Nonidet P40). 3. Some properties of two distinct mitochondrial galactosyltransferases, acting respectively on ovomucoid and ovine asialo-mucin were investigated. 4. Transfer of galactose on ovomucoid was maximal for a pH of 7.6 at 33 degrees C whereas asialo-mucin galactosyltransferase exhibited an optimum pH of 5.6 for an optimal temperature of 46 degrees C. 5. These two distinct membrane-bound enzymes were both inhibited by diacylglycerophospholipids whereas lysophospholipids modulated both enzymes in a different way: at 5 mM lysophosphatidylcholine, asialo-mucin galactosyltransferase was slightly stimulated while ovomucoid galactosyltransferase was markedly activated. 6. The most important activating effect on ovomucoid galactosyltransferase was obtained with a phospholipid containing a long aliphatic side chain linked by an ester bond in sn-1 of glycerol, an hydroxyl group or hydrogen atoms in sn-2 and a phosphorylcholine head group in sn-3.

Animals↗
PubMed1989

Glucosyltransferase activity in mitochondria. Biosynthesis of glucosyl-phosphoryl-dolichol in inner mitochondrial membranes.

1. Inner mitochondrial membranes are able to transfer [14C]glucose from UDP-[14C]glucose onto dolichylmonophosphate. 2. Synthesis of dolichyl-phosphoryl-glucose takes place only in the presence of exogenous dolichyl-monophosphate loaded into phospholipid vesicles. 3. Neutral phospholipids interact preferentially with the membrane-bound enzyme. The effect of phospholipids is not related to the length of fatty acid chains but a correlation between the activation and the degree of unsaturation of fatty acid chains has been found. 4. This enzyme required divalent cations for activity. Such a requirement might be related to lipid-protein interactions which favour a suitable conformation of glycosyltransferase.

Animals↗
PubMed1989

Study of the N-glycoprotein biosynthesis through dolichol intermediates in the mitochondrial membranes.

1. In the mitochondria, the biosynthesis of N-glycoprotein products, through the dolichol intermediates pathway, appears in the outer and in the inner membranes. 2. The biosynthesis of dolichol-pyrophosphoryl-N-acetyl-glucosamine, dolichol-pyrophosphoryl-di-N-acetylchitobiose, dolichol-phosphoryl-glucose and dolichol-phosphoryl-mannose is effective in both membranes. 3. The lipid-linked oligosaccharides biosynthesized in both membranes contain high mannose-type oligosaccharides ranging in size from Man9-GlcNac2 to Man4-GlcNac2. 4. The assembly of the dolichol-pyrophosphoryl-oligosaccharides on the trimannosidic core begins by the elongation of the alpha-1,3 mannose branch in the outer membrane and of the alpha-1,6 mannose branch in the inner membrane.

1-Deoxynojirimycin↗
PubMed1989

Effects of tiaprofenic acid on interleukin 1, phospholipase A2 activity, prostaglandins, neutral protease, and collagenase activity in rheumatoid synovial fluid.

IL-1 and prostaglandin (PGE2, PGF2 alpha, TXB2) concentrations, PLA2 activity, neutral protease activity, and collagenase activity specific for types I and II collagen were determined in the SF of patients suffering from RA, before and after treatment with TA. Active and latent forms of protease and collagenases were regularly detected but were unrelated to IL-1, PLA2, and PGE2. TA induced a significant decrease in tested eicosanoids but IL-1, PLA2, and proteases were unchanged.

Adult↗
PubMed1989

Topological investigations. Study of the trypsin sensitivity of the N-acetylglucosaminyl and mannosyl-transferase activities located in the outer mitochondrial membrane.

The trypsin sensitivity of the mitochondrial N-acetylglucosaminyl and mannosyltransferase activities involved in the N-glycoprotein biosynthesis through dolichol intermediates as well as the N-acetylglucosaminyl-transferase activity involved in direct N-glycosylation were examined in mitochondria and isolated outer mitochondrial membrane preparations. The trypsin action on mitochondrial membrane was checked by measuring the activities of marker enzymes (rotenone-insensitive NADH cytochrome c reductase, adenylate kinase, and monoamine oxidase). Glycosyl-transferase activities of both N-glycosylation pathways were insensitive to trypsin action and consequently were located in the outer mitochondrial membrane. Based on the activator effect of the trypsin on these enzyme activities, the results suggested two distinct orientations of their active sites. As regards the N-glycoprotein biosynthesis pathway through dolichol intermediates, the dolicholphosphoryl-mannose and dolichol-pyrophosphoryl-di-N-acetylchitobiose synthases would be oriented outside while the oligomannosyl-synthase and the oligomannosyl-transferase would be rather oriented inside in the outer membrane. The N-acetylglucosaminyl-transferase involved in the direct transfer of N-acetylglucosamine from its nucleotide donor to a proteinic acceptor would be oriented outside in the outer membrane.

Adenylate Kinase↗