Glycoprotein sialyltransferases in eucaryotic cells.
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Animals↗
Biomedical subjects
P Louisot
Publications and source records attributed to P Louisot.
Peritoneal macrophage response: an in vivo model for the study of synthetic materials.
This study was designed to validate an in vivo model in rat to study cell-implant interactions. Pieces of Dacron and Goretex and polydimethylsiloxane (reference material) precoated or not were implanted in the peritoneal cavity. In some cases the peritoneal cells were stimulated before the implantation. Macrophage phagocytosis and fibrinocoagulolytic activities were investigated in parallel with morphological studies 6 h after implantation. A graded cell response related to the different situations was observed, providing a measure of the material's behaviour. Using this model, other investigations of macrophage/polymer can be conducted, especially to explain implant encapsulation.
5'-Nucleotidase↗
Dietary regulation of intestinal glycosyl-transferase activities: relation between developmental changes and weaning in rats.
Activities of rat intestinal fucosyl-transferase (GDP-fucose: glycoprotein fucosyl-transferase; EC 2.4.1.68) and sialyl-transferase (N-acetylneuraminyl-transferase; EC 2.4.99.1) respectively exhibited a significant increase following weaning and a steady decrease between birth and weaning. The variations of the two glycosyl-transferase activities with age could not be explained by the presence of inhibitory factors in microsomes or cytosol, nor were they due to a natural modification of the milk composition at the end of lactation. The increase in fucosyl-transferase activity that followed weaning was prematurely induced by early weaning. Prolonged nursing prevented the normal increase in fucosyl-transferase activity, and late weaning delayed the increase. The sensitivity to the modification of the time of weaning indicated a major effect of dietary changes, related to the introduction of a solid carbohydrate-rich diet, on the developmental pattern of this enzyme. However, a stress response could not be excluded after early weaning since the corticosterone level of the early weaned rats was enhanced as compared to that of suckling rats. For sialyl-transferase, early weaning caused a slightly greater than normal decrease in the activity, whereas prolonged nursing only weakly diminished the normal decrease in the activity. Moreover, late weaning had no effect on the sialyl-transferase activity. The age-related variations of this enzyme likely are due to factors independent of diet. Thus, the developmental variations of the fucosyl- and sialyl-transferases appear to be differently regulated.
Aging↗
Sialylation processes in mitochondria: evidence for two distinct sialyltransferases located in the outer membrane.
Previous studies have shown that purified mitochondrial outer membrane is able to catalyze the transfer of sialic acid from CMP-Neu5Ac to an exogenous asialoglycoprotein acceptor, asialofetuin. Considering the heterogeneity of the glycan chains borne by this glycoprotein, an investigation of mitochondrial sialyltransferase activities was undertaken. Our data provide evidence for the existence of two distinct sialyltransferases in purified mitochondrial outer membranes. The use of different acceptor substrates, the temperature dependence of these enzymes, and their different sensitivity towards a sulfhydryl reagent, p-CMB, allowed us to discriminate between a galactoside alpha(2-3) sialyltransferase and a galactoside alpha(2-6) sialyltransferase presumably involved in the sialylation of O- and N-glycan chains of glycoprotein, respectively. These results are discussed in terms of mitochondrial autonomy for post-translational events.
Animals↗
Type C Niemann-Pick disease: biochemical aspects and phenotypic heterogeneity.
Within Niemann-Pick diseases, type C has now been demonstrated to be a nosological entity totally distinct from types A and B, and is best characterized at present by unique abnormalities of intracellular translocation of exogenous cholesterol, which are briefly reviewed. Although the primary defect is still unknown in type C Niemann-Pick disease, this discovery has had immediate medical applications, by providing the first strategy for reliable prenatal detection of the disorder and easy diagnosis of patients. From our personal experience of 134 cases, diagnosis is best reached by the combined demonstration of a deficient induction of esterification and of an intravesicular cholesterol storage by cytochemistry after filipin staining. The prevalence of the various clinical forms observed is given, together with a brief report of 6 adult-onset cases. The spectrum of phenotypic heterogeneity in relation to abnormal LDL processing has been defined, resulting in the delineation of three biochemical groups, classical (86%), variant (7%) and intermediate (7%). Correlations between clinical and biochemical phenotypes have been studied. To get further insight into genetic heterogeneity, complementation studies were performed. Preliminary results have yet given no evidence of several complementation groups within type C Niemann-Pick disease. The recognition of the three biochemical phenotypes is however critical for diagnosis and genetic counselling.
Adolescent↗
[Characterization and postnatal evolution of an endogenous protein inhibitor of intestinal fucosyltransferase activities].
An endogenous protein inhibitor was shown to act in vitro on small intestinal fucosyl-transferase activities. In order to support the hypothesis of a physiological role in vivo for this inhibitor in the regulation of the fucosylation process, we characterized this inhibitor and studied its postnatal development. This inhibitor acts differently upon the enzymes of intestinal fucosylation processes. Among the different organs analyzed, the inhibitor was found only in the intestine and the pancreas. Intestinal inhibitory activity decreased 10 fold between the 7th and the 24th day of rat postnatal development; this decrease was inversely correlated with intestinal fucosyl-transferase activities. Two mechanisms of action by which the inhibitor could exert a physiological regulatory role are discussed.
Animals↗
Further characterization of mitochondrial contact sites: effect of short-chain alcohols on membrane fluidity and activity.
Two mitochondrial contact site-enriched fractions were further characterized by freeze-fracture electron microscopy. Examination of the replicas revealed that these two fractions are in the form of vesicles with membrane sheets attached to the vesicles. The physical state of these fractions has been investigated by means of steady-state fluorescence polarization to assess the effects of alcohols on their fluidity properties and activity. Comparison between intact membranes and their extracted lipids shows that butanol and hexanol induce a differential increase of the overall membrane fluidity in the two contact site-enriched fractions. This alteration in the membrane dynamics leads to a complex modulation of cytochrome c oxidase activity in both fractions. These results bring further evidence for the existence of morphologically and functionally distinct domains in mitochondrial membranes.
Butanols↗
Mitochondrial contact sites. Lipid composition and dynamics.
Two membrane fractions of intermediate density between inner and outer mitochondrial membranes were isolated by density gradient centrifugation from osmotically lysed mitochondria and mitoplasts of liver. These fractions were characterized by the presence of both monamine oxidase and cytochrome c oxidase activities and bound hexokinase. 1) The content of the fractions in proteins and lipids was assessed by biochemical determination. Thin-layer and gas-liquid chromatography showed that the two contact site-enriched fractions contain predominantly phosphatidylcholine (31%), phosphatidylethanolamine (27%, half-unsaturated), and cardiolipin (27%, fully unsaturated). 2) The dynamics of the fractions were assessed by fluorescence polarization techniques using 1,6-diphenyl-1,3,5-hexatriene as a probe and by fluorescence decay measurements. We have verified that differences in static anisotropy cannot be exclusively attributed to differences in fluorescence lifetimes. On the contrary, the results indicated an increased lipid mobility in "inner membrane contact sites," which is probably related to a lower cholesterol to phospholipid ratio, as well as a lower saturation of the fatty acyl chains when compared with "outer membrane contact sites." Taken all together, the spectroscopic measurements confirm the biochemical results, leading to the idea that the two populations of contact sites have different physicochemical properties, which are probably mainly determined by the membrane from which they are derived. They constitute microdomains enriched either in inner or outer mitochondrial membranes.
Animals↗
Dolichol kinase activity: a key factor in the control of N-glycosylation in inner mitochondrial membranes.
Inner mitochondrial membranes from liver contain a dolichol kinase which required CTP as a phosphoryl donor. Kinase activity was linear with protein concentration and unlike other reported kinases, activated almost equally well by Mg2+, Mn2+ or Ca2+. Thin-layer chromatography showed that the reaction product co-migrated with authentic dolichyl monophosphate. The phosphorylation of dolichol did not occur in presence of ATP, GTP or UTP but required exogenous dolichol for maximal activity. Newly synthesized [3H]dolichyl monophosphate has been shown to be glycosylated in the presence of GDP[14C]mannose or UDP[14C]glucose. The double labeled lipids formed by the sugar nucleotide-dependent reactions were identified respectively as [14C]mannosylphosphoryl[3H]dolichol and [14C]glucosylphosphoryl [3H]dolichol. These results are discussed in terms of regulation of N-glycosylation processes in inner mitochondrial membranes from liver.
Animals↗
Abnormal cholesterol metabolism in imipramine-treated fibroblast cultures. Similarities with Niemann-Pick type C disease.
Addition of low-density lipoprotein (LDL) to cholesterol-deprived human skin fibroblast cultures treated by imipramine at a 20 microM concentration induced a significant intracellular accumulation of unesterified cholesterol. Intracytoplasmic inclusions were already visible by histochemical filipin staining after 2 h of LDL uptake and were progressively mobilized towards the perinuclear region within 24 h. At this concentration of the drug, the rate of proteolytic 125I-LDL hydrolysis was similar in treated and untreated cells. Treated cells maintained in lipoprotein-deficient medium showed no abnormality, indicating the exogenous origin of the accumulated sterol. Further, the drug induced a drastic dose-dependent impairment of LDL-stimulated cholesterol esterification, not related to an inhibition of acyl CoA:cholesterol acyltransferase, and a significant delay in down-regulation of de novo cholesterol synthesis. However, imipramine did not affect 25-hydroxycholesterol-mediated regulation of the two latter processes. These results resemble those observed in Niemann-Pick type C disease and suggest an impaired mobilization of LDL-derived cholesterol in imipramine-treated cells.
Acetates↗
Triggering of mannosyltransferase activity in inner mitochondrial membranes by dolichyl-monophosphate incorporation mediated through phospholipids or fatty acids.
The activity of GDPmannose:dolichyl monophosphate mannosyltransferase in inner mitochondrial membranes can be triggered by dolichyl-monophosphate incorporation mediated through phospholipids or fatty acids. The efficiency of this incorporation and the efficiency of the enzyme activity are not equivalent. Among a variety of amphiphiles which were tested, the highest mannosyltransferase activity was obtained with the mixture of lipids extracted from the outer mitochondrial membranes. The results presented here appear consistent only with a mechanism involving collisional contacts of the phospholipid vesicles and fusion with the membranes. ESR spectroscopy confirms that (a) the incorporation process is followed by solubilization of dolichyl monophosphate molecules in the lipid phase and (b) the general organization of the inner mitochondrial membranes is not perturbed by the addition of dolichyl monophosphate.
Animals↗
Evidence for the presence of an endogenous cytosolic protein inhibitor of intestinal fucosyltransferase activities.
Soluble endogenous inhibitory activities for glycoprotein: alpha (1-2) and alpha (1-3) fucosyltransferases are demonstrated in rat small intestinal cytosol. These inhibitors are retained on DEAE-cellulose and are eluted as two fractions A and B. Fraction B is non dialyzable, heat stable and pronase-resistant and consists probably of poly-nucleotides. Fraction A is also non-dialyzable, but is thermolabile and pronase-sensitive, suggesting that it contains proteins. The inhibition of fucosyltransferase activity by fraction A is competitive for GDP-fucose and non-competitive for the glycoprotein substrate. Inhibition is not due to interfering enzymatic activities (glycosyl-nucleotide pyrophosphatases, glycosidases or proteases) and is reversible. This protein inhibitor, with a molecular weight of 60,000, is found only in the intestine and the pancreas and appears to be different from the previously reported inhibitors of brain glycolipid glycosyltransferases.
Animals↗
Study of the effect of a glycosaminoglycan-peptide complex on the degradative enzyme activities in human osteoarthritic cartilage.
The in vitro collagenolytic and proteoglycanasic activity from human fibrillated osteoarthritic cartilage was determined using labelled proteoglycans and type II collagen as substrates. In vitro, a glycosaminoglycan-peptide complex (GP-C Rumalon) induced a dose-dependent inhibition of both collagenolytic and proteoglycanasic activities while sodium salicylate and indomethacin had only a weak suppressive effect on proteoglycanase. Phospholipase A2 activity was unmodified by GP-C suggesting that the effect of the drug on degradative enzymes was unrelated to prostaglandin formation.
Aged↗
Assay of serum cardiac myosin heavy chain fragments in patients with acute myocardial infarction: determination of infarct size and long-term follow-up.
To evaluate the correlation between myosin heavy chain release and the necrosis mass, serum levels of myosin heavy chain fragments were determined serially in 55 patients with acute myocardial infarction. Eight of these patients were successfully treated with thrombolytic agents: the others were not treated. The same myosin titration was applied to the sera of 25 dogs with an experimental myocardial infarction. Six of the dogs were successfully treated with thrombolytic agents. The time courses of the myosin concentrations are typical and monophasic for all patients with a noncomplex myocardial infarction. The values for the kinetic parameters of myosin release are comparable to those previously reported. We have now determined that cumulative myosin release significantly correlates with cumulative creatine kinase (CK), CK-MB, and lactate dehydrogenase release, as well as with thallium-201 distribution, as determined for different patient groups. Thrombolytic treatment does not seem to qualitatively upset myosin kinetics. The results obtained in dogs with or without thrombolysis conclusively indicate that myosin release is a quantitative index of the necrosis mass. From a practical point of view, a few serial determinations of serum levels of myosin heavy chains are enough to estimate the necrosed mass in patients with acute myocardial infarction. More generally, serum myosin titration could be useful in detecting any cardiac disturbance involving myocardial injury resulting in membrane leakage of cardiac cells.
Adult↗
Effect of retinoic acid on two glycosyltransferase activities in C6 cultured glioma cells.
1. Activity of two glycosyltransferases was studied in retinoic acid-treated C6 cultured glioma cells. 2. The beta-galactoside alpha 2,3-sialyltransferase transferring N-acetylneuramin onto the O-glycans residues of glycoproteins was activated up to twice after chronic treatment (from 24 to 96 hr) with all-trans retinoic acid. 3. No effect was observed for shorter treatments. 4. On the opposite, the N-glycan galactosyltransferase activity remained unchanged whatever the length of retinoic acid treatment was. 5. The activatory effect was not dependent on isomery, as all-trans and 13-cis retinoic acid isomers were both activators of the C6 glioma cell sialyltransferase. 6. Measurement of adhesion of retinoic acid-treated cells using labelled plasma membranes showed an enhancement of adhesion in correlation with enhancement of sialyltransferase activity.
Animals↗
Comparative study of the N-glycoprotein synthesis through dolichol intermediates in mitochondria, Golgi apparatus-rich fraction and endoplasmic reticulum-rich fraction.
1. Glycosylation of endogenous dolichol acceptors was higher in mitochondria than in C 30,000 g (Golgi apparatus-rich fraction) and C 100,000 g (endoplasmic reticulum-rich fraction). 2. In mitochondria, N-glycoprotein biosynthesized were composed of high mannose type and non-fucosylated biantennary complex type while in C 30,000 g and C 100,000 g preparations, they contained biantennary complex type as tri and tetraantennary complex type oligosaccharides in both fucosylated and non-fucosylated forms.
Animals↗
Cartilage degradative enzymes in human osteoarthritis: effect of a nonsteroidal antiinflammatory drug administered orally.
The activity of stromelysin and collagenase was determined in fibrillated human OA cartilage using labeled proteoglycans and type II collagen as substrates. In vitro paracetamol had no effect on metalloprotease whereas TA induced a significant inhibition of stromelysin. In cartilage and synovium from nine patients treated with TA and nine patients treated with paracetamol during 8 weeks before surgery for hip OA, stromelysin activity was significantly lower in the TA than in the paracetamol group. The results suggest that TA has a potential chondroprotective effect in OA.
Acetaminophen↗
Effects of chronic ethanol administration on plasma-membrane-bound glycosyltransferase activities.
Physiological and membranous modifications induced by a 4-week ethanol administration in mouse liver plasma membrane were studied. Galactosyl- and glucosyltransferase activities were stimulated in the presence of dolichylphosphate alone or with phosphatidyl-choline. The galactosyltransferase activity was inhibited by chronic ethanol administration. These enzymes were modulated by different phospholipids. The phosphatidic acid was the most efficient activator. Ethanol provoked an inhibition of the galactosyltransferase activity whatever the phospholipid used, as well as an inhibition of the glucosyltransferase activity, chiefly in presence of phosphatidyl-inositol. The preincubation of control or treated mouse liver plasma membranes with liposomes loaded by dolichylphosphate and cholesterol greatly enhanced the enzymatic activities without removing the inhibition by ethanol treatment.
Alcoholism↗