Michel Richard

The effects of high molecular weight- and low molecular weight-heparins on superoxide ion production and degranulation by human polymorphonuclear leukocytes

High molecular weight (HMW) and low molecular weight (LMW) heparins affected superoxide ion production and degranulation by polymorphonuclear leukocytes (PMNL) isolated from either chronic hemodialyzed patients or healthy controls. Low concentrations in HMW-heparin, below 1.76 aXa IU/ml for patients and 1.34 aXa IU/ml for controls, increased O2- production started by phorbol myristate acetate. High concentrations above these values decreased it. Increasing LMW-heparin concentrations constantly decreased O2- production using the same stimulus. Myeloperoxidase (MPO) released by PMNL was found to be significantly HMW- and LMW-heparins dose-dependent. The addition of calcium chloride significantly increased MPO release. Lactoferrin release was not dose-dependent of HMW- or LMW-heparins. However, an increase of the percentage of positive responses for lactoferrin release was observed in the simultaneous presence of HMW-heparin and CaCl2 compared to HMW-heparin alone. Lysozyme release was also not dose-dependent of HMW- or LMW-heparins. An increase of the percentage of positive responses for lysozyme release was observed in the presence of CaCl2 alone compared to HMW-heparin.

Evidence for glycosyl-transferases in rat liver nuclei

Abstract Five glycosyl-transferases have been found present in purified hepatocyte nuclei of the rat (mannosyl-, galactosyl-, N-acetyl-glucosaminyl-, N-acetyl-galactosaminyl- and sialyl-transferases); these are capable of fixing specific carbohydrates on to endogenous or exogenous protein acceptors.

Biosynthèse des glycoprotéines. XXI. Étude de la mannosyl-transférase microsomique des splénocytes

Summary An enzymatic system catalyzing mannose transfer to endogenous proteins from the coenzymatically active form of the ose, the GDP-mannose, is present in splenic cellular microsomes. This mannosyltransferase shows an optimum pH of 5.8, an optimum temperature of 30°C, and is highly sensitive to any temperature or ionic strength increase. Its Km for GDP-mannose is 0.1 μ M. The enzyme is activated by Mn++ and Mg++, by mono- and triphosphate nucleosides and inhibited by diphosphate nucleoside and X-100 Triton. Splenic microsomic mannosyl-transferase proves very sensitive to any alternation of subcellular architecture at the microsomic level.

Biosynthesis of glycoproteins in the aortic wall study of intima microsomic mannosyl-transferase activity

Abstract Glycoprotein biosynthesis in the aortic wall has been approached by studying the mannosyl-transferase activity in the intimal microsomes. Studied on “in vitro” acellular systems, the optimal pH is 5.8 and the optimal temperature is 27°C. This enzyme shows thermal inactivation which is compensated by an increased reaction rate, under the influence of its exposure to supra-optimal temperature. The affinity constant for the GDP-mannose substrate is 15 μ m . The enzyme is inhibited by nucleoside-diphosphates, GTP and its structural analogue, β-γ-methylene-GTP at concentration of 10−3 to 10−4 m . Mn2+ and Mg2+ ions are activators, and maximal activity is reached at a 5 × 10−3 m concentrations of Mn2+. Cemulsol, deoxycholate and Triton X-100 are inhibitors in strong concentrations. The same is true for cycloheximide and puromycin. The exposure of the enzyme to supra-optimal temperatures uncovers a varying behaviour as a function of low or high concentrations of substrate. There is an activating effect by the substrate in the presence of a high GDP-mannose concentration, after preincubation at supra-optimal temperature only.

Impaired glycosylation in liver microsomes of orotic-acid-fed rats.

In rats fed orotic acid, the incorporation in liver subcellular fractions of sugars injected intraperitonealy is altered only for mannose, but not for fucose or galactose. Direct determinations of several glycosyltransferases are done in smooth and rough microsomes: fucosyl-, glactosyl-, N-acetylglucosaminyltransferase activities are at quite similar levels in normal and fatty livers. By contrast, sialyltransferase activity is increased (+50%) in smooth microsomes of fatty livers, while mannosyltransferase activity is inhibited by 30%. These alterations are not caused by interfering reactions (pyrophosphatases or proteases). For the mannosyltransferase activity, the inhibition is found in the dolichylphosphorylmannose intermediates. Kinetic studies suggest that there is deficiency of both enzyme and endogenous dolichyl phosphate.

Biosynthese des glycoproteines: XVII. Caracterisation d'une mannosyltransferase d'Asperigillus oryzae

Abstract A microsomal glycoprotein mannosyltransferase utilizing an endogenous acceptor is characterized from Aspergillus oryzae . The enzyme catalyzing the transfer requires Mn 2+ or Mg 2+ (1 or 10 mM) and has a pH optimum of 6 and a temperature optimum of 25°C. Co 2+ does not activate the system while Zn 2+ , Ca 2+ and EDTA act as inhibitors. The enzyme exhibits a K m of 2 μ M for GDP-mannose. The transfer is inhibited by ATP, GTP or its structural analogue, β,γ-methylene GTP. Triton X-100 causes total inhibition at concentrations varying from 1 to 0.1%. The efficiency of the particulate enzyme-acceptor system is discussed.

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.

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.

Biosynthesis of Glycoproteins in the Intestinal Mucosa

To determine the effect of diets on the biosynthesis of glycoproteins in the intestinal mucosa, male Sprague-Dawley rats were fed, ad libitum, high-carbohydrate high-fat and high-protein diets. In in vivo methodology, the animals from each dietary group received an intraperitoneal injection of [14C]-fucose, or [14C]-galactose or [14C]-glucosamine. Incorporated radioactivity was detected in the different subcellular fractions. In in vitro methodology, five glycosyl-transferases were studied on microsomes and cell sap. In vivo, only the high-fat diet induces a decrease in the incorporation of [14C]-glucosamine in the macromolecules. In vitro, only the fucosyl-transferase activity is modified by several diets: as compared with an increase in the activity of the control with the age of the animals, high-protein diet induces a slight activation after 2 weeks of diet, while high-carbohydrate and particularly high-fat diets inhibit the enzyme activity. The inhibition is the same when high-fat diet consists of either oil mixture or triolein or oleic acid or linoleic acid and becomes significant with a supplementation rate of 10%.

Action de divers effecteurs sur l'activité biologique de la mannosyl-transférase microsomique des splénocytes de Rat

The splenic cellular microsomic mannosyltransferase is inhibited by detergent (Triton X 100 and Cemulsol), deoxycholate, dithiotreitol and cyclcheximide, but activated by nucleoside-triphosphates (ATP and GTP) and analogous (β γ-methylene-ATP or GTP); the enzyme is also activated by puromycine. This is good evidence for the sensitivity of the enzyme to any alteration of subcellular architecture at the microsomal level, and for the possibility of good transglycosylation when polypeptide is detached of ribosomes.