M. Richard

Metalloprotease activity, phospholipase A2 activity and cytokine concentration in osteoarthritis synovial fluids

Collagenase, stromelysin and phospholipase A2 (PLA2) activity as well as interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha) and interleukin 6 (IL-6) concentration were determined in the knee joint synovial fluid (SF) of 26 patients with osteoarthritis (OA) and with rheumatoid arthritis (RA). Collagenase and stromelysin were detected in 80.7 and 69.2% of OA SF, respectively. When present, the mean activity of both enzymes was approximately two times lower in OA than in RA SF. PLA2 activity was present in all SF with no significant difference between OA and RA SF. IL-1 beta, TNF alpha and IL-6 were found in 0, 96.1 and 84.6% of OA SF, respectively. Mean TNF alpha and IL-6 concentration was also lower in OA than in RA SF. Metalloprotease activity correlated weakly with IL-6 level and enzymatic activities were unrelated with TNF alpha in OA SF.

Calcitonin inhibits phospholipase A2 and collagenase activity of human osteoarthritic chondrocytes.

Calcitonin (CT) is a known potent inhibitor of bone resorption but its effect on cartilage enzymatic degradation has been incompletely studied. Salmon CT, at a concentration of 0, 0.1, 0.25, 0.5, 2.5 and 50 ng/ml, was added at 24 or 72 h to the culture medium of chondrocytes from human osteoarthritic hips and knees. The spontaneous collagenolytic activity, measured using a radiolabeled type II collagen, was inhibited by CT in a dose-dependent manner. However, CT had no effect on the total collagenolytic activity assayed after APMA activation. Stromelysin and plasmin activity, measured by degradation of casein and a synthetic substrate, were also unaffected by CT. Chondrocyte phospholipase A2 activity, assayed using a labeled specific substrate, was decreased by CT. Chondrocyte pre-incubation with CT significantly decreased the cell binding of labeled TNF alpha, but did not affect IL-1 beta cell binding. Attachment of chondrocytes on fibronectin was markedly stimulated by CT, while attachment to type II collagen was not. Significant effects were obtained using at least 2 or 5 ng/ml of CT. CT appears to decrease collagenolytic activity by decreasing its activation and/or increasing its inhibition by tissue inhibitors of metalloproteinases (TIMP). CT might act on osteoarthritic chondrocyte activation via mechanisms such as phospholipase A2 activity, human necrosis factor-alpha or fibronectin receptor expression.

Serotonin-stimulated phospholipase A2 and collagenase activation in chondrocytes from human osteoarthritic articular cartilage

We have previously described several receptors on the chondrocyte membrane. In an attempt to further characterize the coupling mechanisms of serotoninergic receptors, here we examined the involvement of serotonin in the phospholipase A2 activity. Serotonin dose‐dependently stimulated phospholipase A2. This activation enhanced collagenase type II activity and had no effect on proteoglycanase activity.

Occurrence of monosialosyl pentahexaosylceramide GalNAc-GM1 as specific tumor-associated ganglioside of human head and neck squamous cell carcinomas

In a recent study of the ganglioside profiles of human head and neck squamous cell carcinomas versus normal tissue, one unidentified GX ganglioside was found exclusively in tumor extracts, migrating between GM1 and GD3 by thin-layer chromatography. To determine the chemical structure of this ganglioside which accounted for 3-8% of the total gangliosides, the lipid samples were pooled and separated by high-pressure liquid chromatography to obtain individual ganglioside species purified to homogeneity. The tumor-associated GX ganglioside was analyzed by gas-liquid chromatography, mass spectrometry and immunostaining on thin-layer plates with mouse monoclonal antibodies after enzymatic cleavage. The data allowed the identification of GX ganglioside as GalNAc-GM1 that has been reported as a very minor brain ganglioside in humans. Thus, GalNAc-GM1 is a specific tumor-associated ganglioside in human head and neck squamous cell carcinomas that could be potentially valuable for clinicians.

Calmodulin-dependent collagenase and proteoglycanase activities in chondrocytes from human osteoarthritic cartilage.

Chondrocyte metalloproteinases appear to play a major role in the development of osteoarthritis. The intracellular post-traductional mechanisms regulating collagenase and proteoglycanase are not known. Calmodulin antagonists including phenothiazine and sulfonamide derivatives significantly increased proteoglycanase activity and decreased collagenase activity. H-7, a specific inhibitor of protein kinase C, had no effect on the two metalloproteinase activities, and calmodulin was ineffective in in vitro assays upon metalloproteinase activities. We postulate that collagenase and proteoglycanase activities are controlled by calmodulin-dependent regulation.

Screening of degradative enzymes from articular cartilage in experimental osteoarthritis

SummarySixteen rabbits were killed 12 weeks after sectioning of the right knee anterior cruciate ligament. The left unoperated knee served as a control. The surface area of fibrillated cartilage from femoral condyles and tibial plateau was evaluated and expressed as a percentage of articular surfaces area. Cartilage from the femoro-patellar surfaces was homogenized for the quantification of several degradative activities, based on the release of digested products. Acid phosphatase, several glycosidases and neutral protease activity from the operated joint cartilage were significantly elevated, while collagenolytic activity was unmodified. The percentage of fibrillated cartilage correlated positively with arylsulfatase, glucosidase and neutral protease but negatively with mannosidase and fucosidase. The results may be consistent with the hypothesis of a sequential degradative process leading to cartilage destruction.

Glycosyltransferase Activities in the Rat Intestinal Mucosa: Comparison between Standard Commercial and Semi-Synthetic Diets

Two groups of rats were fed either a commercial (C group) or a semi-synthetic (S group) diet of very similar quantitative composition, which induce similar growth in the animals. Glycosyltransferase activities are very different in the two groups: fucosyltransferase is lower in S group than in C group, regardless of the expression of the results (specific activity versus protein or DNA). The other activities (galactosyl-, N-acetylglucosaminyl-, N-acetylgalactosaminyltransferases) are modified when expressed versus DNA content; N-acetylneuraminyltransferase specific activity is not altered. Since the weight of mucosa is significantly lower in the S group than in the C group, all the total activities are decreased in the S group. The fucosylation process is further characterized by partial purification of the enzyme and study of the synthesis of GDP-fucose. In each case, interfering reactions (glycosyl-nucleotide pyrophosphatases and proteinases) are controlled. The results give evidence that glycosyltransferases are very sensitive to the qualitative composition of the diet.

Regulation of a soluble intestinal glycoprotein: Fucosyl-transferase by fatty acids in vitro

Abstract 1. 1. A soluble intestinal fucosyl-transferase (EC 2.4.1.68), partially purified by DEAE-cellulose chromatography, is inhibited by fatty acids in vitro . By testing the action of 25 compounds, the structural features needed are defined. 2. 2. The inhibition is due neither to pH alterations, nor to differential solubility of the fatty acids, nor to modifications of glycosyl-nucleotide pyrophosphatases. 3. 3. The inhibition requires at least a double bond and is stronger when the double bond is in a cis configuration. 4. 4. The mechanism of the inhibition is different for the two substrates: competitive for the glycoproteic exogenous acceptor (desialyzed fetuin) and noncompetitive for the other substrate GDP-fucose.

étude du mécanisme de I'effet inhibiteur des lipides sur la fucosyl-transférase intestinale de rat

Abstract(2 figures)High-fat diets decrease microsomic and soluble fucosyl-transferase activities in rat intestinal mucosa. This inhibition is not due to qualitative (pH1) or quantitative (relative activities) changes in three isoenzymes, nor is it caused by alterations in the kinetic behaviour of these enzymes (Km, V). It is also not due to a direct effect of the fatty acids on the enzyme activities. It seems reasonable to suggest that a decreased biosynthesis of the fucosyl-transferase occurs as a result of the high-fat diets.

Purification and separation of two soluble fucosyltransferase activities of small intestinal mucosa

1. Rat small intestinal soluble fucosyltransferase is purified more than 2000-fold using chromatographic procedures with DEAE-cellulose, CM-cellulose, GDP-Sepharose and Concanavalin A-Sepharose. 2. Chromatography on Sephadex G15 of the final enzymatic fraction clearly separates two activities: a first peak incorporates fucose on asialoserotransferrin and a second peak on asialofetuin. 3. The use of small saccharidic acceptors (phenylgalactose, lactose, lacto-N-fucopentaose I) and the analysis of fucosylated asialoglycoproteins indicate that the first activity corresponds to an alpha-(3/4)-fucosyltransferase and the second one to an alpha-(1-2)-fucosyltransferase. 4. Protein analysis by polyacrylamide gel electrophoresis in the presence of SDS for each enzyme shows two bands corresponding to a mol. wt of about 65,000 and 70,000. The two enzymes have the same sensitivity to the action of N-ethylmaleimide.