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Biomedical subjects

P Louisot

Publications and source records attributed to P Louisot.

At least 127 records · Page 7Linked to original sources

Purification of liver plasma membranes and localization of a protein galactosyltransferase.

Plasma membranes were isolated from mouse liver. The purification was achieved by a modified method of Ray (1970). The validity of this fractionation procedure is controlled by measurements of specific enzymatic activities and by electron microscopy. The purified plasma membranes were found to contain galactosyltransferase which transferred galactose from UDP-galactose onto endogenous acceptors. This activity requires Mn2+ for its catalytic activity.

Animals↗

Enzymatic characterization of UDP-galactose:FdSdG galactosyltransferase in purified liver plasma membrane.

This study yields evidence for the localization of a galactosyltransferase in purified liver plasma membranes, with the following results. The purified plasma membranes contained galactosyltransferases which transfered galactose from UDP-galactose into desialylated-degalactosylated fetuine. A suitable system of glycosylation reaction was defined. It contained: 10 mM Tris-HCl pH 7.4, 5 mM Mn2+, 4 mM NaF. For the assay with exogenous acceptors, 1.6 mg/ml of desialylated-degalactosylated fetuine and 0.1% of triton X-100 were added. The apparent Km values for UDP-galactose were about 10(-5) M. Enzymatic activities required Mn2+. This cation appeared to be a linear non-competitive activator when used at concentrations below 5 mM. Mg2+ and Ca2+ had an inhibitory effect on Mn2+-dependant-galactosyltransferase.

Animals↗

Distribution of glycosyltransferase activities in different compartments of mitochondria.

The liver mitochondria were submitted to a first swelling which allowed to get outer membranes. The mitoplasts obtained in these conditions were subject to a second swelling. The separation of submitochondrial membranes on a discontinuous sucrose gradient revealed three membrane fractions, an outer membrane rich fraction, an inner membrane rich fraction and a fraction enriched with contact sites between the two membranes. The various glycosyltransferase systems involved in the biosynthesis of N-glycoproteins were located in these fractions.

Animals↗

Phospholipase A2 activity in human osteoarthritic cartilage.

Phospholipase A2 (PLA2) activity was measured in articular cartilage from normal and osteoarthritic (OA) human femoral heads. Protoglycanase and collagenase activity was determined in the same specimens using radiolabelled human proteoglycan subunit and type II collagen, respectively. Grossly normal and fibrillated OA cartilage samples showed a significant increase in PLA2 activity which was not found in osteophytic cartilage. PLA2 activity was found to be correlated with proteoglycanase but unrelated to collagenase activity. Tiaprofenic acid induced in vitro a concomitant increase in PLA2 and a decrease in proteoglycanase activity. PLA2 which may be activated by cytokines as well as mechanical factors is suggested as a key enzyme in chondrocyte metabolism regulation. Tiaprofenic acid is shown as a potential chondroprotective nonsteroidal anti-inflammatory drug.

Aged↗

[The neoartery, myth or reality? Study of 79 explanted arterial prostheses].

This study represents the third stage of a personal study on the long term fate of reconstruction and arterial restoration materials presented to the Academie de Chirurgie. 79 explanted prostheses were studied. First of all by an in depth histological study (8 cases selected from 70 reinterventions carried out before 1980), and then by a more complete protocol: scanning electron microscopy, resistance testing, programmed differential calorimetry, X-ray diffraction and biochemical analysis (71 reinterventions carried out after 1980). Our findings agree with the studies of R. Guidoin. With the exception of structural manufacturing defects or defects related to surgical manipulation, mechanical fatigue in a Dacron (PET) arterial prosthesis is inevitable: on average by the 7th to 10th year, a prosthesis looses one half of its mechanical resistance. The covering tissue, like that of the external capsule, which has poor mechanical properties, cannot compensate for prosthetic deterioration which may be considered to be complete by the 25th year. Knitted arterial prostheses, especially aortic, more rapidly undergo dilatation than woven prostheses which are much more resistant. On the other hand, the poor compliance of the latter compared with the recipient artery, perhaps favorises late anastomotic rupture. At internal capsule or pseudo-intimal level, endothelium is never present, but rather there is a permanent turnover with a variable time course, the mechanisms of which are poorly understood (role of prostaglandins?). Rather than a neo-artery, the vascular surgeon is only capable of producing an imperfect arterial tube, fragile from many points of view, and having an evolution which remains poorly understood.

Aneurysm↗

Galactosyltransferase activities in mitochondrial outer membrane: biosynthesis of dolichylmonophosphate-galactose.

Mitochondrial outer membranes were prepared from mouse liver homogenates by swelling purified mitochondria in phosphate buffer and were purified on a discontinuous sucrose gradient. Assays for marker enzymes and controls in electron microscopy confirmed the purity and homogeneity of this subfraction. Mitochondrial outer membranes had significant galactosyltransferase activity when incubated with UDP-[14C]galactose: 14C-labelling was found in products extractable with organic solvents and in a residual precipitate. Addition of exogenous dolichylmonophosphate loaded into phosphatidylcholine liposomes strongly enhanced the incorporation of [14C]galactose into chloroform/methanol (2:1, v/v) -extractable products. Thin-layer chromatography of these 2:1 extracts showed that the increase of [14C]galactose incorporation was attributable to the synthesis of a new galactosylated lipid, 'lipid L'. This 'lipid L' has been purified on silicic acid columns by elution with chloroform/methanol (1:1, v/v). The purified 'lipid L' was labile in acid and released [14C]galactose. It had the same chromatographic behaviour as dolichylmonophosphate-mannose in neutral, acid and alkaline solvent systems. Upon incubation in presence of [3H]dolichylmonophosphate and UDP-[14C]galactose, purified 'lipid L' contained both 3H- and 14C-labelling. 'Lipid L', synthesized by mitochondrial outer membranes, was therefore characterized as dolichylmonophosphate-galactose.

Animals↗

Characterization of a glucosyltransferase activity in liver plasma membrane: modulation by cations and lipidic effectors.

1. Glucosyltransferase activity incorporating [14C]glucose from UDP-[14C]glucose onto endogenous lipidic acceptors was localized primarily in the plasma membrane of liver. 2. Incubation of plasma membrane by phosphatidyl-choline liposomes loaded with dolichyl-phosphate stimulated the enzymatic activity. 3. This enzyme required Mg2+ for maximal catalitic activity. Ca2+ could substitute Mg2+. 4. Mn2+ acted as a partial non-competitive inhibitor of the Mg2+-activated glucosyltransferase. 5. This enzyme can be modulated by neutral and acidic phospholipids; the most efficient were phosphatidyl-serine and phosphatidyl-inositol. 6. The enzymatic activity was not significantly changed by cholesterol alone but it is greatly enhanced by liposomes loaded with dolichyl-phosphate and cholesterol.

Animals↗

[CK-MB isoenzyme in the diagnosis of myocardial infarction after myocardial revascularization surgery].

The usefulness of measuring serum MB creatine kinase activity (CK-MB) for the diagnosis of per- and postoperative myocardial infarction (MI) was assessed in 104 patients undergoing coronary artery bypass grafts. In each patient, 15 samples were taken during the week which followed the surgical procedure. New Q waves were considered to be a criteria of MI. 19 patients developed new Q waves (MI group), whereas 57 had no significant ECG changes (control group); 13 showed only ST changes, whilst 15 had unassessable recordings. In the MI group, CK-MB was greater than in the control group, both at the first peak (8 to 10 h after induction of anaesthesia) and at the greater peak (13 to 21 h after induction) (p less than 0.05). Significant differences were also seen between both groups between 8 and 32 h after induction, but there was also a large overlap. An area under the curve (AUC) greater than 50,000 IU.l-1.min-1 had a positive predictive value of 0.64, and an AUC less than 50,000 IU.l-1.min-1 a negative predictive value of 0.89 if all the groups of patients were taken into account. An AUC greater than 65,000 IU.l-1.min-1 was always seen in MI patients, but only 25% of MI patients had a value greater than this threshold. There were no significant differences between the patient groups in the first peak time, nor in the CK-MB/total CK ratio. CK-MB appeared therefore as a less reliable criterium of per- and postoperative MI during coronary artery bypass operations than previously reported, especially when intermediate values are found.

Adult↗

Iodinated contrast agents: effect on acetylcholine hydrolase. In vitro study.

Several conventional and new contrast media were studied in vitro in order to evaluate their effects on acetylcholine hydrolase. Iohexol proved to be the most potent inhibitor of cholinesterase; all other conventional ionic agents are not inhibitors or they are weak inhibitors at the concentrations that are apparently achieved locally in clinical practice. The full clinical significance of the relationship between cholinesterase inhibition in vitro and contrast media toxicity must be reviewed.

Animals↗

Study of the fucosyltransferase system in intestinal microsomes.

Fucosyltransferase activity of rat small intestine microsomes is solubilized by 0.5% Triton X-100. The solubilized activity can be purified up to 8,300-fold using DEAE-cellulose and affinity chromatography on GDP-Sepharose. At this step, chromatography on Sephadex G15 separates different specificities: N-acetylglucosaminide-alpha-(1,3)-fucosyltransferase acting on asialoserotransferrin, and galactoside-alpha-(1,2)-fucosyltransferase acting on O-glycans of asialofetuin. The use of small saccharidic acceptors also indicates the presence of a N-acetylglucosaminide-alpha-(1,4)-fucosyltransferase and of a very weak glucose-alpha-(1,3)-fucosyltransferase activity. These activities are tightly bound to concanavalin A-Sepharose, suggesting that they are supported by N-glycosylproteins.

Animals↗

[Lactates, amino acids, other derivatives and provocation tests].

Myocardial metabolism is a dynamic process which, by using lipids, carbohydrates, proteins and heterocyclic compounds, translates the energy liberated by the broken bonds in terms of adenosine triphosphate (ATP). The dynamic character of this metabolism demands extremely precise controlling mechanisms of five main types: availability of substrates, transcriptional and translational effects, post-translational changes, allosteric and non-allosteric effects and shuttle systems. Within this very complex model with its multiple interferences, static analysis of fixed metabolites seems to be quite futile: it can only give a summary and global assessment at blood flow or even cardiac level of a tremendous number of contradictory reactions, and the sophistication of biological analysis does not succeed in hiding our conceptual ignorance. These pessimistic comments are aimed especially at the lactate and other similar tests.

Allosteric Regulation↗

Glycosyl-transferase activities in pancreas: comparison between semi-synthetic and commercial diets.

The glycosyl-transferase activities in the rat pancreas have been previously demonstrated to be modified by the quantity of different dietary components (lipids or proteins). To evaluate the role of the qualitative composition of the diet on such enzymic systems, two groups of rats were fed either a semi-synthetic diet or a commercial diet of very similar quantitative composition but differing in the quality of their components. The two diets induce a quite similar growth of animals, although the pancreas weight of the commercial-diet-fed rats is slightly higher. The galactosyl-, fucosyl- and mannosyl-transferase activities are more or less highly enhanced by the commercial diet according to the enzyme studied. The highest increase is observed for the biosynthesis of mannosyl-phosphoryl-dolichol. This enhancement by the commercial diet disappears when exogenous phosphoryl-dolichol is added. Such results indicate that the mannose transfer is probably modified by an increased level of the pancreatic lipidic acceptor.

Animals↗

Purification and properties of a mannosyltransferase solubilized from mitochondrial outer membranes.

The enzyme GDPmannose: dolichyl monophosphate mannosyltransferase has been solubilized and purified from mice liver mitochondrial outer membranes. The purification combines detergent extraction of purified outer membranes using Nonidet P-40, with subsequent ion-exchange chromatography on DEAE-cellulose. At this stage, a 400-fold purification is obtained. The partially purified mannosyltransferase is activated by choline-containing lipids such as phosphatidylcholine, lysophosphatidylcholine and sphingomyelin. The reaction is dependent upon the addition of exogenous dolichyl monophosphate. The sole reaction product has been identified as dolichyl phosphate-mannose. The partially purified mannosyltransferase exhibits a Km of 1.33 microM for GDPmannose. Enzyme activity, eluted from DEAE-cellulose, could be further purified after incorporation into sphingomyelin vesicles containing dolichyl monophosphate followed by a sucrose density gradient centrifugation. The mannosyltransferase activity is completely associated with the liposomes at the top of the gradient. Significant stabilization and purification (approx. 1600-fold) of enzyme activity associated with these liposomes is obtained. Furthermore, the reconstitution of this purified enzyme within specific liposomes provides a good model membrane to investigate the molecular requirement of this mitochondrial mannosyltransferase.

Animals↗

Presence of immunoreactive material in Niemann-Pick type A placenta using anti-sphingomyelinase rabbit gammaglobulins.

Human placental sphingomyelinase was highly purified through an original six-step scheme in order to raise a specific rabbit anti-sphingomyelinase antibody. Pure enzyme preparations showed specific activities ranging between 100 and 150 mumol/h per mg protein and gave two constant silver-stained bands (Mr 70,000 and 57,000) on acrylamide after electrophoresis under denaturing conditions. These two bands were the sole areas detected by the described antibody on Western blots from normal placental preparations at various stages of purification. A similar procedure was applied to the separate study of placental sphingomyelinase from two prenatally diagnosed foetuses with confirmed Niemann-Pick disease type A. During purification, the mutant enzyme could be followed owing to its minute but measurable level of catalytic activity, and behaved normally at the various chromatographic steps. In the purified preparations, specific activities of 0.18 and 0.49 mumol/h per mg protein, respectively, were reached. No alteration of the Km value (19 mumol/l) was observed, while the Vmax was 0.5-1% of normal. With immunostaining of Western blots obtained as above, results similar to those described for normal tissue were found, leading to the conclusion that immunoreactive sphingomyelinase is present in Niemann-Pick disease type A.

Animals↗

Characterization of the tissue proliferated at the blood interface of carbon/ceramic composites.

The present study focuses on cell adhesion/differentiation and material stability of surfaces of the three carbon/ceramic composites implanted in intra-atrial position in dogs for 1 year. Before implantation their surface was characterized by scanning electron microscopy. After harvesting, the tissue proliferated on the blood interface was examined by histology, scanning, and transmission electron microscopy, wavelength dispersive and x-ray spectrometry, electrophoretic and enzymatic characterization of glycosaminoglycans (GAGs) which were compared to endocardiac tissue as control samples. One year after implantation, the pattern of GAGs in the newly developed tissue was characterized by: 1) a constant increase of the total GAGs present on all carbon composites, 2) a significant increase of dermatan sulfate (p less than 0.05), 3) a significant increase of chondroitin sulfate (p less than 0.05), 4) a significant decrease of heparan sulfate in Group 1, whereas this GAG fraction was increased in Groups 2 and 3. Cellular surface differentiation towards endothelial-like cells occurred in places particularly in groups 1 and 3, whereas only fibrous tissue was found covering the implants in Group 2. Fibroblastic cells with dense intracellular deposits, which produced emission of Si, Ca, and C energy as well as extracellular lipidic containing inclusions were observed. The macromolecular modifications were associated with 1) the absence of endothelial lining, 2) the migration of carbon and silicon particles, and 3) the occurrence of calcifications and lipidic inclusions. These results suggest that the relative smoothness of these materials could be responsible for the development of a tissue that did not adhere to the biomaterial, indicating that cell adhesion and functional differentiation are in intimal relationship with the physical-chemical structure of the material surface.

Animals↗

Screening of degradative enzymes from articular cartilage in experimental osteoarthritis.

Sixteen 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.

Animals↗

Study of circadian correlations between acetylcholine muscarinic receptor and brain glycosyltransferases by multivariate analysis.

Circadian variations of the acetylcholine muscarinic receptor and some glycosyltransferases were studied in brain using multivariate analysis. Highly significant correlations exist between fucosyltransferase, sialyltransferase and galactosyltransferase and to a lesser extent between both of these enzymes and acetylcholine receptor. No correlation appeared between these enzymes and dolichol phosphate mannose synthase.

Analysis of Variance↗

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.

Animals↗