Biomedical subjects

P Louisot

Publications and source records attributed to P Louisot.

At least 55 records · Page 3Linked to original sources

PubMed1995

Effects of specific dietary sugars on the incorporation of 13C label from dietary glucose into neutral sugars of rat intestine and serum glycoproteins.

Although theoretically all glycoprotein sugars can be derived from glucose, it may be hypothesized that specific dietary sugars could be preferential substrates for glycoprotein synthesis. To test this hypothesis, groups of rats received either continuously (continuous-labelling experiment) or for a single nutritional period (pulse-labelling experiment) a 13C-rich diet containing either maize starch or artificially labelled [13C]glucose. Some groups of rats were also provided during a single nutritional period with low amounts (20-200 mg/animal) of low-13C dietary sugars (mannose, galactose, fucose or fructose). If specific dietary sugars were preferentially incorporated into glycoproteins instead of glucose-derived labelled sugars, a decrease would be expected in the intestinal or serum glycoprotein-sugar 13C enrichment monitored by gas chromatography-isotope-ratio mass spectrometry (GC-IRMS). Contrary to this hypothesis the results showed no significant decrease with any of the specific dietary sugars. Furthermore, with dietary low-13C mannose or galactose, a significant increase in 13C enrichment of glycoprotein-sugars was observed compared with some other nutritional groups. Moreover, in the pulse-labelling experiment, dietary mannose and galactose induced similar patterns of 13C enrichment in intestinal and serum glycoprotein-sugars. Therefore, although specific dietary sugars do not appear to be preferential substrates for glycosylation under conditions and doses relevant to current concepts of nutrition, regulatory roles of some specific dietary sugars in relation to glycoprotein-sugar metabolism might be hypothesized. These findings could lead to similar studies using stable-isotope methodology in man which could have practical consequences, especially in parenteral nutrition where glucose is the only sugar provided to the metabolism.

Animals↗

PubMed1995

Differential incorporation of 13C label from dietary glucose into neutral sugars of rat intestine macromolecules.

Using gas chromatography-isotope ratio mass spectrometry, the detection, with a good reproducibility and in a single step, of the 13C content of glycoprotein neutral sugars, glycogen glucose, and ribonucleic acid ribose allowed us to trace incorporation into intestinal macromolecules of sugars derived from dietary glucose, labeled either naturally (as corn starch) or artificially. The 13C enrichment of glycoprotein neutral sugars was strong and rapid and plateaued up to the end of the experiment, whereas the 13C content of ribose increased linearly with time. By contrast, with artificially enriched dietary glucose, no significant 13C enrichment was detected in glycogen, suggesting that this macromolecule does not directly derive from dietary glucose. These results suggest differential control of sugars involved in the synthesis of intestinal macromolecules and demonstrate that this technique would be sensitive and reliable enough to be applied in man for serum glycoprotein sugar determinations.

Animals↗

PubMed1995

Study of retinoic acid effect upon retinoic acid receptors beta (RAR-beta) in C6 cultured glioma cells.

Using monoclonal antibodies against the RAR-alpha and RAR-beta retinoic receptors, we demonstrated that these receptors were present together in C6 glioma cells as two isoforms of 50 and 55 kDa. For RAR-beta, the 50 kDa isoform predominated (60 to 80% of the total of the two isoforms). After a treatment for 48 h with retinoic acid 10 microM, the 55 kDa form was enhanced, while no effect was observed either on RAR-alpha isoforms from C6 cells and on both RAR-alpha and RAR-beta forms from neuroblastoma SKN SH SY5Y used as a control. Using purified neuronal and glial rat brain nuclei, we showed that the 55 kDa isoform from RAR-beta predominated in glial cells. These results suggest that retinoic acid treatment of C6 cells led to a partial differentiation, the enhancement of the heavy form of RAR-beta being a marker of this phenomenon.

Animals↗

PubMed1994

Ontogenic and nutritional modifications in the intestinal fucosylation process at the weaning period. Influence of dietary fibers.

In the rat small intestine, the glycosylation changes which normally take place at the weaning period are characterized by a shift from sialylation to fucosylation. The introduction of dietary fibers at weaning is one of the more striking nutritional modification so that some authors have suggested that the presence of fibers and the development of colonic fermentation might be important for the development of the small intestine, as for the colon. In order to define the respective contribution of ontogenic and nutritional factors to the intestinal glycosylation changes at this period, some aspects of the intestinal glycosylation were studied in five groups of rats (16-day-old suckling rats, prolonged nursing 23-day-old rats, 23-day-old rats weaned at day 19 with either a fiber-free, a cellulose or a pectin diet). Intestinal glycoproteins of suckling rats are characterized by a low fucose content and a high proportion of mannose. The amounts of the neutral sugars (fucose, mannose and galactose), expressed either per gram of intestine or for one intestine, are always higher in the fiber-fed groups than in the prolonged-nursing group or the group fed the fiber-free diet. Activities which promote fucosylation process (GDP-fucose production and fucosyltransferase activities) and those which are opposed to fucosylation (endogenous inhibitor of fucosyltransferase and GDP-fucose pyrophosphatase) are strongly modified in opposite ways at day 23 as compared to day 16. These modifications depend on the age of the animal (ontogenic factors) with additional modifications induced by the dietary factors. In particular, similar sugar contents and patterns are obtained with cellulose and pectin diets though the enzymatic activities of the fucosylation pathway are very different. No correlation was found between the caecal content of short chain fatty acids and any of the parameters under study. Thus, dietary fibers induce metabolic changes in the small intestine glycosylation in short-term experiments independently of colonic fermentation. Besides, these results point out that the consideration of fucosyl-transferase activities alone are not sufficient to predict glycoprotein fucose content and that other regulatory sites are involved. Dietary manipulations at the weaning period could represent a good model for the study of glycosylation regulation.

Animals↗

PubMed1994

Purification and partial amino acid sequence of fuctinin, an endogenous inhibitor of fucosyltransferase activities.

A powerful endogenous protein inhibitor of fucosyltransferase activities, called fuctinin, was purified to homogeneity from rat small-intestinal mucosa. The purification scheme involved DEAE-cellulose ion-exchange chromatography, ammonium sulfate fractionation, hexyl-agarose hydrophobic chromatography and size-exclusion HPLC. Active native fuctinin has an isoelectric point of 4.55 and apparent molecular mass approximately 66 kDa, whereas a single protein band with a molecular mass of approximately 24 kDa was obtained by denaturing polyacrylamide gel electrophoresis, suggesting that fuctinin is an oligomeric protein. Two-dimensional polyacrylamide gel electrophoresis displayed eight spots in this single band. Comparisons of the N-terminal amino acid sequences of each spot support the idea of the existence of three related polypeptides and suggest a proteolytic N-terminal cleavage despite the use of an efficient protease inhibitor throughout the purification. In spite of the presence of an N-glycosylation site, fuctinin is not glycosylated. One of the three polypeptides, peptide 3, possesses two consensus sequences for phosphorylation and a consensus sequence for myristoylation. The sequences of functinin-related peptides, especially peptide 3, exhibit high similarity to the N-terminal domain of the Set protein and a putative human leukocyte antigen-associated protein. The possible implications of these results are discussed.

Amino Acid Sequence↗

PubMed1994

Free sphingoid bases in tissues from patients with type C Niemann-Pick disease and other lysosomal storage disorders.

The 20-fold increase of free sphingoid bases found in liver from a murine model of Niemann-Pick type C (NPC) combined to the NPC-like phenotype induced by addition of sphinganine to normal fibroblast cultures prompted us to investigate the potential involvement of these compounds in the human disease. The contents of sphingosine and sphinganine were measured in liver, spleen, brain and skin fibroblast cultures by a sensitive HPLC method. In liver and spleen from NPC patients, a 6- to 24-fold elevation of sphingosine and sphinganine already prominent at the fetal stage of the disease was observed, while no clear increase could be evidenced in brain tissue. A significant increase, not modulated by the intralysosomal content of free cholesterol, also occurred in skin fibroblast cultures. To investigate the specificity of these findings, other lysosomal storage disorders were studied. A striking accumulation was found in liver and spleen (24- to 36-fold) from patients with Niemann-Pick disease type A and B (sphingomyelinase-deficient forms), and in cerebral cortex of type A Niemann-Pick disease. A significant storage also occurred in Sandhoff disease, while several other sphingolipidoses showed a moderate elevation. In all cases but Sandhoff disease brain, the sphingosine/sphinganine ratio remained unchanged, suggesting that the accumulated free sphingoid bases derived from sphingolipid catabolism. Formation of complexes between sphingosine and the lipid material accumulated in lysosomes might be a general mechanism in lysosomal lipidoses. In NPC, however, an increase of free sphingoid bases disproportionate to the degree of lysosomal storage and a specific involvement of cultured fibroblasts suggested a more complex or combined mechanism.

Brain Chemistry↗

PubMed1994

Further characterization of mitochondrial outer membrane: evidence for the presence of two endogenous sialylated glycoproteins.

The distribution of sialic acid-containing glycoproteins was investigated in highly purified mitochondrial membranes using labeled Sambucus nigra agglutinin as a detection system. Two sialylated glycoproteins were shown to be true components of the mitochondrial outer membrane. Relative to monoamine oxidase activity, these glycoproteins were found to be preferentially located in the "free" outer membrane fraction. As sialic acid is thought to be involved in molecular recognition, a role for these glycoproteins in mediating the interactions between mitochondria and other sub-cellular organelles is considered.

Animals↗

PubMed1994

Creatinine metabolism impairment by an anticonvulsant drug, phenacemide.

OBJECTIVE: To report two cases of increased true serum creatinine (Scr) without renal failure caused by an anticonvulsant drug, phenacemide, and to discuss the possible mechanisms. CASE SUMMARY: Two patients treated with phenacemide were investigated for markedly increased Scr and decreased creatinine clearance (Clcr) values. Glomerular filtration rates, as determined by 125I-iothalamate clearance, were normal in both patients and analytical interferences with the Jaffé reaction were excluded. After discontinuation of the drug, phenacemide concentrations became undetectable within 2 days but it took 7-14 days for Scr and Clcr to return to normal values. DISCUSSION: The Scr increase with phenacemide (120-170 percent) was higher than that reported with cimetidine or trimethoprim (10-40 percent) and could not be explained solely by inhibition of the tubular secretion of creatinine. The hypothesis of an overproduction of creatinine caused by phenacemide was ruled out by experimental studies in rats. Creatinine increase in tissues was lower than that in the serum of rats given phenacemide. In vitro creatinine influx into red blood cells was inhibited in a dose-dependent way by phenacemide. CONCLUSIONS: Increased Scr concentrations in these patients could be related to an inhibition of transport and a decrease in creatinine volume of distribution. Creatinine concentrations should not be considered when dosage adjustments of renally eliminated drugs are being calculated for patients with such metabolic interferences.

Adult↗

PubMed1994

Study of the succinate dehydrogenase activation in permeabilized mitochondria through the Ca(2+)-stimulated phospholipase A2.

The development of a mitochondrial membrane permeability triggered by the Ca(2+)-stimulation of PLA2 (phospholipase A2; EC 3.1.1.4.) and based on swelling, polyunsaturated fatty acids release and calcium influx, induced the activation of SDH (succinate dehydrogenase; EC 1.3.9.9.) without damaging mitochondria structures. The activity of SDH increased within the length of permeabilization treatment before reaching a plateau. The study of Km and Vm showed that the affinity of SDH for succinate and the maximal velocity were increased. Based on these results, the change of SDH activity triggered under these conditions could be explained by a substrate activation of SDH taking account that the succinate content was significantly enhanced.

Animals↗

PubMed1994

Comparative effects of dietary corn, fish and Krill oils on intestinal glycosylation.

Antarctic Krill is considered as a valuable protein resource for animal and human nutrition. Due to the high content of long chain polyunsaturated fatty acids of the n-3 family, Krill consumption could be also interesting in cardiovascular diseases. In the search for the demonstration of the absence of toxicity of Krill, we studied the effect of Krill oil, as compared to fish and corn oil, on the rat intestinal fucosylation process at weaning, a very sensitive model of the influence of nutritional factors. Krill oil containing diets were very well tolerated as compared to other currently used oils and induced only slight modification in fucose and mannose proportions in intestinal glycoprotein sugars. These modifications were not reflected in the enzymatic activities involved in the fucosylation pathway. These results confirm the harmlessness of Krill derived products and their possible use in human nutrition.

Animals↗

PubMed1993

Involvement of mitochondrial contact sites in the subcellular compartmentalization of phospholipid biosynthetic enzymes.

The concerted synthesis of phospholipids derived from serine involving two microsomal enzymes (phosphatidylserine synthase and phosphatidylethanolamine N-methyltransferase) and a mitochondrial one (phosphatidylserine decarboxylase) occurs in reconstituted cell-free systems. Subfractionation of crude mitochondria after swelling and separating on a sucrose density gradient resulted in the isolation of two contact site-enriched fractions from total outer membranes and inner membranes, respectively. Estimation of marker enzyme activities shows a high recovery of glucose-6-phosphate phosphatase (a marker for the endoplasmic reticulum) associated with contact site-enriched fractions. Accordingly, the linked synthesis of phosphatidylserine, phosphatidylethanolamine, and at a lesser extent phosphatidylcholine can occur. This biosynthetic pathway was absent from purified contact site-enriched fractions correlative with the absence of glucose-6-phosphate phosphatase activity. Reconstitution experiments, including contact site-enriched fractions incubated with endoplasmic reticulum-rich fraction, led to the restoration of the linked synthesis of phospholipids, thereby demonstrating that a reversible association between these two fractions can occur. These functional interactions between the endoplasmic reticulum and mitochondria are confirmed at the ultrastructural level using either chemical or physical fixation before resin embedding. These results show that the interorganelle trafficking of lipids may involve only highly specialized microdomains of both membranes, thereby allowing the maintenance of a specific lipid composition and distribution within membranes.

Animals↗

PubMed1993

Further evidence for both functional and structural microcompartmentation within the membranes of two associated organelles, mitochondrion and endoplasmic reticulum.

We previously demonstrated that the translocation of phospholipids between the mitochondrion and the endoplasmic reticulum occurs via highly specialized membrane microdomains of both organelles that are in situ closely associated. As understanding of the interactions between both organelles requires characterization of the translocation sites organization, we first analysed the amino acid compositions of these sites. Using principal component analysis, we have shown that the translocation sites exhibit characteristic patterns when compared with the membranes from which they are derived. The results are discussed in terms of both functional and structural microcompartmentation within the membranes of mitochondria and endoplasmic reticulum.

Amino Acids↗

PubMed1993

Preparation of an anti-acid sphingomyelinase monoclonal antibody for the quantitative determination and polypeptide analysis of lysosomal sphingomyelinase in fibroblasts from normal and Niemann-Pick type A patients.

An anti-acid sphingomyelinase monoclonal antibody has been prepared using an in vitro booster technique. The antigen, acid sphingomyelinase, was purified from human placentas by sequential chromatographic steps in the presence of the non-ionic detergent Nonidet P40. This monoclonal antibody (MAB 236) precipitates specifically the enzyme activity by immunoadsorption techniques and presents the same specificity to normal and mutated sphingomyelinase in Niemann-Pick type A patients. MAB 236 is the first antibody able to precipitate the protein in the presence of detergent thereby permitting the quantitative determination of normal and mutated sphingomyelinase in tissue and cell extracts. Polypeptide analysis and quantitative determination experiments using this monoclonal antibody showed no difference between patients and normal controls.

Antibodies, Monoclonal↗

PubMed1993

Dacron vascular biomaterial triggers macrophage ectoenzyme activity without change in cell membrane fluidity.

Biomaterials induce an inflammatory reaction characterized by a rapid recruitment at the implantation site of polymorphonuclear cells and macrophages. In the course of the inflammatory response, the cellular activation triggers expression of a number of enzymes, such as 5'-nucleotidase, which is widely distributed in animal cell membranes as an ectoenzyme. It is now well established that 5'-nucleotidase activity decreases following the contact of inflammatory cells with foreign particles. In this paper we investigate a possible correlation between the enzymatic activities and the dynamic properties of the cell membrane bilayer. Dacron pieces were introduced into rats' peritoneal cavities for a period of 6 h, after which the peritoneal cells were harvested, and various enzyme assays performed, including those for cytoplasmic, lysosomal, and ectoenzymes. In parallel, we studied cell membrane fluidity, using fluorescence polarization of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), and cellular ultrastructural alteration resulting from the cell-biomaterial interactions using scanning and transmission electron microscopy. Our results show that: 1) macrophages spread around the Dacron fibers with cytoplasmic finger-like projections, but no phagolysosomes, 2) 5'-nucleotidase levels decrease with surgical trauma in comparison with the resident cell exudate, 3) implantation of biomaterials slightly modify the 5'-nucleotidase levels observed in the sham animal, 4) no differences in the anisotropy values indicating that membrane lipid order within the cells could not account for the observed decrease of 5'-nucleotidase activity. Thus, we can suggest that 5'-nucleotidase expression may reflect a particular feature of cell activation without a phagocytic process.

5'-Nucleotidase↗

PubMed1993

Use of Percoll gradients for isolation of human placenta mitochondria suitable for investigating outer membrane proteins.

Human mitochondria were isolated from placenta by a combination of differential and Percoll gradient centrifugation, resulting in a highly pure and intact preparation as assessed by marker enzyme analysis and electron microscopy. The advantages over previous methods are the rapidity of the procedure and the excellent resolution of mitochondria and lysosomes. Moreover, the high extent of intactness of the mitochondria so obtained made them particularly well suited for investigating outer membrane proteins. Taking advantage of this method, we have purified human mitochondrial porin. The purified protein consists of a single unglycosylated polypeptide of molecular mass 33 kDa.

Cell Fractionation↗

PubMed1993

A nondenaturing preparative gel electrophoresis system for the recovery of functional proteins. Application to the identification of an endogenous protein inhibitor of fucosyl-transferase activities.

Electroelution of protein bands resolved by nondenaturing polyacrylamide gel electrophoresis was performed to identify an endogenous protein inhibitor of fucosyltransferase activities, called fuctinin, through its biological activity. After electrophoresis protein bands were negatively stained with zinc acetate, indicating that this staining technique can be also applied to nondenaturing polyacrylamide gels. However, even under appropriate electroelution conditions, a strong fucosyl-transferase inhibitory activity was eluted from the polyacrylamide gel itself that impeded the measure of fuctinin activity. As an alternative to electroelution, collection of proteins resolved by nondenaturing polyacrylamide gel electrophoresis as they are electrophoresed off the end of the gel was assayed. In the commercially available preparative electrophoretic systems, an elution chamber is limited by a semipermeable membrane on which proteins were adsorbed in the low-ionic-strength buffer used in nondenaturing electrophoresis. We demonstrate that the simple and inexpensive electrofractionation system described by Shain et al. (Anal. Biochem. 200, 47-51, 1992) can be successfully applied under nondenaturing conditions to purify and identify fuctinin through its biological activity. However, caution must be taken in the design of the apparatus in order to avoid local heating and thermal denaturation of proteins. The utility of this nondenaturing preparative electrophoresis system for the study of functional proteins is also demonstrated by the recovery of enzymatic activities of alkaline phosphatase and beta-galactosidase.

Electrophoresis, Polyacrylamide Gel↗

PubMed1993

Deletion of arginine (608) in acid sphingomyelinase is the prevalent mutation among Niemann-Pick disease type B patients from northern Africa.

There is a high incidence of Niemann-Pick type B disease in the Maghreb region of North Africa, which includes Morocco, Algeria and Tunisia. A hypothesis that there may well be a common, predominant mutant acid sphingomyelinase allele responsible for the type B phenotype in this population has been tested. A deletion of an arginine codon at amino acid residue 608 was found in one patient. The same mutation was also observed in another of our cases. An original screening procedure using 3'-end digoxigenin-labeled allele-specific oligonucleotides and chemiluminescent detection was developed and used parallel to the conventional assay with 5'-end radiolabeled oligonucleotides. Of the 15 non-related, non-Jewish North African type B patients studied, 12 were homozygous and two compound heterozygous for this deletion (26 delta R608 alleles/30 mutant alleles). Among type B patients from other geographic regions (France, UK, Italy, Czechoslovakia), this mutation was observed in only one of the 16 alleles studied. Our results indicate that deletion of arginine 608 in the acid sphingomyelinase gene is the highly prevalent mutation underlying Niemann-Pick type B disease in the population of Maghreb. A varying severity of the clinical and enzymatic expression within the non-neuronopathic phenotype has however been observed in patients homozygous for the mutation.

Adolescent↗

PubMed1993

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.

Adult↗