Characterization of alpha,alpha-trehalase released in the intestinal lumen by the probiotic Saccharomyces boulardii.

Objective. Trehalose intolerance due to alpha,alpha-trehalase deficiency has scarcely been studied. The purpose of this study was to measure alpha,alpha-trehalase activity in intestinal biopsy samples from 200 consecutive patients over a period of 6 months, and to characterize alpha,alpha-trehalase released by the probiotic Saccharomyces boulardii (S. boulardii). Material and methods. Enzyme activities were measured in human and rat intestinal mucosal samples using the micromethod of Messer & Dalqvist. alpha,alpha-trehalase from S. boulardii was immunoprecipitated and Western blotted using an IgG purified antibody raised against a 23 amino acid peptide of alpha,alpha-trehalase of S. cerevisiae. Results. Among 200 patients, most of whom complained of abdominal symptoms and diarrhoea, 18 (9%) had total alpha,alpha-trehalase deficiency (0-12 U/g mucosa) and 39 had partial deficiency (3-12 U/g mucosa). Only 4 patients (2%) presented selective alpha,alpha-trehalase deficiency with otherwise normal disaccharidases. Expressed per gram of powder, alpha,alpha-trehalase from S. boulardii delivered in vitro an activity 175 times higher than that of human trehalase per gram of intestinal mucosa. V(max) (22+/-0.43 micromol) and K(m) (5 mM) were close to that of the human enzyme, whereas Western blot revealed a signal of two subunits of 82 kDa. Finally, treatment of rats with S. boulardii resulted in increases in alpha,alpha-trehalase activities of 25 to 45% (p<0.01) in endoluminal fluid and intestinal mucosa compared with in controls. Conclusions. Our data suggest that alpha,alpha-trehalase deficiency is more common than is believed and that oral administration of S. boulardii could be beneficial in patients with digestive symptoms caused by trehalose intolerance

La dimension "travail", un élément clé pour le maintien de nos systèmes laitiers

Les agriculteurs sont les principaux architectes du milieu rural wallon. En effet, ils assurent la gestion de près de la moitié de la superficie de la Wallonie. On observe néanmoins, depuis quelques années, une régression lente (3%/an) et constante de leur effectif. Confrontés à un contexte changeant, de plus en plus concurrentiel, les agriculteurs sont face à des choix stratégiques difficiles en vue d’optimiser l’efficience de leur exploitation. Pour y parvenir, ils doivent mener une réflexion globale de leur mode de production et intégrer tous les aspects assurant la durabilité de leur exploitation. Pour les aider dans cette démarche, une analyse de la durabilité de 90 exploitations « 100% Lait » (>95% de vaches laitières et pas de culture de rente) a été réalisée dans le cadre de DuraLait et de DuraLait Plus. Ces études sont subsidiées par la DGARNE, Direction de la Qualité. Les piliers économique et social de la durabilité ont été plus particulièrement étudiés dans ces projets. L’organisation du travail a été spécifiquement traitée car elle constitue un enjeu essentiel pour l’avenir de l’agriculture. En effet, les agriculteurs souhaitent soulager la pénibilité de leur travail. Dans un contexte économique difficile, cette dimension est essentielle pour envisager le maintien de l’agriculture dans notre région. Le présent article s’intéresse uniquement aux données relatives à l’organisation du travail

Insulin signal transduction in rat small intestine: role of MAP kinases in expression of mucosal hydrolases

The postreceptor events regulating the signal of insulin downstream in rat intestinal cells have not yet been analyzed. Our objectives were to identify the nature of receptor substrates and phosphorylated proteins involved in the signaling of insulin and to investigate the mechanism(s) by which insulin enhances intestinal hydrolases. In response to insulin, the following proteins were rapidly phosphorylated on tyrosine residues: 1) insulin receptor substrates-1 (IRS-1), -2, and -4; 2) phospholipase C-isoenzyme-gamma; 3) the Ras-GTPase-activating protein (GAP) associated with Rho GAP and p62(Src); 4) the insulin receptor beta-subunit; 5) the p85 subunits of phosphatidylinositol 3-kinase (PI 3-kinase); 6) the Src homology 2 alpha-collagen protein; 7) protein kinase B; 8) mitogen-activated protein (MAP) kinase-1 and -2; and 9) growth receptor-bound protein-2. Compared with controls, insulin enhanced the intestinal activity of MAP kinase-2 and protein kinase B by two- and fivefold, respectively, but did not enhance p70/S6 ribosomal kinase. Administration of an antireceptor antibody or MAP-kinase inhibitor PD-98059 but not a PI 3-kinase inhibitor (wortmannin) to sucklings inhibited the effects of insulin on mucosal mass and enzyme expression. We conclude that normal rat enterocytes express all of the receptor substrates and mediators involved in different insulin signaling pathways and that receptor binding initiates a signal enhancing brush-border membrane hydrolase, which appears to be regulated by the cascade of MAP kinases but not by PI 3-kinase

Saccharomyces boulardii produces in rat small intestine a novel protein phosphatase that inhibits Escherichia coli endotoxin by dephosphorylation.

Using a polyclonal antibody raised against a highly conserved sequence of 38 amino acids containing the activation site (VTDSAAGAT) common to mammalian and yeast alkaline phosphatases (AP), we identified in decapsidated Saccharomyces boulardii a protein phosphatase detected by autoradiography as a single signal (63 kD). Using an affinity chromatography column, the protein phosphatase could be concentrated 39.1-fold and presented as a doublet of two subunits. Compared with rat and bovine purified intestinal AP, the enzyme from S. boulardii had a greater ability to dephosphorylate the lipopolysaccharide (LPS) of Escherichia coli 055B5. When tested in vivo, intraperitoneal injection of intact LPS to rats produced, after 9 h, 100 ng/mL of circulating tumor necrosis factor-alpha with inflammatory lesions and apoptotic bodies in the liver and the heart, whereas rats injected with partially dephosphorylated LPS produced only 40 ng/mL tumor necrosis factor-alpha without organic lesions. In conclusion, S. boulardii is able to inhibit toxicity of E. coli surface endotoxins by the release of a protein phosphatase exhibiting a great capacity of dephosphorylation

Saccharomyces boulardii enhances N-terminal peptide hydrolysis in suckling rat small intestine by endoluminal release of a zinc-binding metalloprotease.

Saccharomyces boulardii (S. boulardii), a biotherapeutic agent effective in acute and chronic enterocolopathies, produces trophic intestinal effects at least in part mediated by the endoluminal release of polyamines. However, the effects of the yeast on peptide hydrolysis have not yet been studied. The objectives of this study were to assess in suckling rats the endoluminal and mucosal aminopeptidase activities in response to S. boulardii treatment and to analyze their related mechanisms. Peptidase activities were assayed on yeast cells by using several L-amino acid-p-nitroanilide substrates in the pH range of 2 to 10. A marked hydrolytic activity was found for L-leucine-p-nitroanilide that peaked at pH = 8 (K(m) = 0.334 mM, V(max) = 44.7 micromol.min(-1).g(-1) protein). N-terminal peptide hydrolysis was confirmed using as substrate L-Leu-Gly-Gly (K(m) = 4.71 mM, V(max) = 18.08 micromol.min(-1).g(-1) protein). Enzyme reactions were inhibited in the presence of 1 mM Zn(2+). Oral treatment of sucklings with S. boulardii significantly enhanced jejunal and ileal mucosal leucine-aminopeptidase activities by 24 and 34%, respectively, over controls. In concordance, aminopeptidase activity was enhanced in jejunal and ileal endoluminal fluid samples by 47 and 105%, respectively. By use of an IgG-purified antibody raised against the zinc-binding domain common to metalloproteases, the yeast aminopeptidase was immunoprecipitated and detected as an heteromeric enzyme of 108 and 87-kD subunits. S. boulardii, when given orally to suckling rats, is able to significantly enhance hydrolysis of N-terminal oligopeptides in both endoluminal fluid and intestinal mucosa by the endoluminal release of a leucine aminopeptidase that appears to be a zinc-binding metalloprotease belonging to the M1 family of peptidases

Ontogeny of MAP kinases in rat small intestine: premature stimulation by insulin of BBM hydrolases is regulated by ERKs but not by p-38 MAP kinase.

Although mitogen-activating protein (MAP) kinases are crucial signal transduction molecules regulating cellular proliferation, differentiation, and morphology, their ontogenic changes in the small intestine have not been analyzed. Also, it remains unknown which pathway of activated MAP kinases regulates the expression of brush border membrane hydrolases during growth. Therefore, we have analyzed the mucosal distribution, ontogeny, and responses to insulin and to inhibitors of p44, p42, and p38 MAP kinases in immature and mature enterocytes using Western blot analysis and autoradiography after immunoprecipitation, immunohistochemistry, and in vitro phosphorylation assays. Between d 10 and 40 postpartum, diphosphorylated active p44/p42 extracellular regulated protein kinases (ERKs) increased in abundance compared with total immunoprecipitated ERKs, and were highly responsive to exogenous insulin. In concordance, ERK total activity increased by 4-fold during the same period of growth and was further enhanced 2-fold by exogenous insulin. In weaning rats, ERKs were mainly located in membranes of villus cells and with less intensity in crypt cells. By contrast, p38 MAP kinase was unresponsive to insulin and was confined to nuclei. Administration to sucklings of PD 098059, a specific inhibitor of ERKs, not only inhibited the premature stimulation of sucrase, lactase, and maltase total activities in response to exogenous insulin, but also depressed the natural expression of these brush border membrane enzymes in the absence of insulin stimulation. In concordance, administration of SB 203580, a specific inhibitor of p38 MAP kinase, failed to inhibit both the response of brush border membrane hydrolases to insulin and their natural expression in the absence of insulin stimulation. We conclude that the ontogenic expression of brush border membrane hydrolases and their premature stimulation by insulin are regulated at least in part by the activation of p44/p42 ERKs but not by p38 MAP kinase