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-␣ 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-␣ 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. S accharomyces boulardii, a nonpathogenic yeast, exerts therapeutic properties in acute and chronic enterocolopathies, antibiotic-associated diarrhea, and enterotoxigenic Clostridium difficile infections (1-4). In human volunteers (4,5) and in growing rats (4), several studies have documented that oral treatment with a lyophilized preparation of S. boulardii produces trophic intestinal effects, including increases in the activities of BBM enzymes (4,5) and enhanced secretion of s-IgA in intestinal fluid (6). After oral treatment of rats with S. boulardii, there is a marked stimulation of sodium-dependent D-glucose uptake into BBM vesicles with a corresponding accumulation of the sodium D-glucose co-transporter-1 (SGLT-1) (7). These trophic effects are, at least in part, mediated by endoluminal release of polyamines, as yeast cells contain substantial amounts of spermine and spermidine (4,8,9). In a recent work (10), we found that S. boulardii enhanced N-terminal peptide hydrolysis in suckling rat small intestine by endoluminal release of a zinc-binding metalloprotease. In the present study, we have analyzed whether oral treatment with S. boulardii could enhance the endoluminal activity of IAP. We also have purified a protein phosphatase secreted by S. boulardii in the rat intestinal lumen and have compared some of its properties with rat and bovine IAP. Finally, we have assessed whether the protein phosphatase released from S. boulardii can inhibit the toxicity of LPS from O55B5 Escherichia coli by dephosphorylation of its two phosphorylation sites. METHODSMedia and culture conditions. S. boulardii cells were inoculated in YPD (yeast extract, 0.5%; peptone, 2%; glucose, 2%; DIFCO, Detroit, MI) media and were grown at 30°C with moderate shaking to exponential growth as described (10).To disrupt the external capside, yeast cells were concentrated (1.45-1.50 ϫ 10 10...
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 (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 ϭ 8 (K m ϭ 0.334 mM, V max ϭ 44.7 mol·min Ϫ1 ·g -1 protein). N-terminal peptide hydrolysis was confirmed using as substrate L-Leu-Gly-Gly (K m ϭ 4.71 mM, V max ϭ 18.08 mol·min Ϫ1 ·g -1 protein). Enzyme reactions were inhibited in the presence of 1 mM Zn 2ϩ . 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. Saccharomyces boulardii (S. boulardii) is a nonpathogenic yeast exerting therapeutic properties in acute and chronic enterocolopathies, antibiotic-associated diarrheas, and enterotoxigenic Clostridium difficile overgrowth (1-4).In human volunteers (5, 6) and in growing rats (5), several studies have documented that oral treatment with a lyophilized preparation of S. boulardii produces trophic intestinal effects including increases in the specific and total activities of brushborder membrane (BBM) enzymes (5), enhanced secretion of s-IgA in intestinal fluid (7), and enhanced production of the receptor for polymeric Ig in villus and crypt cells (7). In addition, after oral treatment of rats with S. boulardii, there is a marked stimulation of sodium-dependent D-glucose uptake into BBM vesicles with a corresponding accumulation of the BBM sodium D-glucose cotransporter-1 (SGLT-1, 75 kD) (8). These trophic effects are, at least in part, mediated by endoluminal release of polyamines, as yeast cells contain substantial amounts of putrescine, spermidine, and spermine (9, 10). In addition, polyamine concentrations in mucosa and endoluminal fluid were found to be increased in proportion to the amount of spermine and spermidine supplied by the yeast ...
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.
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.
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