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.

A chemogenomic analysis of the human proteome: application to enzyme families.

Sequence-based phylogenies (SBP) are well-established tools for describing relationships between proteins. They have been used extensively to predict the behavior and sensitivity toward inhibitors of enzymes within a family. The utility of this approach diminishes when comparing proteins with little sequence homology. Even within an enzyme family, SBPs must be complemented by an orthogonal method that is independent of sequence to better predict enzymatic behavior. A chemogenomic approach is demonstrated here that uses the inhibition profile of a 130,000 diverse molecule library to uncover relationships within a set of enzymes. The profile is used to construct a semimetric additive distance matrix. This matrix, in turn, defines a sequence-independent phylogeny (SIP). The method was applied to 97 enzymes (kinases, proteases, and phosphatases). SIP does not use structural information from the molecules used for establishing the profile, thus providing a more heuristic method than the current approaches, which require knowledge of the specific inhibitor's structure. Within enzyme families, SIP shows a good overall correlation with SBP. More interestingly, SIP uncovers distances within families that are not recognizable by sequence-based methods. In addition, SIP allows the determination of distance between enzymes with no sequence homology, thus uncovering novel relationships not predicted by SBP. This chemogenomic approach, used in conjunction with SBP, should prove to be a powerful tool for choosing target combinations for drug discovery programs as well as for guiding the selection of profiling and liability targets.

Streamlined system for purifying and quantifying a diverse library of compounds and the effect of compound concentration measurements on the accurate interpretation of biological assay results.

As part of an overall systems approach to generating highly accurate screening data across large numbers of compounds and biological targets, we have developed and implemented streamlined methods for purifying and quantitating compounds at various stages of the screening process, coupled with automated "traditional" storage methods (DMSO, -20 degrees C). Specifically, all of the compounds in our druglike library are purified by LC/MS/UV and are then controlled for identity and concentration in their respective DMSO stock solutions by chemiluminescent nitrogen detection (CLND)/evaporative light scattering detection (ELSD) and MS/UV. In addition, the compound-buffer solutions used in the various biological assays are quantitated by LC/UV/CLND to determine the concentration of compound actually present during screening. Our results show that LC/UV/CLND/ELSD/MS is a widely applicable method that can be used to purify, quantitate, and identify most small organic molecules from compound libraries. The LC/UV/CLND technique is a simple and sensitive method that can be easily and cost-effectively employed to rapidly determine the concentrations of even small amounts of any N-containing compound in aqueous solution. We present data to establish error limits for concentration determination that are well within the overall variability of the screening process. This study demonstrates that there is a significant difference between the predicted amount of soluble compound from stock DMSO solutions following dilution into assay buffer and the actual amount present in assay buffer solutions, even at the low concentrations employed for the assays. We also demonstrate that knowledge of the concentrations of compounds to which the biological target is exposed is critical for accurate potency determinations. Accurate potency values are in turn particularly important for drug discovery, for understanding structure-activity relationships, and for building useful empirical models of protein-ligand interactions. Our new understanding of relative solubility demonstrates that most, if not all, decisions that are made in early discovery are based upon missing or inaccurate information. Finally, we demonstrate that careful control of compound handling and concentration, coupled with accurate assay methods, allows the use of both positive and negative data in analyzing screening data sets for structure-activity relationships that determine potency and selectivity.

Incidence and risk factors of oral antibiotic-associated diarrhea in an outpatient pediatric population.

BACKGROUND: Little information is available on the epidemiologic characteristics of antibiotic-associated diarrhea (AAD) in children. The authors' aim was to evaluate the incidence of AAD in an outpatient pediatric population and to identify risk factors. METHODS: Children aged 1 month to 15.4 years treated with oral antibiotics for a proven or suspected infection were enrolled from an ambulatory pediatric practice during an 11-month period. Parents recorded the daily frequency and characteristics of stools using a diary during the antibiotic treatment and for 1 week after it was stopped. An episode of diarrhea was defined by at least 3 soft or liquid stools/d for at least 2 consecutive days. Risk factors for AAD-age, type of antibiotic treatment, type of combined treatment, and site of infection-were analyzed. RESULTS: Of 650 children included, 11% had an episode of AAD, lasting a mean of 4.0 +/- 3.0 days, beginning a mean of 5.3 +/- 3.5 days after the start of antibiotic treatment. No child was hospitalized because of AAD. The incidence of AAD was higher in children less than 2 years (18%) than in those more than 2 years (3%; P < 0.0001). The incidence of AAD was particularly high after administration of certain antibiotics (amoxicillin/clavulanate, 23%; P = 0.003 compared with other antibiotics). The type of combined treatment and site of infection did not influence the onset of AAD. CONCLUSIONS: Antibiotic-associated diarrhea was common in these outpatient children, especially for those aged less than 2 years and after the prescription of certain antibiotics, particularly, the combination of amoxicillin/clavulanate.