Estimating the prevalence of congenital disaccharidase deficiencies using allele frequencies from gnomAD.

BACKGROUND: There are currently three known congenital disaccharidase deficiencies: congenital lactase deficiency (CLD), congenital sucrase-isomaltase deficiency (CSD), and congenital trehalase deficiency (CTD). No congenital deficiency has been described for maltase-glucoamylase (MGAM). METHODS: A literature search was performed in PubMed for the pathogenic variants CLD, CSD, and CTD and the articles retrieved were analyzed to estimate the prevalence of congenital disaccharidase deficiencies. RESULTS: Based on reported variants, the estimated prevalence was 1.3 per 106 births (95% CI: 1.1-1.7) for CLD, and 31.4 per 106 births (95% CI: 28.3-34.8) for CSD. Using data on previously reported variants and variants predicted to be loss-of-function in gnomAD, the overall estimated prevalence was 2.3 per 106 births (95% CI: 1.9-2.9) for CLD, 57.6 per 106 births (95% CI:52.5-63.2) for CSD, and 9.2 per 106 births (95% CI: 2.5-3.7) for CTD. CONCLUSION: The prevalence of CSD was found to be relatively high, while for other congenital disaccharidase deficiencies, the estimated prevalence was very low.

Development of a Novel Cell Surface Attachment System to Display Multi-Protein Complex Using the Cohesin-Dockerin Binding Pair.

Autodisplay of a multimeric protein complex on a cell surface is limited by intrinsic factors such as the types and orientations of anchor modules. Moreover, improper folding of proteins to be displayed often hinders functional cell surface display. While overcoming these drawbacks, we ultimately extended the applicability of the autodisplay platform to the display of a protein complex. We designed and constructed a cell surface attachment (CSA) system that uses a noncovalent protein-protein interaction. We employed the high-affinity interaction mediated by an orthogonal cohesin-dockerin (Coh-Doc) pair from Archaeoglobus fulgidus to build the CSA system. Then, we validated the orthogonal Coh-Doc binding by attaching a monomeric red fluorescent protein to the cell surface. In addition, we evaluated the functional anchoring of proteins fused with the Doc module to the autodisplayed Coh module on the surface of Escherichia coli. The designed CSA system was applied to create a functional attachment of dimeric α-neoagarobiose hydrolase to the surface of E. coli cells.

Production of Ethanol from Agarose by Unified Enzymatic Saccharification and Fermentation in Recombinant Yeast.

The unified saccharification and fermentation (USF) system was developed for direct production of ethanol from agarose. This system contains an enzymatic saccharification process that uses three types of agarases and a fermentation process by recombinant yeast. The pGMFα-HGN plasmid harboring AGAH71 and AGAG1 genes encoding ß-agarase and the NABH558 gene encoding neoagarobiose hydrolase was constructed and transformed into the Saccharomyces cerevisiae 2805 strain. Three secretory agarases were produced by introducing an S. cerevisiae signal sequence, and they efficiently degraded agarose to galactose, 3,6-anhydro- L-galactose (AHG), neoagarobiose, and neoagarohexose. To directly produce ethanol from agarose, the S. cerevisiae 2805/pGMFα-HGN strain was cultivated into YP-containing agarose medium at 40°C for 48 h (for saccharification) and then 30°C for 72 h (for fermentation). During the united cultivation process for 120 h, a maximum of 1.97 g/l ethanol from 10 g/l agarose was produced. This is the first report on a single process containing enzymatic saccharification and fermentation for direct production of ethanol without chemical liquefaction (pretreatment) of agarose.

Personalized medicine in functional gastrointestinal disorders: Understanding pathogenesis to increase diagnostic and treatment efficacy.

There is overwhelming evidence that functional gastrointestinal disorders (FGIDs) are associated with specific mechanisms that constitute important targets for personalized treatment. There are specific mechanisms in patients presenting with functional upper gastrointestinal symptoms (UGI Sx). Among patients with UGI Sx, approximately equal proportions (25%) of patients have delayed gastric emptying (GE), reduced gastric accommodation (GA), both impaired GE and GA, or neither, presumably due to increased gastric or duodenal sensitivity. Treatments targeted to the underlying pathophysiology utilize prokinetics, gastric relaxants, or central neuromodulators. Similarly, specific mechanisms in patients presenting with functional lower gastrointestinal symptoms, especially with diarrhea or constipation, are recognized, including at least 30% of patients with functional constipation pelvic floor dyssynergia and 5% has colonic inertia (with neural or interstitial cells of Cajal loss in myenteric plexus); 25% of patients with diarrhea-predominant irritable bowel syndrome (IBSD) has evidence of bile acid diarrhea; and, depending on ethnicity, a varying proportion of patients has disaccharidase deficiency, and less often sucrose-isomaltase deficiency. Among patients with predominant pain or bloating, the role of fermentable oligosaccharides, disaccharides, monosaccharides and polyols should be considered. Personalization is applied through pharmacogenomics related to drug pharmacokinetics, specifically the role of CYP2D6, 2C19 and 3A4 in the use of drugs for treatment of patients with FGIDs. Single mutations or multiple genetic variants are relatively rare, with limited impact to date on the understanding or treatment of FGIDs. The role of mucosal gene expression in FGIDs, particularly in IBS-D, is the subject of ongoing research. In summary, the time for personalization of FGIDs, based on deep phenotyping, is here; pharmacogenomics is relevant in the use of central neuromodulators. There is still unclear impact of the role of genetics in the management of FGIDs.

Coimmobilization of ß-Agarase and α-Neoagarobiose Hydrolase for Enhancing the Production of 3,6-Anhydro-l-galactose.

Here we report a simple and efficient method to produce 3,6-anhydro-l-galactose (l-AHG) and agarotriose (AO3) in one step by a multienzyme system with the coimmobilized ß-agarase AgWH50B and α-neoagarobiose hydrolase K134D. K134D was obtained by AgaWH117 mutagenesis and showed improved thermal stability when immobilized via covalent bonds on functionalized magnetic nanoparticles. The obtained multienzyme biocatalyst was characterized by Fourier transform infrared spectroscopy (FTIR). Compared with free agarases, the coimmobilized agarases exhibited a relatively higher agarose-to-l-AHG conversion efficiency. The yield of l-AHG obtained with the coimmobilized agarases was 40.6%, which was 6.5% higher than that obtained with free agarases. After eight cycles, the multienzyme biocatalyst still preserved 46.4% of the initial activity. To the best of our knowledge, this is the first report where two different agarases were coimmobilized. These results demonstrated the feasibility of the new method to fabricate a new multienzyme system onto magnetic nanoparticles via covalent bonds to produce l-AHG.

Fusion of agarase and neoagarobiose hydrolase for mono-sugar production from agar.

In enzymatic saccharification of agar, endo- and exo-agarases together with neoagarobiose hydrolase (NABH) are important key enzymes for the sequential hydrolysis reactions. In this study, a bifunctional endo/exo-agarase was fused with NABH for production of mono-sugars (D-galactose and 3,6-anhydro-L-galactose) from agar using only one fusion enzyme. Two fusion enzymes with either bifunctional agarase (Sco3476) or NABH (Zg4663) at the N-terminus, Sco3476-Zg4663 (SZ) and Zg4663-Sco3476 (ZS), were constructed. Both fusion enzymes exhibited their optimal agarase and NABH activities at 40 and 35 °C, respectively. Fusions SZ and ZS enhanced the thermostability of the NABH activity, while only fusion SZ showed a slight enhancement in the NABH catalytic efficiency (K cat/K M) from 14.8 (mg/mL)-1 s-1 to 15.8 (mg/mL)-1 s-1. Saccharification of agar using fusion SZ resulted in 2-fold higher mono-sugar production and 3-fold lower neoagarobiose accumulation when compared to the physical mixture of Sco3476 and Zg4663. Therefore, this fusion has the potential to reduce enzyme production cost, decrease intermediate accumulation, and increase mono-sugar yield in agar saccharification.

Bacillus licheniformis trehalose-6-phosphate hydrolase structures suggest keys to substrate specificity.

Trehalose-6-phosphate hydrolase (TreA) belongs to glycoside hydrolase family 13 (GH13) and catalyzes the hydrolysis of trehalose 6-phosphate (T6P) to yield glucose and glucose 6-phosphate. The products of this reaction can be further metabolized by the energy-generating glycolytic pathway. Here, crystal structures of Bacillus licheniformis TreA (BlTreA) and its R201Q mutant complexed with p-nitrophenyl-α-D-glucopyranoside (R201Q-pPNG) are presented at 2.0 and 2.05 Å resolution, respectively. The overall structure of BlTreA is similar to those of other GH13 family enzymes. However, detailed structural comparisons revealed that the catalytic site of BlTreA contains a long loop that adopts a different conformation from those of other GH13 family members. Unlike the homologous regions of Bacillus cereus oligo-1,6-glucosidase (BcOgl) and Erwinia rhapontici isomaltulose synthase (NX-5), the surface potential of the BlTreA active site exhibits a largely positive charge contributed by the four basic residues His281, His282, Lys284 and Lys292. Mutation of these residues resulted in significant decreases in the enzymatic activity of BlTreA. Strikingly, the (281)HHLK(284) motif and Lys292 play critical roles in substrate discrimination by BlTreA.

Molecular characterization of a novel trehalose-6-phosphate hydrolase, TreA, from Bacillus licheniformis.

An unidentified Bacillus licheniformis trehalose-6-phosphate hydrolase (BlTreA) gene was cloned and heterologously expressed in Escherichia coli M15 cells. The over-expressed BlTreA was purified to apparent homogeneity by metal-affinity chromatography and its molecular mass was determined to be approximately 65.9 kDa. The temperature and pH optima for BlTreA were 30 °C and 8.0, respectively. The enzyme hydrolyzed p-nitrophenyl-α-d-glucopyranoside (pNPG) and trehalose-6-phosphate efficiently, but it was inactive toward five other p-nitrophenyl derivatives. Steady-state kinetics with pNPG showed that BlTreA had a K(M) value of 5.2mM and a k(cat) value of 30.2s(-1). Circular dichroism analysis revealed that the secondary structures of BlTreA did not altered by 5-10% acetone and 10-20% ethanol, whereas 5-10% SDS had a detrimental effect on the folding of the enzyme. Thermal unfolding of this enzyme was found to be highly irreversible. The native enzyme started to unfold beyond ~0.14 M guanidine hydrochloride (GdnHCl) and reached the unfolded intermediates, [GdnHCl](0.5,N-I) and [GdnHCl](0.5,I-U), at 1.02 and 2.24 M, respectively. BlTreA was unfolded completely by 8M urea with [urea](0.5,N-U) of 4.98 M, corresponding to a free energy change of 4.29 kcal/mol for the N→U process. Moreover, the enzyme was unfolded by GdnHCl through a reversible pathway and the refolding reaction exhibited an intermediate state. Taken together, the characterization data provide a foundation for the future structure-function studies of BlTreA, a typical member of glycoside hydrolase family 13.

Insulin deficiency induces abnormal increase in intestinal disaccharidase activities and expression under diabetic states, evidences from in vivo and in vitro study.

Structural and functional alterations in the gastrointestinal tract of diabetic patients are often accompanied by increase in absorption of intestinal glucose and activities of brush-border disaccharidases. The purpose of this study was to investigate the role of insulin in regulating intestinal disaccharidases using in vivo and in vitro experiments. Streptozotocin-induced diabetic rats and normal rats received protamine zinc insulin (10 IU/kg) subcutaneously twice daily for 5 weeks. Disaccharidase activities and sucrase-isomaltase (SI) complex protein and mRNA expression in intestinal regions were assessed. In addition, Caco-2 cells were cultured in medium containing glucose, insulin or insulin plus some pharmacological inhibitors for 7 days, disaccharidase activities, sucrase-isomaltase (SI) complex and Cdx2 mRNA levels were measured. The animal experiments showed that diabetes increased intestinal disaccharidase activities, accompanied by high mRNA and protein expression of SI complex. Insulin treatment reversed the increases induced by diabetes. The cellular results showed that insulin suppressed disaccharidase activities and down-regulated SI complex and Cdx2 mRNA expression in a concentration-dependent manner. The inhibitor of MAPK signal pathway PD-98059 blocked the suppression of disaccharidase activities and expression of SI complex and Cdx2 mRNA induced by insulin. In conclusion, insulin deficiency induces abnormal increase in intestinal disaccharidase activities and expression under diabetic states. Insulin plays an essential role in regulation disaccharidase activities and expression, at least in part, via the MAPK-dependent pathway.

Crystal structure of a key enzyme in the agarolytic pathway, α-neoagarobiose hydrolase from Saccharophagus degradans 2-40.

In agarolytic microorganisms, α-neoagarobiose hydrolase (NABH) is an essential enzyme to metabolize agar because it converts α-neoagarobiose (O-3,6-anhydro-alpha-l-galactopyranosyl-(1,3)-d-galactose) into fermentable monosaccharides (d-galactose and 3,6-anhydro-l-galactose) in the agarolytic pathway. NABH can be divided into two biological classes by its cellular location. Here, we describe a structure and function of cytosolic NABH from Saccharophagus degradans 2-40 in a native protein and d-galactose complex determined at 2.0 and 1.55 Å, respectively. The overall fold is organized in an N-terminal helical extension and a C-terminal five-bladed ß-propeller catalytic domain. The structure of the enzyme-ligand (d-galactose) complex predicts a +1 subsite in the substrate binding pocket. The structural features may provide insights for the evolution and classification of NABH in agarolytic pathways.

Increased thermal and osmotic stress resistance in Listeria monocytogenes 568 grown in the presence of trehalose due to inactivation of the phosphotrehalase-encoding gene treA.

The food-borne pathogen Listeria monocytogenes is a problem for food processors and consumers alike, as the organism is resistant to harsh environmental conditions and inimical barriers implemented to prevent the survival and/or growth of harmful bacteria. One mechanism by which listeriae mediate survival is through the accumulation of compatible solutes, such as proline, betaine and carnitine. In other bacteria, including Escherichia coli, the synthesis and accumulation of another compatible solute, trehalose, are known to aid in the survival of stressed cells. The objective of this research was to investigate trehalose metabolism in L. monocytogenes, where the sugar is thought to be transferred across the cytoplasmic membrane via a specific phosphoenolpyruvate phosphotransferase system and phosphorylation to trehalose-6-phosphate (T6P). The latter is subsequently broken down into glucose and glucose-6-phosphate by α,α-(1,1) phosphotrehalase, the putative product of the treA gene. Here we report on an isogenic treA mutant of L. monocytogenes 568 (568:ΔTreA) which, relative to the wild-type strain, displays increased tolerances to multiple stressors, including heat, high osmolarity, and desiccation. This is the first study to examine the putative trehalose operon in L. monocytogenes, and we demonstrate that lmo1254 (treA) in L. monocytogenes 568 indeed encodes a phosphotrehalase required for the hydrolysis of T6P. Disruption of the treA gene results in the accumulation of T6P which is subsequently dephosphorylated to trehalose in the cytosol, thereby contributing to the stress hardiness observed in the treA mutant. This study highlights the importance of compatible solutes for microbial survival in adverse environments.

Isolation of genes involved in biofilm formation of a Klebsiella pneumoniae strain causing pyogenic liver abscess.

BACKGROUND: Community-acquired pyogenic liver abscess (PLA) complicated with meningitis and endophthalmitis caused by Klebsiella pneumoniae is an emerging infectious disease. To investigate the mechanisms and effects of biofilm formation of K. pneumoniae causing PLA, microtiter plate assays were used to determine the levels of biofilm formed by K. pneumoniae clinical isolates and to screen for biofilm-altered mutants from a transposon mutant library of a K. pneumoniae PLA-associated strain. METHODOLOGY/PRINCIPAL FINDINGS: The biofilm formation of K. pneumoniae was examined by microtiter plate assay. Higher levels of biofilm formation were demonstrated by K. pneumoniae strains associated with PLA. A total of 23 biofilm-decreased mutants and 4 biofilm-increased mutants were identified. Among these mutants, a biofilm-decreased treC mutant displayed less mucoviscosity and produced less capsular polysaccharide (CPS), whereas a biofilm-increased sugE mutant displayed higher mucoviscosity and produced more CPS. The biofilm phenotypes of treC and sugE mutants also were confirmed by glass slide culture. Deletion of treC, which encodes trehalose-6-phosphate hydrolase, impaired bacterial trehalose utilization. Addition of glucose to the culture medium restored the capsule production and biofilm formation in the treC mutant. Transcriptional profile analysis suggested that the increase of CPS production in ΔsugE may reflect elevated cps gene expression (upregulated through rmpA) in combination with increased treC expression. In vivo competition assays demonstrated that the treC mutant strain was attenuated in competitiveness during intragastric infection in mice. CONCLUSIONS/SIGNIFICANCE: Genes important for biofilm formation by K. pneumoniae PLA strain were identified using an in vitro assay. Among the identified genes, treC and sugE affect biofilm formation by modulating CPS production. The importance of treC in gastrointestinal tract colonization suggests that biofilm formation contributes to the establishment and persistence of K. pneumoniae infection.

The -13914G>A variant upstream of the lactase gene (LCT) is associated with lactase persistence/non-persistence.

BACKGROUND: Adult-type hypolactasia (lactase non-persistence) is a common cause of gastrointestinal symptoms. Several DNA sequence variants have been identified for the lactase-persistence/non-persistence (LP/LNP), the most common being the C to T residing -13910 bp upstream of the lactase gene (LCT). We have analysed sequence variants of LP/LNP in subjects originating from Northern Russia. METHODS: A total of 148 subjects with gastrointestinal complaints were genotyped covering about 400 bp around the -13910C/T variant using direct PCR-sequencing. All patients were interviewed about milk-related symptoms using the questionnaire. Disaccharidase activities were measured from intestinal biopsy specimens of the index person. RESULTS: The prevalence of the -13910C/C genotype among 148 patients was 28.4%. A G to A variant residing 13914 bp upstream from the LCT gene (-13914G>A) was identified in one participant carrying the -13910C/C genotype. In two biopsy specimens her lactase activity was above the generally accepted cut off level for adult-type hypolactasia, 10U/g protein. Three other family members also carried the -13914G>A genotype. Among eight family members five had the LNP genotype -13910C/C. CONCLUSION: A rare variant G to A residing 13914 bp upstream of the LCT gene was identified in a subject carrying the more frequent variant -13910C/C. The -13914G>A variant in heterozygous state was associated with increased lactase activity, suggesting that the increased lactase activity is most likely to be associated with the -13914G>A variant. Further studies need to be done to confirm the functional role of this variant.

[Effect of Weichang'an pill on intestinal digestion enzymes and the AQP4 concentration in proximal colon in IBS-D rats].

OBJECTIVE: To investigate the influence of Weichang'an pill on the treatment of diarrhea-predominant irritable bowel syndrome (IBS-D) in model rats. METHOD: Animal model of compound diarrhea was induced by a lactose enriched diet in the Wistar rat, combining with restraint stress. Twenty four female Wistar rats were randomly divided into normal group, model group and 60 mg x kg(-1) x d(-1) Weichang'an pill group. The rate of weight increase, the incubation period of diarrhea and the diarrhea index were observed. Then 45 female Wistar rats randomly divided into five groups: control group, model group and Weichang'an pill groups of high, medium and low doses (80, 60, 40 mg x kg(-1) x d(-1)). The indexes of thymus and spleen were calculated. The activities of LDH, MDH and disaccharidase in intestinal organization were inspected. Serum D-xylose content and the AQP4 concentration in proximal colon were detected. RESULT: After taking Weichang'an pill for 4 days, the rate of weight increase in Weichang'an pill group was higher than the model group's. While the rate of diarrhea was lower significantly. So the best cycle of taking medicine was 4 days. The indexes of thymus and spleen of model group were decreased than that of control group. And the activities of LDH, MDH and disaccharidase in intestinal organization were also decreased. But the AQP4 concentration in proximal colon was increased. Compared with the model group, the indexes of thymus and spleen increased remarkably in the group of medium doses. Meanwhile, the activities of LDH, MDH and disaccharidase increased. But the AQP4 concentration didn't change. CONCLUSION: Weichang'an pill has the effect of antidiarrhea. It can adjust the sugar's catabolism through increasing the activity of intestinal digestive ferment.

Crystallization and preliminary X-ray analysis of neoagarobiose hydrolase from Saccharophagus degradans 2-40.

Many agarolytic bacteria degrade agar polysaccharide into the disaccharide unit neoagarobiose [O-3,6-anhydro-alpha-L-galactopyranosyl-(1-->3)-D-galactose] using various beta-agarases. Neoagarobiose hydrolase is an enzyme that acts on the alpha-1,3 linkage in neoagarobiose to yield D-galactose and 3,6-anhydro-L-galactose. This activity is essential in both the metabolism of agar by agarolytic bacteria and the production of fermentable sugars from agar biomass for bioenergy production. Neoagarobiose hydrolase from the marine bacterium Saccharophagus degradans 2-40 was overexpressed in Escherichia coli and crystallized in the monoclinic space group C2, with unit-cell parameters a = 129.83, b = 76.81, c = 90.11 A, beta = 101.86 degrees . The crystals diffracted to 1.98 A resolution and possibly contains two molecules in the asymmetric unit.

Computational and experimental analyses of furcatin hydrolase for substrate specificity studies of disaccharide-specific glycosidases.

Disaccharide-specific glycosidases (diglycosidases) are unique glycoside hydrolases, as their substrate specificities differ from those of monosaccharide-specific beta-glycosidases (monoglycosidases), in spite of similarities in their sequences and reaction mechanisms. Diglycosidases selectively hydrolyse the beta-glycosidic bond between glycone and aglycone of disaccharide glycosides, but do not cleave the bond between two saccharides, and barely hydrolyse monosaccharide glycosides. We analysed the substrate recognition mechanisms of diglycosidases by computational and experimental methods, using furcatin hydrolase (FH) (EC 3.2.1.161) derived from Viburnum furcatum. Amino acid sequence comparisons and model structure building revealed two residues, Ala419 and Ser504 of FH, as candidates determining the substrate specificity. These residues were specifically conserved in the diglycosidases. The model structure suggested that Ala419 is involved in the aglycone recognition, whereas Ser504 recognizes the external saccharide of the glycone. Mutations at these sites drastically decreased the diglycosidase activity. The mechanism by which the diglycosidases acquired their substrate specificity is discussed, based on these observations.

The sim operon facilitates the transport and metabolism of sucrose isomers in Lactobacillus casei ATCC 334.

Inspection of the genome sequence of Lactobacillus casei ATCC 334 revealed two operons that might dissimilate the five isomers of sucrose. To test this hypothesis, cells of L. casei ATCC 334 were grown in a defined medium supplemented with various sugars, including each of the five isomeric disaccharides. Extracts prepared from cells grown on the sucrose isomers contained high levels of two polypeptides with M(r)s of approximately 50,000 and approximately 17,500. Neither protein was present in cells grown on glucose, maltose or sucrose. Proteomic, enzymatic, and Western blot analyses identified the approximately 50-kDa protein as an NAD(+)- and metal ion-dependent phospho-alpha-glucosidase. The oligomeric enzyme was purified, and a catalytic mechanism is proposed. The smaller polypeptide represented an EIIA component of the phosphoenolpyruvate-dependent sugar phosphotransferase system. Phospho-alpha-glucosidase and EIIA are encoded by genes at the LSEI_0369 (simA) and LSEI_0374 (simF) loci, respectively, in a block of seven genes comprising the sucrose isomer metabolism (sim) operon. Northern blot analyses provided evidence that three mRNA transcripts were up-regulated during logarithmic growth of L. casei ATCC 334 on sucrose isomers. Internal simA and simF gene probes hybridized to approximately 1.5- and approximately 1.3-kb transcripts, respectively. A 6.8-kb mRNA transcript was detected by both probes, which was indicative of cotranscription of the entire sim operon.

Correlation of intestinal disaccharidase activities with the C/T-13910 variant and age.

AIM: To correlate the C/T(-13910) variant, associated with lactase persistence/non-persistence (adult-type hypolactasia) trait, with intestinal disaccharidase activities in different age groups of the adult population. METHODS: Intestinal biopsies were obtained from 222 adults aged 18 to 83 years undergoing upper gastrointestinal endoscopy because of unspecified abdominal complaints. The biopsies were assayed for lactase, sucrase and maltase activities and genotyped for the C/T(-13910) variant using PCR-minisequencing. RESULTS: There was a significant correlation between lactase activity and the C/T(-13910) variant (P < 0.00001). The mean level of lactase activity among subjects with C/C(-13910) genotype was 6.86 +/- 0.35 U/g, with C/T(-13910) genotype 37.8 +/- 1.4 U/g, and with T/T(-13910) genotype 57.6 +/- 2.4 U/g protein, showing a trimodal distribution of this enzyme activity. Significant differences were also observed in maltase activities among individuals with different C/T(-13910) genotypes (P = 0.005). In contrast, in sucrase activity, no significant differences emerged between the C/T(-13910) genotypes (P = 0.14). There were no statistical differences in lactase (P = 0.84), sucrase (P = 0.18), or maltase activity (P = 0.24) among different age groups. In the majority (> 84%) of the patients with the C/C(-13910) genotype associated with lactase non-persistence, the lactase activity was less than 10 U/g protein. CONCLUSION: Our study demonstrates a statistically significant correlation between the C/T(-13910) genotype and lactase activity and this correlation is not affected by age in adults but the cut-off value of 20 U/g protein used for the diagnosis of lactase non-persistence might be too high.

Disaccharide digestion: clinical and molecular aspects.

Sugars normally are absorbed in the small intestine. When carbohydrates are malabsorbed, the osmotic load produced by the high amount of low molecular weight sugars and partially digested starches in the small intestine can cause symptoms of intestinal distention, rapid peristalsis, and diarrhea. Colonic bacteria normally metabolize proximally malabsorbed dietary carbohydrate through fermentation to small fatty acids and gases (ie, hydrogen, methane, and carbon dioxide). When present in large amounts, the malabsorbed sugars and starches can be excreted in the stool. Sugar intolerance is the presence of abdominal symptoms related to the proximal or distal malabsorption of dietary carbohydrates. The symptoms consist of meal-related abdominal cramps and distention, increased flatulence, borborygmus, and diarrhea. Infants and young children with carbohydrate malabsorption show more intense symptoms than adults; the passage of undigested carbohydrates through the colon is more rapid and is associated with detectable carbohydrates in copious watery acid stools. Dehydration often follows feeding of the offending sugar. In this review we present the clinical and current molecular aspects of disaccharidase digestion.

Characterization of the tre locus and analysis of trehalose cryoprotection in Lactobacillus acidophilus NCFM.

Freezing and lyophilization are common methods used for preservation and storage of microorganisms during the production of concentrated starter cultures destined for industrial fermentations or product formulations. The compatible solute trehalose has been widely reported to protect bacterial, yeast and animal cells against a variety of environmental stresses, particularly freezing and dehydration. Analysis of the Lactobacillus acidophilus NCFM genome revealed a putative trehalose utilization locus consisting of a transcriptional regulator, treR; a trehalose phosphoenolpyruvate transferase system (PTS) transporter, treB; and a trehalose-6-phosphate hydrolase, treC. The objective of this study was to characterize the tre locus in L. acidophilus and determine whether or not intracellular uptake of trehalose contributes to cryoprotection. Cells subjected to repeated freezing and thawing cycles were monitored for survival in the presence of various concentrations of trehalose. At 20% trehalose a 2-log increase in survival was observed. The trehalose PTS transporter and trehalose hydrolase were disrupted by targeted plasmid insertions. The resulting mutants were unable to grow on trehalose, indicating that both trehalose transport into the cell via a PTS and hydrolysis via a trehalose-6-phosphate hydrolase were necessary for trehalose fermentation. Trehalose uptake was found to be significantly reduced in the transporter mutant but unaffected in the hydrolase mutant. Additionally, the cryoprotective effect of trehalose was reduced in these mutants, suggesting that intracellular transport and hydrolysis contribute significantly to cryoprotection.