Characterization of Neoagarooligosaccharide Hydrolase BpGH117 from a Human Gut Bacterium Bacteroides plebeius.

α-Neoagarobiose (NAB)/neoagarooligosaccharide (NAO) hydrolase plays an important role as an exo-acting 3,6-anhydro-α-(1,3)-L-galactosidase in agarose utilization. Agarose is an abundant polysaccharide found in red seaweeds, comprising 3,6-anhydro-L-galactose (AHG) and D-galactose residues. Unlike agarose degradation, which has been reported in marine microbes, recent metagenomic analysis of Bacteroides plebeius, a human gut bacterium, revealed the presence of genes encoding enzymes involved in agarose degradation, including α-NAB/NAO hydrolase. Among the agarolytic enzymes, BpGH117 has been partially characterized. Here, we characterized the exo-acting α-NAB/NAO hydrolase BpGH117, originating from B. plebeius. The optimal temperature and pH for His-tagged BpGH117 activity were 35 °C and 9.0, respectively, indicative of its unique origin. His-tagged BpGH117 was thermostable up to 35 °C, and the enzyme activity was maintained at 80% of the initial activity at a pre-incubation temperature of 40 °C for 120 min. Km and Vmax values for NAB were 30.22 mM and 54.84 U/mg, respectively, and kcat/Km was 2.65 s-1 mM-1. These results suggest that His-tagged BpGH117 can be used for producing bioactive products such as AHG and agarotriose from agarose efficiently.

Expression of sodium-glucose co-transporter and brush border disaccharidases in Giardia lamblia infected rat intestine.

The absorption of D-glucose and brush border membrane disaccharidases in the intestine of rat during infection by Giardia lamblia has been studied. The level of mRNA encoding Na+/glucose co-transporter (SGLT1) and brush border sucrase and lactase activities were also analyzed. At the peak of infection, i.e, day 7, 11 and 15 post-infection, there was a marked decrease in the signal of 4.5 kb and 2.8 kb mRNAs encoding SGTL1 compared to the controls. A similar decrease in sucrase and lactase mRNA's (6.5 kb and 6.8 kb respectively) was also observed under these conditions. This corresponds to observed decrease in the rate of Na(+)-dependent D-glucose uptake and low activities of brush border sucrase and lactase under these conditions. There was no change in Na(+)-independent D-glucose uptake in giardia infected rat intestine. These findings suggest that the down regulation of the expression of SGLT1 and brush border sucrase and lactase activities may be responsible for the observed malabsorption in G. lamblia infection.

Induction of RpoS-dependent functions in glucose-limited continuous culture: what level of nutrient limitation induces the stationary phase of Escherichia coli?

treA and osmY expression and RpoS protein levels were investigated in glucose-limited continuous culture. The level of induction of these stationary-phase markers became as high during growth at a D of 0.1 to 0.2 h(-1) as in carbon-starved batch cultures but only in rpoS+ bacteria. The stress protectant trehalose was actually produced at higher levels at low growth rates than in stationary-phase cultures. The pattern of induction of RpoS-dependent activities could be separated from those regulated by cyclic AMP (cAMP) or endoinduction, and the induction occurred at extreme glucose limitation. Escherichia coli turns to a protective stationary-phase response when nutrient levels fall below approximately 10(-7) M glucose, which is insufficient to saturate scavenger transporters regulated by cAMP plus endoinducers, and this response is optimally expressed at 10(-6) M glucose. The high-level induction of protective functions also explains the maintenance energy requirement of bacterial growth at low dilution rates.

The prenatal development and glucocorticoid control of brush-border hydrolases in the pig small intestine.

The development of brush-border enzymes and the possible regulatory role of cortisol were investigated in the small intestine of the fetal and neonatal pig. With the sows under pentobarbitone anesthesia, osmotic minipumps containing either saline or cortisol were inserted s.c. into 25 fetuses from 10 pregnant sows (82-96 d gestation). Six d later, the infused fetuses were removed by cesarean section and samples of the proximal, middle, and distal intestine taken for analysis. Samples were also obtained from 48 piglets that did not undergo an operation (controls) and that were removed at intervals from 82 d gestation until term (114 +/- 2 d). In the proximal and middle intestine, the mean levels of lactase-phlorizin hydrolase (EC 3.2.1.23-62), maltaseglucoamylase (EC 3.2.1.20), aminopeptidase N (EC 3.4.11.2), and aminopeptidase A (EC 3.4.11.7) increased during the last 10-15 d before term, correlated positively with log10 plasma cortisol values, and were higher in cortisol-infused than in saline-infused fetuses (p < 0.05). Activity of sucrase-isomaltase (EC 3.2.1.48-10) was low in fetal pigs, and this enzyme and dipeptidyl peptidase IV (EC 3.4.14.5) were not significantly affected by fetal age or exogenous cortisol. Maltase (EC 3.2.1.48-10 and EC 3.2.1.20) activity was significantly decreased in the middle and distal intestine of cortisol-infused fetuses. The results suggest that the prepartum rise in endogenous cortisol secretion stimulates the prenatal expression of certain brush-border enzymes in the pig small intestine at this critical time. However, the effects of cortisol on the developing intestine were highly idiosyncratic for particular enzymes and intestinal regions.

Characterization of intestinal gamma-glucoamylase deficiency in CBA/Ca mice.

In previous work, we found that CBA/Ca mice display only 20% of the maltase activity present in other mouse strains. In this study, we characterized more fully the maltase deficiency in CBA/Ca mice. Virtually all of the intestinal maltase activity of CBA/Ca mice was inactivated at 50 degrees C, indicating that it was due only to the sucrase-isomaltase complex. High-performance liquid chromatographic analysis revealed that CBA/Ca mice had undetectable maltase activity displaying the molecular mass characteristic of murine gamma-glucoamylase (gamma-GA) (530 kDa). Gel electrophoretic analysis confirmed that CBA/Ca mice lacked maltase activity with molecular mass of 530 kDa corresponding to gamma-GA. Two-dimensional electrophoretic analysis revealed that the gamma-GA deficiency in CBA/Ca mice was due to the failure to synthesize the enzyme and not to the synthesis of an inactive protein. gamma-GA maltase activity could not be induced in CBA/Ca mice by a diet rich in starch, whereas the activity of other disaccharidases were readily increased. gamma-GA-deficient CBA/Ca mice appear to lack any gross metabolic abnormality resulting from this defect.

Secretin induces precocious cessation of intestinal macromolecular transmission and maltase development in the suckling rat.

The effect of endogenous and exogenous secretin on the intestinal closure of macromolecular transmission and maltase development were investigated in suckling rats. The increase in secretin-like immunoreactivity with orogastric infusion of HCl solution in 14 day-old pups was confirmed. By the repeated oral administration of HCl, pancreatic hyperplasia, suppression of intestinal bovine IgG transmission, and precocious induction of maltase activity were occurred. By repeated subcutaneous injection of secretin, dose-dependent suppression of intestinal bovine IgG absorption and increase in maltase activity were observed. The suppression of IgG absorption with secretin treatment was also observed in adrenalectomized pups. These results suggest that secretin affects the maturation of gastrointestinal function in suckling rats.

Use of inducible disaccharidases to assess the importance of different carbohydrate sources for Bacteroides ovatus growing in the intestinal tracts of germfree mice.

Patterns of disaccharidase expression were used to determine which polysaccharides were the major sources of carbohydrate for Bacteroides ovatus growing in the intestinal tracts of monocolonized germfree mice. Results indicate that B. ovatus grows on a variety of different carbohydrates, which are present in low concentrations, rather than relying on one type of carbohydrate as the major carbohydrate source.

Disaccharidase activities in small intestine of rotavirus-infected suckling mice: a histochemical study.

A histochemical study of the time course of the appearance and location of lactase and alpha-glucosidase (used to detect sucrase and maltase) activities was carried out on control and rotavirus-infected mice from 7 to 14 days old. The overall pattern of enzyme activity was in agreement with previous quantitative studies on the activities of these enzymes. No evidence was obtained to support the idea that lactase deficiency was the result of repopulation of villi (denuded of lactase-producing villus cells) with immature lactase-negative cells. Low lactase activity was more likely to reflect profound changes in metabolically crippled cells, and recovery of lactase activity with recovery of normal metabolic functions. The location of enzyme activity to brush border regions rather than the cytoplasm of villus enterocytes enhances the significance of previous quantitative studies on these enzymes. The timing and duration of diminished lactase activities were such that they were unlikely to cause the induction or perpetuation of diarrhea in murine rotavirus diarrhea. The appearance in infected animals of alpha-glucosidase 3 days earlier than normal indicates that, in addition to reversible changes seen with lactase, developmental changes were accelerated that affected both crypt and villus cells.

Effect of sympathectomy on ontogeny of small intestinal disaccharidases in the rat.

In the present study the effect of chemical sympathectomy on the development of small intestinal enzymes in the rat was analyzed. Eight doses of guanethidine sulfate were administered subcutaneously every 48 h to 34 newborn rats, starting at birth. The last dose was given at 14 days of age. Twenty-two littermates served as controls. Intestinal lactase, maltase, sucrase, and alkaline phosphatase were determined at 15, 17, 20, 23, and 25 days of age. Sympathectomy was demonstrated by reduction of the number of perikarya in the superior cervical ganglia in treated rats as compared with control rats. A normal developmental pattern of activities of the disaccharidases in the small intestine was observed in both groups. The activity of alkaline phosphatase was significantly lower (p less than 0.01) in the 15- and 17-day-old treated animals.

Enterocyte protein processing and synthesis.

Until recently, structural information regarding large brush border proteins was obtained largely by protein purification and protein chemistry. Molecular biology has added the techniques of cell-free translation and cDNA cloning to provide additional information. In addition, the regulation of protein synthesis was largely deduced by the use of inhibitors of protein or nucleic acid synthesis. The ability to assess mRNA content by cell-free translation or by hybridization with isolated cDNA has expanded these studies. Moreover, polysomal isolation and organ explants can now be used to determine synthetic and secretion rates directly. Future research in these areas will depend upon the isolation of cDNA clones encoding specific enterocyte proteins to simplify structural analysis, as well as the use of intestinal cell lines to simplify measurement of synthetic rates and protein processing.

Selective expression of brush border hydrolases by mouse Peyer's patch and jejunal villus enterocytes.

Developmental profiles describing the expression of lactase, alpha-glucosidase, and alkaline phosphatase activities have been determined quantitatively in mouse jejunal enterocytes during migration over villi and Peyer's patch lymphoid tissue. The predicted maximal lactase and alpha-glucosidase activities expressed by enterocytes migrating over Peyer's patch follicles were about one-quarter and one-half of values found in control villi. Alkaline phosphatase activity was, on the other hand, one third greater in Peyer's patch compared with villus enterocytes. Expression of lactase and alpha-glucosidase activities was initially less in enterocytes migrating along interfollicular compared with control villi. Subsequent increase in hydrolase activities occurred during the later stages of enterocyte migration over interfollicular villi. Lactase activity in athymic mice Peyer's patch enterocytes was identical to that recorded for control mice. The corresponding value for villus lactase was, however, only half that found in control tissue. Factors produced locally in lymphoid follicles are probably responsible for selective effects on enterocyte differentiation.

Effect of various sugars on the induction of chick embryonic intestinal disaccharidases in the organ culture system.

Disaccharidases activities in 20-day-old chick embryonic intestine were induced by the addition of sucrose, maltose, fructose and glucose to the culture medium. However, maltitol, which cannot be digested by intestinal enzymes, showed no effect on the induction of disaccharidase activity. Kinetic study of the enzymes demonstrated that the maximum velocity (Vmax) and the Michaelis constant (Km) of sucrose induced disaccharidases activities of the explants showed changes similar to those observed in the chick of same developmental stage in vivo. Namely, Vmax values of sucrase and maltase were increased. Km values of sucrase did not change, but that of maltase showed a significant decrease during development.

Expression of alpha-D-mannosidase in man-hamster somatic cell hybrids.

Two types of alpha-D-mannosidase isozymes are present in human white blood cells, human diploid fibroblasts, and HeLa cells. One of these (the S isozyme) constitutes the major alpha-D-mannosidase of the human cells, has a pH optimum of 4.4, and is associated with lysosomes. The other (the F isozyme) is most active at pH 6, is acid labile, and is located in the soluble portion of the cytoplasm. The expression of human lysosomal alpha-D-mannosidase was examined in man-hamster hybrid clones, and was found to be concordant with that of phosphohexose isomerase in 54 of 55 primary clones. A locus specifying human lysosomal alpha-D-mannosidase has therfore been assigned to chromosome 19.

alpha-Mannosidase and mannanase of some wood-rotting fungi.

Cultivation media from 11 wood-rotting fungi contained alpha-mannosidase and mannanase activity, alpha-Mannosidase was studied in more detail in Phellinus abietis and mannanase was studied more intimately in basidiomycetes Phellinus abietis, Trametes sanguinea and Pholiota aurivella. Suitable cultivation conditions and optimum conditions for the production of alpha-mannosidase and mannanase were determined. Both enzymes are constitutive; mannanase is extracellular, alpha-mannosidase was found in both mycelium and cultivation medium.

Induction of chick embryonic intestinal disaccharidases by hydrocortisone and sucrose in the organ culture system.

The effect of hydrocortisone and sucrose on the development of chick intestinal disaccharidases was studied using the organ culture system. When intestines of 15- and 17-day-old embryos were cultured in the presence of hydrocortisone, there was significant enhancement of disaccharidases activity compared with the control. However, there was no effect in the 20-day embryonic intestines. On the other hand, the disaccharidase activity of cultured intestines from 20-day-old chick embryos were significantly stimulated by the addition of sucrose. The observed increase in disaccharidase activity induced by the administration of hydrocortisone in 17-day-old embryos in vitro was sensitive to actinomycin D and cycloheximide. The activity induced by the administration of sucrose in 20-day-old embryos in vitro was sensitive to cycloheximide but insensitive to actinomycin D.

The structural gene for alpha-mannosidase-1 in Dictyostellium discoideum.

We have isolated 4 independent mutations affecting alpha-mannosidase-1, a developmentally regulated activity in Dictyrostelium discoideum. Three of these result in a thermolabile alpha-mannosidase-1 activity. One mutation also affects the substrate affinity (Km) of the activity. In diploids these mutations show a gene dosage effect and are all alleles. The structural gene for alpha-mannosidase-1, as defined by these mutations, defines a new linkage group, linkage group VI. alpha-mammosidase 1 is probably a homopolymer with subunits of 54,000 daltons. We have also mapped two temperature-sensitive-for-growth mutations onto two previously defined linkage groups.