Comparative Genomics Analysis of Streptococcus Isolates from the Human Small Intestine Reveals their Adaptation to a Highly Dynamic Ecosystem

The human small-intestinal microbiota is characterised by relatively large and dynamic Streptococcus populations. In this study, genome sequences of small-intestinal streptococci from S. mitis, S. bovis, and S. salivarius species-groups were determined and compared with those from 58 Streptococcus strains in public databases. The Streptococcus pangenome consists of 12,403 orthologous groups of which 574 are shared among all sequenced streptococci and are defined as the Streptococcus core genome. Genome mining of the small-intestinal streptococci focused on functions playing an important role in the interaction of these streptococci in the small-intestinal ecosystem, including natural competence and nutrient-transport and metabolism. Analysis of the small-intestinal Streptococcus genomes predicts a high capacity to synthesize amino acids and various vitamins as well as substantial divergence in their carbohydrate transport and metabolic capacities, which is in agreement with observed physiological differences between these Streptococcus strains. Gene-specific PCR-strategies enabled evaluation of conservation of Streptococcus populations in intestinal samples from different human individuals, revealing that the S. salivarius strains were frequently detected in the small-intestine microbiota, supporting the representative value of the genomes provided in this study. Finally, the Streptococcus genomes allow prediction of the effect of dietary substances on Streptococcus population dynamics in the human small-intestine.     

Morphologic changes in the liver as affected by small intestine fistulae

Rat experiments have revealed the relationship between the liver pathomorphosis and the small-intestinal fistulae situated at various levels (high, low, or medium-height). Vacuolar dystrophy of the hepatocytes was found to be the major pathology in fistulae of the small intestine.     

Intestinal glycosidase activities in one adult and two suckling echidnas: absence of a neutral lactase (beta-D-galactosidase)

The activities of various glycosidases in homogenates of the small-intestinal mucosa of one adult and two suckling echidnas, Tachyglossus aculeatus, were investigated. The activities of lactase (beta-D-galactosidase), beta-N-acetylglucosaminidase, neuraminidase and alpha-L-fucosidase were higher in the sucklings than in the adult animal. Maltase and isomaltase activities were lower. Sucrase and cellobiase activities were absent or present in trace amounts only. The lactase activity had a pH optimum of 4.0-4.5, was predominantly in the soluble fraction following ultracentrifugation and was inhibited by p-chloromercuribenzene sulfonate, suggesting that it was due to a lysosomal acid beta-galactosidase and not a brush-border neutral lactase. The maltase activity of the sucklings also had the characteristics predominantly of a lysosomal acid hydrolase. It is proposed that in suckling echidnas, the oligosaccharides (mainly neuraminyllactose and fucosyllactose) of the mother's milk are digested intracellularly by lysosomal enzymes, rather than at the brush border, of the epithelial cells of the small-intestinal mucosa.     

A cytokinetic study of small-intestinal and colonic mucosa after resection of 70% of the small intestine

Pulse-labelling with tritiated thymidine and a fraction of labelled mitoses experiments have been performed in order to investigate the proliferative changes induced at various sites in the hyperplastic small-intestinal mucosa of rats previously subjected to resection of 70% of the small intestine. Proliferative activity in the colon was also studied. In the distal ileum there is a significant reduction in cell cycle time (Tc) of cells at all levels within the crypt and the growth fraction falls. In the jejunum and proximal ileum the crypts contain an increased number of proliferating cells, but as the size of the maturation zone is also increased, there is no significant alteration in the relative number of proliferating cells per crypt. Nor does the distribution of proliferating cells in these crypts seem to alter. There is no general reduction in Tc at these sites, but there does appear to be a significant reduction in Tc on the part of the cells in the stem-cell zone at the crypt base. In neither proximal nor distal colon was there any significant proliferative change apparent after small-intestinal resection.     

A Cytokinetic Study of Small-Intestinal and Colonic Mucosa After Resection of 70% of the Small Intestine

Abstract. Pulse-labelling with tritiated thymidine and a fraction of labelled mitoses experiments have been performed in order to investigate the proliferative changes induced at various sites in the hyperplastic small-intestinal mucosa of rats previously subjected to resection of 70% of the small intestine. Proliferative activity in the colon was also studied.
In the distal ileum there is a significant reduction in cell cycle time (T_c) of cells at all levels within the crypt and the growth fraction falls. In the jejunum and proximal ileum the crypts contain an increased number of proliferating cells, but as the size of the maturation zone is also increased, there is no significant alteration in the relative number of proliferating cells per crypt. Nor does the distribution of proliferating cells in these crypts seem to alter. There is no general reduction in T_c at these sites, but there does appear to be a significant reduction in T_c on the part of the cells in the stem-cell zone at the crypt base.
In neither proximal nor distal colon was there any significant proliferative change apparent after small-intestinal resection.     

Antigenic and structural features of goblet-cell mucin of human small intestine.

With the use of a newly developed solid-phase radioimmunoassay method, the major antigenic determinants of human small-intestinal goblet-cell mucin were investigated and related to the overall tertiary structure of the mucin. Preliminary hapten inhibition studies with various oligosaccharides of known sequence and structure suggested that the determinants did not reside in carbohydrate. Exhaustive thiol reduction, however, almost abolished antigenicity, caused breakdown of the mucin into small heterogeneous glycopeptides, and liberated a 'link' peptide of Mr 118000. Western 'blots' of reduced mucin from polyacrylamide gels on to nitrocellulose sheets showed that a small amount of residual antigenicity remained in large-Mr glycopeptides (Mr greater than 200000). The 'link' peptide was not antigenic. Timed Pronase digestion of native mucin resulted in a progressive loss of antigenic determinants. Gel electrophoresis revealed that after 8h of digestion the 118000-Mr peptide had disappeared, whereas antigenicity, which was confined to large-Mr glycopeptides, was destroyed much more slowly with time (70% by 24h, 100% by 72h). Despite the loss of antigenicity, 72h-Pronase-digested glycopeptides retained all of the carbohydrate of the native mucin. Therefore the antibody to human small-intestinal mucin appears to recognize a 'naked' (non-glycosylated and Pronase-susceptible) peptide region(s) of mucin glycopeptides. For full antigenicity, however, disulphide bonds are required to stabilize a specific three-dimensional configuration of the 'naked' region.     

In vitro total-gas, CH4, H2, volatile fatty acid, and lactate kinetics studies on luminal contents from the small intestine, cecum, and colon of the pig.

Two experiments were conducted to assess differences in fermentative activities of digesta obtained from various regions of the pig gastrointestinal tract. In experiment 1, the contents of small intestines, ceca, and colons of 110-kg pigs were collected, diluted twofold, and incubated for 2 h at 37 degrees C. In experiment 2, colonic samples from 16,100-kg pigs were similarly treated, except that the incubation period was 5 h. Total gas (gas pressure), CH4, H2, lactate, formate, acetate, propionate, butyrate, valerate, and isovalerate were measured in experiment 1. Only the gas variables were measured in experiment 2. Statistically significant differences (P greater than 0.05) were not observed among the gas production rate estimates across the small-intestinal, cecal, and colonic regions in experiment 1. Furthermore, all the small-intestinal samples and half the cecal samples assayed in experiment 1 were nonmethanogenic. The mean methanogenic and total-gas production rate estimates for the colonic samples in experiment 1 were 0.052 ml g of wet contents-1 h-1 and 1.7 ml of total gas g of wet contents-1 h-1, respectively. No differences in the methanogenic rate estimates were detected between the proximal, middle, and distal thirds of the pig colons (P greater than 0.05). The volatile fatty acid and lactate molar percentages measured in experiment 1 were consistent with previously published observations. Hydrogen accumulated to the greatest extent (7 microM on average) in the in vitro incubations of small-intestinal contents, whereas the H2 concentrations ranged from 0.5 to 1 microM for the incubated cecal and colonic samples in experiment 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

A recombinant matriptase causes an increase in caspase-3 activity in a small-intestinal epithelial IEC-6 line cultured on fibronectin-coated plates

Matriptase is an epithelial-derived type-II transmembrane serine protease. This protease is expressed prominently in the villus tip of small-intestinal epithelia at which senescent cells undergo shedding and/or apoptosis. The basement membrane of epithelial cells, including small-intestinal epithelial cells, contains extracellular matrix (ECM) proteins such as fibronectin and laminin. We found previously that high concentrations of a recombinant matriptase catalytic domain (r-MatCD) (e.g. 1 μM) caused an increased detachment of and increases in the activity of apoptotic effector caspase-3 in a rat small-intestinal epithelial IEC-6 line cultured on laminin-coated plates and proposed that at sites with its high level of expression, matriptase contributes to promoting shedding and/or detachment-induced death of epithelial cells through a mechanism mediating loss of cell-ECM adhesion. In this study, we found that even without increasing cell detachment, a high concentration of r-MatCD causes an increase in caspase-3 activity in IEC-6 cells cultured on fibronectin-coated plates, suggesting that the recombinant matriptase can cause apoptosis by a mechanism unrelated to cell detachment. Also, r-MatCD-treated IEC-6 cells on fibronectin were found to display spindle-like morphological changes. We suggest that r-MatCD causes apoptosis of IEC-6 on fibronectin by a mechanism involving the disruption of cell integrity.     

Regulatory role for l-arginine in the utilization of amino acids by pig small-intestinal bacteria

We recently reported that bacteria from the pig small intestine rapidly utilize and metabolize amino acids (AA). This study investigated the effect of L-arginine on the utilization of AA by pure bacterial strains (Streptococcus sp., Escherichia coli and Klebsiella sp.) and mixed bacterial cultures derived from the pig small intestine. Bacteria were incubated at 37°C for 3 h in anaerobic AA media containing 0-5 mmol/L of arginine to determine the effect of arginine on the bacterial utilization of AA. Amino acids in the medium plus cell extracts were analyzed by high-performance liquid chromatography. Results indicated concentration-dependent increases in the bacterial utilization of arginine and altered fluxes of arginine into ornithine and citrulline in the bacteria. Net glutamine utilization increased in pure bacterial strains with increased concentrations of arginine. With the addition of arginine, net utilization of threonine, glycine, phenylalanine and branched-chain AA increased (P<0.05) in Streptococcus sp. and Klebsiella sp., but decreased in E. coli. Net utilization of lysine, threonine, isoleucine, leucine, glycine and alanine by jejunal or ileal mixed bacteria decreased (P<0.05) with the addition of arginine. Complete utilization of asparagine, aspartate and serine were observed in pig small-intestinal bacteria after 3 h of incubation. Overall, the addition of arginine affected the metabolism of the arginine-family of AA and the serine- and aspartate-family of AA in small-intestinal bacteria and reduced the utilization of most AA in ileal mixed bacteria. These novel findings indicate that arginine exerts its beneficial effects on swine nutrition partially by regulating AA utilization and metabolism in the small-intestinal microbiota.     

In vitro total-gas, CH4, H2, volatile fatty acid, and lactate kinetics studies on luminal contents from the small intestine, cecum, and colon of the pig

Two experiments were conducted to assess differences in fermentative activities of digesta obtained from various regions of the pig gastrointestinal tract. In experiment 1, the contents of small intestines, ceca, and colons of 110-kg pigs were collected, diluted twofold, and incubated for 2 h at 37 degrees C. In experiment 2, colonic samples from 16,100-kg pigs were similarly treated, except that the incubation period was 5 h. Total gas (gas pressure), CH4, H2, lactate, formate, acetate, propionate, butyrate, valerate, and isovalerate were measured in experiment 1. Only the gas variables were measured in experiment 2. Statistically significant differences (P greater than 0.05) were not observed among the gas production rate estimates across the small-intestinal, cecal, and colonic regions in experiment 1. Furthermore, all the small-intestinal samples and half the cecal samples assayed in experiment 1 were nonmethanogenic. The mean methanogenic and total-gas production rate estimates for the colonic samples in experiment 1 were 0.052 ml g of wet contents-1 h-1 and 1.7 ml of total gas g of wet contents-1 h-1, respectively. No differences in the methanogenic rate estimates were detected between the proximal, middle, and distal thirds of the pig colons (P greater than 0.05). The volatile fatty acid and lactate molar percentages measured in experiment 1 were consistent with previously published observations. Hydrogen accumulated to the greatest extent (7 microM on average) in the in vitro incubations of small-intestinal contents, whereas the H2 concentrations ranged from 0.5 to 1 microM for the incubated cecal and colonic samples in experiment 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gland formation induced in the allantoic and small-intestinal endoderm by the proventricular mesenchyme is not coupled with pepsinogen expression

Abstract. Allantoic and small-intestinal endoderms of chick and quail embryos were associated with the proventricular mesenchyme of chick embryos and then cultivated on chorioallantoic membrane. This resulted in the induction of complex glands, but the recombinates never produced embryo-specific pepsinogens; also, glandular cells developed a brush border, expressed sucrase antigen on their apical surface, and sometimes differentiated into goblet cells, thus indicating that both endoderms have the tendency to differentiate into an intestinal epithelium. In the recombinates composed of allantoic endoderm and proventricular mesenchyme, acid-protease activity was detected, but biochemical analysis revealed that this activity was not due topepsinogens. These results indicate that the gland formation induced in allantoic and small-intestinal endoderms by the proventricular mesenchyme is not accompanied by the expression of pepsinogens, suggesting that independent mechanisms are responsible for the morphogenesis and cyto chemical differentiation of the endoderm.     

The lipid messenger OEA links dietary fat intake to satiety

The association between fat consumption and obesity underscores the need to identify physiological signals that control fat intake. Previous studies have shown that feeding stimulates small-intestinal mucosal cells to produce the lipid messenger oleoylethanolamide (OEA) which, when administered as a drug, decreases meal frequency by engaging peroxisome proliferator-activated receptors-alpha (PPAR-alpha). Here, we report that duodenal infusion of fat stimulates OEA mobilization in the proximal small intestine, whereas infusion of protein or carbohydrate does not. OEA production utilizes dietary oleic acid as a substrate and is disrupted in mutant mice lacking the membrane fatty-acid transporter CD36. Targeted disruption of CD36 or PPAR-alpha abrogates the satiety response induced by fat. The results suggest that activation of small-intestinal OEA mobilization, enabled by CD36-mediated uptake of dietary oleic acid, serves as a molecular sensor linking fat ingestion to satiety.     

Identification of an NAD(P)+-dependent ''malic'' enzyme in small-intestinal-mucosal mitochondria.

An NAD(P)+-dependent 'malic' enzyme is shown to be present in mitochondria from small-intestinal mucosa. The intracellular location, activity and regulatory kinetic properties of the enzyme suggest that it participates in the major energy-producing pathway for net oxidation of glutamine-derived tricarboxylic acid-cycle intermediates.     

Critical carboxyl group(s) in Na+-dependent cotransporters of the intestinal brush-border membrane

We have previously provided functional evidence for a role of carboxyl group(s) in the mechanism of coupling of Na+ and D-glucose fluxes by the small-intestinal cotransporter(s) (Kessler, M. and Semenza, G. (1983) J. Membrane Biol. 76, 27-56). We present here a study on the inactivation of the Na+-dependent transport systems, but not of the Na+-independent ones, in the small-intestinal brush-border membrane, by hydrophobic carbodiimides. Although marginal or insignificant protection by the substrates or by Na+ was observed, the parallelism between Na+-dependence and inactivation by these carbodiimides strongly indicates the role of carboxyl group(s) previously indicated. Contrary to the carboxyl group identified by Turner [1986) J. Biol. Chem. 261, 1041-1047) in the sugar binding site of the renal Na+/D-glucose cotransporter, the carboxyl group(s) studied here probably occur elsewhere in the cotransporter molecule.     

Immunocytochemical demonstration of the lysosomal enzyme alpha-glucosidase in the brush border of human intestinal epithelial cells

In investigations on the intracellular transport route(s) of lysosomal enzymes in polarized epithelial cells, we used immunocytochemical methods to localize lysosomal alpha-glucosidase in human small-intestinal epithelial cells. Two monoclonal antibodies which can discriminate between different biosynthetic forms of this enzyme were used. One monoclonal antibody, 43D1, which recognizes all forms of the enzyme, showed labeling of the Golgi apparatus, the lysosomes and, unexpectedly, of the brush border of the cells. Multivesicular bodies were free of label. In contrast, monoclonal antibody 43G8, which recognizes all forms except the 110,000 Da precursor of alpha-glucosidase, showed labeling of the lysosomes only. This leads us to conclude that the 110,000 Da precursor form of alpha-glucosidase is present in the Golgi apparatus and the brush border of human small-intestinal epithelial cells. Moreover, biochemical experiments show that this precursor copurifies with sucrase, a typical brush-border marker, when a partially purified microvilli fraction is prepared.     

The biguanide inhibition of d-glucose transport in membrane vesicles from small intestinal brush borders

Biguanides inhibit d-glucose uptake in vesicles from small-intestinal brush border membranes. Evidence is presented that this inhibition is due to a reduced concentration of Na⁺ in the microenvironment of the carrier(s) for d-glucose. Biguanides do not inhibit the uptake of either d-fructose or l-glucose.     

Identification and partial characterization of phospholipid methylation in rat small-intestinal brush-border membranes

An earlier study (Biochim. Biophys. Acta 46 (1961) 205-216) failed to detect the enzymatic synthesis of phosphatidylcholine (PC) from phosphatidylethanolamine (PE) via a transmethylation pathway in rat small-intestinal microsomal membranes. This pathway was therefore assumed to be absent from this organ. Recently, however, in our laboratory it has been demonstrated that this pathway for the synthesis of phosphatidylcholine is present in rat colonic brush-border and basolateral membranes. It was therefore of interest to examine whether phospholipid methylation activity was present in rat small-intestinal brush-border membranes. The results of the present experiments demonstrate for the first time that this pathway for the synthesis of phosphatidylcholine exists in these plasma membranes. Evidence to support the enzymatic nature of this reaction include: loss of activity by heat denaturation and at 0 degree C, significant inhibition by S-adenosyl-L-homocysteine and saturation kinetics. The predominant product of this brush-border membrane phospholipid methyltransferase is phosphatidyl-N-monomethylethanolamine. This enzymatic activity has an apparent Km for S-adenosyl-L-methionine of 40 microM, a Vmax of 8.4 pmol/mg protein per 5 min, and a pH optimum of 8.0.     

Critical carboxyl group(s) in Na-dependent cotransporters of the intestinal brush-border membrane

We have previously provided functional evidence for a role of carboxyl group(s) in the mechanism of coupling of Na⁺ and d-glucose fluxes by the small-intestinal cotransporter(s) (Kessler, M. and Semenza, G. (1983) J. Membrane Biol. 76, 27–56). We present here a study on the inactivation of the Na⁺-dependent transport systems, but not of the Na⁺-independent ones, in the small-intestinal brush-border membrane, by hydrophobic carbodiimides. Although marginal or insignificant protection by the substrates or by Na⁺ was observed, the parallelism between Na⁺-dependence and inactivation by these carbodiimides strongly indicates the role of carboxyl group(s) previously indicated. Contrary to the carboxyl group identified by Turner ((1986) J. Biol. Chem. 261, 1041–1047) in the sugar binding site of the renal Na⁺/d-glucose cotransporter, the carboxyl group(s) studied here probably occur elsewhere in the cotransporter molecule.     

Application of immunohistochemistry to the isolated mucosa of the mouse gastrointestinal tract, with special reference to somatostatin cells

In the present study, in order to easily grasp whole images of somatostatin (D) cells, the isolated mucosa of the mouse gastrointestinal tract was immunohistochemically treated. The present study revealed that: (1) in the stomach, small-intestinal villi and colon, about 20% of the D cells extrude basal cytoplasmic processes, showing terminal expansions in many cases; on the other hand, in the crypts of the small intestine, few D cells possess basal processes, and (2) in the stomach, there is no determined tendency in the direction of the basal processes of the D cells; on the other hand, in the small-intestinal villi and colon, most D cell basal processes run toward the villus base and colon crypt bottoms. The direction of the basal processes of the D cells in the gastrointestinal tract seems to be mostly in favor of the migration pattern of epithelial cells described previously. It is likely that, if the targets of the D cells are near the D cells, the basal process is not necessary for local secretion of somatostatin. During migration, however, D cells might extrude basal processes to keep relationships with their targets.     

Hyaluronic acid in human articular cartilage. Age-related changes in content and size.

A specific lactotransferrin receptor was identified in the mouse small-intestinal brush-border membrane and the binding features were investigated in homologous and heterologous systems. The receptor was found to be specific for lactotransferrins isolated from milk of various species, but the affinity was higher toward the homologous ligand (Ka = 3.5 x 10(6) M-1 compared with 2.6 x 10(6) M-1 for both human and bovine lactotransferrins). However, the number of binding sites (n) was the same for the three lactotransferrins, namely 0.53 x 10(12)/micrograms of membrane protein. The binding of mouse lactotransferrin to its receptor was found to be pH-dependent, with an optimal binding at pH 5.5, and seemed unlikely to be carbohydrate-mediated. The receptor was demonstrated to be devoid of any affinity for human and mouse serotransferrins or for a 'serotransferrin-like' protein isolated from mouse milk. The receptor was solubilized with 1% Triton X-100 with good yield. The solubilized receptor was found to retain lactotransferrin-binding activity and sensitivity to pH.