Galectin-2 at the enterocyte brush border of the small intestine

The brush border of pig small intestine is a local hotspot for β-galactoside-recognizing lectins, as evidenced by its prominent labeling with fluorescent lectin PNA. Previously, galectins 3-4, intelectin, and lectin-like anti-glycosyl antibodies have been localized to this important body boundary. Together with the membrane glycolipids these lectins form stable lipid raft microdomains that also harbour several of the major digestive microvillar enzymes. In the present work, we identified a lactose-sensitive 14-kDa protein enriched in a microvillar detergent resistant fraction as galectin-2. Its release from closed, right-side-out microvillar membrane vesicles shows that at least some of the galectin-2 resides at the lumenal surface of the brush border, indicating that it plays a role in the organization/stabilization of the lipid raft domains. Galectin-2 was released more effectively from the membrane by lactose than was galectin-4, and surprisingly, it was also released by the noncanonical disaccharides sucrose and maltose. Furthermore, unlike galectin-4, galectin-2 was preferentially coimmunoisolated with sucrase-isomaltase rather than with aminopeptidase N. Together, these results show that the galectins are not simply redundant proteins competing for the same ligands but rather act in concert to ensure an optimal cross-linking of membrane glycolipids and glycoproteins. In this way, they offer a maximal protection of the brush border against exposure to bile, pancreatic enzymes and pathogens.     

Endocrine cells in the human fetal small intestine

Summary In this report we describe the time of appearance and ultrastructural features of enteroendocrine cells (EECs) in the human fetal small intestine (SB) between 9 and 22 weeks gestation. Thirteen distinctive EECs were identified in fetal SB. Two of these, not found in normal adult SB, appeared within the stratified epithelium of the proximal SB at 9–10 weeks. They were arbitrarily termed primitive and precursor cells. As in all fetal EECs, the pale cytoplasm of the primitive cell contains a distinctive population of secretory granules (SGs). Primitive cell SGs average 200–330 nm; some have dense cores with lucent halos while others are filled with a homogeneous dense or flocculent material. The SGs of the precursor cells are larger, averaging up to 1 m in diameter and their contents vary in electron density. A third group of cells not described in normal adult SB was arbitrarily termed transitional cells. These have two populations of SGs; one resembles the SGs of the precursor cells, and the other resembles the SGs of some of the specific adult type EECs. Transitional EC, S, I and G cells are seen. In addition, mature appearing EC, S, G, I, L, D, and D₁ cells were identified by 12 weeks of gestation. The primitive, precursor, and transitional cells may represent sequential developmental precursors of adult type EECs.Supported by Research Grant AM-17537 from the National Institutes of Health, Besthesda, MarylandThe authors would like to thank Ms. Linda Barstein for her excellent technical assistance     

Leptin and the obesity receptor (OB-R) in the small intestine and colon: a colocalization study.

Leptin is a hormone that plays an important role in overall body energy homeostasis, and the obesity receptor, OB-R, is widely distributed in the organism. In the intestine, a multitude of leptin actions have been reported, but it is currently unclear to what extent the hormone affects the intestinal epithelial cells by an endocrine or exocrine signaling pathway. To elucidate this, the localization of endogenous porcine leptin and OB-R in enterocytes and colonocytes was studied. By immunofluorescence microscopy, both leptin and OB-R were mainly observed in the basolateral membrane of enterocytes and colonocytes but also in the apical microvillar membrane of the cells. By electron microscopy, coclustering of hormone and receptor in the plasma membrane and localization in endosomes was frequently detected at the basolateral surface of the epithelial cells, indicative of leptin signaling activity. In contrast, coclustering occurred less frequently at the apical cell surface, and subapical endosomal localization was hardly detectable. We conclude that leptin action in intestinal epithelial cells takes place at the basolateral plasma membrane, indicating that the hormone uses an endocrine pathway both in the jejunum and colon. In contrast, the data obtained did not provide evidence for an exocrine, lumenal action of the hormone in the intestine.     

In situ absorption of aflatoxins in rat small intestine

To evaluate the rate at which the four main aflatoxins (aflatoxins B₁, B₂, G₁ and G₂) are able to cross the luminal membrane of the rat small intestine, a study about intestinal absorption kinetics of these mycotoxins has been made. In situ results obtained showed that the absorption of aflatoxins in rat small intestine is a very fast process that follows first-order kinetics, with an absorption rate constant (k _a ) of 5.84±0.05 (aflatoxin B₁), 4.06±0.09 (aflatoxin B₂), 2.09±0.03 (aflatoxin G₁) and 1.58±0.04 (aflatoxin G₂) h^–1, respectively.     

Expression of TLR2 and TLR4 in murine small intestine during postnatal development

The important role played by the gut microbiota in host immunity is mediated, in part, through toll-like receptors (TLRs). We evaluated the postnatal changes in expression of TLR2 and TLR4 in the murine small intestine and assessed how expression is influenced by gut microbiota. The expression of TLR2 and TLR4 in the murine small intestine was highly dynamic during development. The changes were especially profound during the suckling period, with the maximal mRNA levels detected in the mid-suckling period. Immunohistochemical and flow-cytometric analyses indicated that the changes in TLR2 and TLR4 expression involve primarily epithelial cells. The germ-free mice showed minor changes in TLR2/TLR4 mRNA and TLR2 protein during the suckling period. This study demonstrated that the postnatal expression of TLR2 and TLR4 in small intestinal epithelial cells is dynamic and depends on the presence of commensal intestinal microbiota.     

Protein metabolism in the small intestine of the ethanol-fed rat

The effects of chronic ethanol feeding on the small intestine were investigated in young rats. Rats were fed a nutritionally-adequate liquid diet, containing 36 per cent of total energy as ethanol (treated, n = 7), or isovolumetric amounts of the same diet in which ethanol was substituted by isocaloric glucose (controls, n = 7). After six weeks the wet weight and total tissue contents of protein, RNA and DNA were significantly reduced by 21 per cent, 23 per cent, 16 per cent and 28 per cent respectively, (p less than 0.014). Rates of protein synthesis were measured with L[4(3H)]phenylalanine and fractional rates (defined as the percentage of constituent tissue protein synthesised each hour, i.e. ks, % h-1) were calculated from the specific radioactivity of free phenylalanine in both tissue homogenates and plasma. Ethanol-feeding reduced ks by approx 10 per cent (p less than 0.181). The amount of protein synthesized unit-1 RNA was also reduced by approx 15 per cent (p less than 0.059) but the amount of protein synthesis unit-1 DNA was unaffected by ethanol-feeding (p less than 1.000). In contrast, the absolute rates of protein synthesis were reduced by approximately 30 per cent (p less than 0.022). It was concluded that, as the small intestine contributes to approx. 20-25 per cent of whole body synthesis these results may have an important effect on whole body nitrogen homeostasis and may have implications for the gastrointestinal effects of ethanol seen during chronic alcoholic abuse.     

Immunohistochemical characterization of cell types expressing the cellular prion protein in the small intestine of cattle and mice

The gastrointestinal tract is thought to be the main site of entry for the pathological isoform of the prion protein (PrP^Sc). Prion diseases are believed to result from a conformational change of the cellular prion protein (PrP^c) to PrP^Sc. Therefore, PrP^c expression is a prerequisite for the infection and spread of the disease to the central nervous system. However, the distribution of PrP^c in the gut is still a matter of controversy. We therefore investigated the localization of PrP^c in the bovine and murine small intestine. In cattle, most PrP^c positive epithelial cells were detected in the duodenum, while a few positive cells were found in the jejunum. PrP^c was expressed in serotonin producing cells. In bovine Peyer’s patches, PrP^c was distributed in extrafollicular areas, but not in the germinal centre of the jejunum and ileum. PrP^c was expressed in myeloid lineage cells such as myeloid dendritic cells and macrophages. In mice, PrP^c was expressed in some epithelial cells throughout the small intestine as well as in cells such as follicular dendritic cell in the germinal centre of Peyer’s patches. In this study, we demonstrate that there are a number of differences in the localization of PrP^c between the murine and bovine small intestines.     

Expression of the monocarboxylate transporter 1 (MCT1) in cells of the porcine intestine

Uptake of energy into cells and its allocation to individual cellular compartments by transporters are essential for tissue homeostasis. The present study gives an analysis of MCT1 expression and its cellular occurrence in the porcine intestine. Tissue portions from duodenum, jejunum, ileum, colon ascendens, colon transversum and colon descendens were collected and prepared for immunohistochemistry, Western blot and real time RT-PCR. A 169bp porcine MCT1 cDNA fragment was amplified and published. MCT1 mRNA expression in the large intestine was 20 fold higher compared to the small intestine. Western blot detected a single protein band of 41kDa at a much higher amount of MCT1 protein in the large intestine vs. the small intestine. MCT1 protein was detected in mitochondrial fractions of the large but not the small intestine. Immunohistochemistry in the small intestine showed that immune cells in the lamina propria and in the lymphoid follicles primarily expressed MCT1 while in the colon epithelial cells were the main source of MCT1. In summary, cellular expression of MCT1 differs between epithelial cells in the colon and small intestine. A possible role of MCT1 for uptake of butyrate into immune cells and the overall role of MCT1 for intestinal immune cell function remains elusive.     

应用基因芯片技术检测肝组织的基因表达

为检测正常肝组织中的基因表达状况,采用cDNA microarray技术对正常肝组织中表达的1500个基因进行定量。结果为97 个基因较高表达,1010个基因中度表达,380个基因低表达。结果是cDNA microarray技术是大规模检测基因表达的有效方法。     

Investigation of the protective effects of crocin on acrylamide induced small and large intestine damage in rats

We investigated repair of acrylamide (AA) induced damage in intestines by administration of crocin. We used 40 male Wistar rats in four groups of 10 animals: control, AA, crocin, and AA + crocin groups. We investigated biochemical and histological changes to small and large intestine. AA ingestion decreased glutathione (GSH) levels and total antioxidant status (TAS) in the intestine compared to the control group, while superoxide dismutase (SOD) and catalase (CAT) activities, and total oxidant status (TOS) and malondialdehyde (MDA) levels were increased. Villi were shortened and villus degeneration was observed in ileum of the AA group. Degeneration of surface epithelium and Liberkühn crypts were observed in colon sections. GSH and TAS levels increased after administration of AA together with crocin, while SOD and CAT levels and TOS and MDA levels decreased; significant recovery of histological damage also was observed. We found that crocin exhibits protective effects on AA induced small and large intestine damage by inhibiting oxidative stress.     

Developmental control of the expression of two ten-sugar branched chain fucolipids in rat small intestine

Two branched decaglycosylceramides, apparently identical to those identified in the small intestine of adult rats [Breimer ME, Falk K-E, Hansson GC, Karlsson K-A (1982) J Biol Chem 257:50–59], were absent during the three weeks following birth. They appeared abruptly at around 21 days. After their appearance, their tissue concentration and their base composition did not change during development. Their fatty acids were non-hydroxylated and the percentage of C₂₂–C₂₄ fatty acids, which was low at 24 days, increased and reached 48.6% by 27 days.Nomenclature Gal1-4Gal1-4GlcCer Globotriaosylceramide (GbOse₃Cer) - Il³NeuAc-LacCer M_M3-ganglioside - GalNAc1-3Gal1-4Gal1-4GlcCer globoside (globotetraosylceramide, GbOse₄Cer)     

Changes in surviving nerve fibers associated with submucosal arteries following extrinsic denervation of the small intestine

Summary The neuropeptide content of nerve fibers associated with submucosal arteries in the small intestine of guinea pigs was studied in whole-mount preparations using immunohistochemical methods. Tissues were obtained from normal animals or animals in which the small intestine had been extrinsically denervated. In normal animals, submucosal arteries are innervated by extrinsic sensory nerve fibers which contain both substance P and calcitonin gene-related peptide, and by sympathetic noradrenergic nerve fibers. In preparations obtained from animals 5–9 days after denervation, nerve fibers which contained substance P without detectable calcitonin gene-related peptide were associated with a few submucosal arteries. Nerve fibers which contained vasoactive intestinal peptide were also associated with some arteries. By 42–48 days after extrinsic denervation, substance P-containing fibers (without calcitonin gene-related peptide) and vasoactive intestinal peptide-containing fibers were associated with nearly every blood vessel. The extrinsic sympathetic nerve fibers did not regenerate during the course of this study. The nerve fibers associated with submucosal arteries in denervated tissues were not sensitive to capsaicin treatment.The alteration in the innervation of submucosal arterioles that follows extrinsic denervation of the gut may reflect either an increase in the neuropeptide content of the fibers, synthesis of a new peptide, or an increase in the number of fibers as a result of axonal sprouting.     

Effects of ceftriaxone‐induced intestinal dysbacteriosis on dendritic cells of small intestine in mice

Intestinal microflora plays a pivotal role in the development of the innate immune system and is essential in shaping adaptive immunity. Dysbacteriosis of intestinal microflora induces altered immune responses and results in disease susceptibility. Dendritic cells (DCs), the professional antigen‐presenting cells, have gained increasing attention because they connect innate and adaptive immunity. They generate both immunity in response to stimulation by pathogenic bacteria and immune tolerance in the presence of commensal bacteria. However, few studies have examined the effects of intestinal dysbacteriosis on DCs. In this study, changes of DCs in the small intestine of mice under the condition of dysbacteriosis induced by ceftriaxone sodium were investigated. It was found that intragastric administration of ceftriaxone sodium caused severe dysteriosis in mice. Compared with controls, numbers of DCs in mice with dysbacteriosis increased significantly (P = 0.0001). However, the maturity and antigen‐presenting ability of DCs were greatly reduced. In addition, there was a significant difference in secretion of IL‐10 and IL‐12 between DCs from mice with dysbacteriosis and controls. To conclude, ceftriaxone‐induced intestinal dysbacteriosis strongly affected the numbers and functions of DCs. The present data suggest that intestinal microflora plays an important role in inducing and maintaining the functions of DCs and thus is essential for the connection between innate and adaptive immune responses.     

Effects of porcine bile on survival of Bacillus cereus vegetative cells and Haemolysin BL enterotoxin production in reconstituted human small intestine media

AIMS: To determine the effects of porcine bile (PB) on Bacillus cereus vegetative cells and Haemolysin BL (HBL) enterotoxin production in reconstituted small intestine media (IM). METHODS AND RESULTS: The effects of PB on the growth of B. cereus vegetative cells in reconstituted IM at PB concentrations ranging between 0 and 3.0 g l(-1) were examined. Four gastric media (GM) named GM-J broth (JB), GM-chicken, GM-milk and GM-pea were prepared by mixing equal volumes of a gastric electrolyte solution containing pepsin with JB, chicken, semi-skimmed milk and pea soup, respectively. Bacillus cereus was inoculated at approx. 2 x 10(4) CFU ml(-1) into each GM at pH 5.0 for 30 min at 37 degrees C, then mixed to the same volume of double-strength JB (IM) and PB to give concentrations of between 0 and 3.0 g of PB per litre at pH 6.5 and incubated at 37 degrees C. The diarrhoeal B. cereus strain F4430/73 grew in IM-JB, IM-chicken and IM-milk at PB concentrations of up to 0.6, 1.5 and 1.2 g l(-1), respectively. Growth was observed in IM-pea at all concentrations tested. The highest PB concentrations allowing a 3 log B. cereus increase in IM-JB, IM-chicken, IM-milk and IM-pea after a 7-10 h incubation period were 0.3, 0.9, 0.9 and 3.0 g l(-1), respectively. The effect of PB on B. cereus cells was strongest in IM-JB, followed by IM-chicken, IM-milk and IM-pea. Haemolysin BL enterotoxin was detectable in IM-chicken, IM-whole milk, IM-semi-skimmed milk and IM-pea up to PB concentrations of only 0.6, 0.6, 0.3 and 0.9 g l(-1), respectively. The diarrhoeal B. cereus strain F4433/73 behaved similarly to B. cereus strain F4430/73, whereas the food strain TZ415 was markedly more susceptible to bile. CONCLUSIONS: The tolerance of B. cereus cells to PB strongly depends on the type of food contained in the IM. Bile tolerance is also subject to strain variation. SIGNIFICANCE AND IMPACT OF THE STUDY: The probability that B. cereus cells will grow in the small intestine, produce toxins and cause diarrhoea is likely to depend on the food they are ingested with, on the bile tolerance of the B. cereus strain, and on bile concentration.     

IL-22 promotes the formation of a MUC17 glycocalyx barrier in the postnatal small intestine during weaning

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Immunohistochemical localization of microtubule-associated proteins in the nervous system of the small intestine of guinea pig

Summary Layers containing Auerbach's and Meissner's plexuses were dissected from the small intestine of guinea pig and immunostained with affinity-purified antibodies against brain-specific microtubule-associated proteins (MAPs): MAP1, MAP2 and tau and a MAP with a molecular weight of 190000 dalton purified from bovine adrenal cortex (190-kDa MAP). MAP1 antibody stained the network of nerve fibers and the cell bodies of enteric neurons in both Auerbach's and Meissner's plexuses. Staining with anti-tau antibody gave the same results. Antibody against MAP2 stained neuronal cell bodies and short thin processes extending from them. Interganglionic strands composed mainly of long processes were unstained. Anti-190-kDa MAP antibody stained both the neuronal cell bodies and bundles of nerve fibers. However, the staining was less intense than that with anti-MAP1 and tau antibodies. Differentiation in the structure of the cytoskeleton probably exists in the neuronal processes of the enteric neurons as is shown in the dendrites and axons in some neurons of the central nervous system. Thus, enteric neurons possess axon-like processes containing MAP1, tau and probably lower amounts of 190-kDa MAP. Cell bodies and dendrite-like structures of these neurons contain MAP2 in addition to MAP1, tau and 190-kDa MAP.     

Induction of metamorphosis by thyroid hormone in anuran small intestine cultured organotypically in vitro

Summary We have developed an organ culture system of the anuran small intestine to reproduce in vitro the transition from larval to adult epithelial form which occurs during spontaneous metamorphosis. Tubular fragments isolated from the small intestine ofXenopus laevis tadpoles were slit open and placed on membrane filters in culture dishes. In 60% Leibovitz 15 medium supplemented with 10% charcoal-treated serum, the explants were maintained in good condition for at least 10 days without any morphologic changes. Addition of triiodothyronine (T₃) at a concentration higher than 10⁻⁹ M to the medium could induce cell death of larval epithelial cells, but T₃ alone was not sufficient for proliferation and differentiation of adult epithelial cells. When insulin (5 μg/ml) and cortisol (0.5 μg/ml) besides T₃ were added, the adult cells proliferated and differentiated just as during spontaneous metamorphosis. On Day 5 of cultivation, the adult cells rapidly proliferated to form typical islets, whereas the larval ones rapidly degenerated. At the same time, the connective tissue beneath the epithelium suddenly increased in cell density. These changes correspond to those occurring at the onset of metamorphic climax. By Day 10, the adult cells differentiated into a simple columnar epithelium which possessed the brush border and showed the adult-type lectin-binding pattern. Therefore, the larval epithelium of the small intestine responded to the hormones and transformed into the adult one. This organ culture system may be useful for clarifying the mechanism of the epithelial transition from larval to adult type during metamorphosis.     

Lysine transport across the small intestine. Stimulating and inhibitory effects of neutral amino acids

Summary The ordinary aliphatic, neutral amino acids and phenylalanine have been examined for cis-inhibition of influx of alanine (J _mc ^ala ) and lysine (J _mc ^lys ) and trans-stimulation ofJ _mc ^lys across the brush border membrane of rat small intestines: and their effects on the unidirectional mucosa-to-serosa flux (J _ms ^lys ) across the short circuited intestine have been studied. The effects of alanine, -amino-n-butyric acid, leucine, and methionine on the steady-state epithelial uptake of lysine [Lys] _c have also been measured. In addition the trans-effects of alanine and leucine have been examined for sodium-dependence, and alanine was tested as trans-stimulator of influx of galactose across the brush border membrane (J _mc ^gal ).All the neutral amino acids were found to be competitive cis-inhibitors ofJ _mc ^lys , and all, except isoleucine, were trans-stimulators ofJ _mc ^lys . The magnitude of the trans-effect was unrelated to the efficiency of the amino acid as cis-inhibitor. As illustrated by alanine, the trans-effects are probably completely sodium-dependent. Alanine was also effective as trans-stimulator ofJ _mc ^gal . With respect to effects on [Lys] _c andJ _ms ^lys the neutral amino acids fall into two groups: One which reduces [Lys] _c and stimulatesJ _ms ^lys , and one which increases [Lys] _c and relatively inhibitsJ _ms ^lys . These effects are not correlated with the affinities of the neutral amino acids for the two carriers involved.It is proposed that the trans-effects onJ _mc ^lys are induced by an electrogenic, sodium-coupled efflux of the neutral amino acid across the brush border membrane, that the stimulation ofJ _ms ^lys is brought about by a selective stimulation (of unknown nature) of efflux of lysine across the basolateral membrane (J _cs ^lys ), assisted by competitive inhibition of lysine efflux across the brush border membrane (J _cm ^lys ), and that the amino acids which do not stimulateJ _cm ^lys increase [Lys] _c by competitively inhibitingJ _cs ^lys andJ _cm ^lys .The inhibitory effect of the neutral amino acids onJ _mc ^lys support the view that the carrier of basic amino acids serves as a second carrier of these amino acids.     

Current-voltage relations of sodium-coupled sugar transport across the apical membrane ofNecturus small intestine

Summary The current-voltage (I–V) relations of the rheogenic Na-sugar cotransport mechanism at the apical membrane ofNecturus small intestine were determined from the relations between the electrical potential difference across the apical membrane, ^mc , and that across the entire epithelium, ^ms , when the latter was varied over the range ±200 mV, (i) under steady conditions in the presence of galactose and (ii) after the current across the apical membrane carried by the cotransporter,I _SNa ^m , is blocked by the addition of phloridzin to the mucosal solution.I _SNa ^m was found to be strongly dependent upon ^mc over the range –50 mV < ^mc <E _SNa ^m whereE _SNa ^m is the zero current or reversal potential. Over the range of values of ^mc encountered under physiological conditions the cotransporter may be modeled as a conductance in series with an electromotive force so thatI _SNa ^m =g _SNa ^m (E _SNa ^m – ^mc ) whereg _SNa ^m is the contribution of this mechanism to the conductance of the apical membrane and is near constant. In several instancesI _SNa ^m saturated at large hyperpolarizing or depolarizing values of ^mc .The values ofE _SNa ^m determined in the presence of 1, 5, and 15mm galactose strongly suggest that if the Na-galactose cotransporters are kinetically homogeneous, the stoichiometry of this coupled process is unity.Finally, the shapes of the observedI–V relations are consistent with the predictions of a simple kinetic model which conforms with current notions regarding the mechanico-kinetic properties of this cotransport process.     

Effects of Enterococcus faecium NCIMB 10415 as probiotic supplement on intestinal transport and barrier function of piglets

Abstract

Many studies report positive effects of probiotic supplementation on the performance and health of piglets. The intention of this study was to describe the effects of Enterococcus faecium NCIMB 10415 on the transport and barrier functions of pig small intestine to improve our understanding of the underlying mechanisms of this probiotic. Ussing chamber studies were conducted with isolated jejunal epithelia of piglets at the age of 14, 28, 35 and 56 days. Jejunal tissues of the control group were compared with epithelia of piglets that had received a diet supplemented with the probiotic Enterococcus faecium NCIMB 10415. Transport properties (absorption and secretion) of the epithelia were examined by mucosal addition of glucose or L-glutamine or by serosal addition of PGE₂. Electrophysiology of the epithelia was continuously recorded and the change in short circuit current (I_sc) was determined. Paracellular permeability was measured by measuring the flux rates of mannitol. The increase of I_sc caused by mucosal addition of glucose was, at all glucose concentrations, higher in the probiotic group compared with the control group. However, the difference (up to 100% of the control) was not significant. The increase of I_sc after the mucosal addition of L-glutamine (12 mmol/l) was higher in the tissues of the probiotic group but did not reach significance. Serosal PGE₂ induced a significantly higher increase of I_sc in tissues of the probiotic group at the age of 28 days. No consistent differences were observed in mannitol transport rates between the feeding groups.

Significant age-dependent alterations of absorptive and secretory properties of the jejunal epithelium were observed; these were independent of the treatment. A probiotic supplementation seems to influence transport properties of small intestine epithelium. The increased absorption of glucose could be interpreted as a positive effect for the animal.