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With the capacity for rapid self-renewal and regeneration, the intestinal epithelium is stereotypical of stem cell--supported tissues. Yet the pattern of stem cell turnover remains in question. Applying analytical methods from population dynamics and statistical physics to an inducible genetic labeling system, we showed that done size distributions conform to a distinctive scaling behavior at short times. This result demonstrates that intestinal stem cells form an equipotent population in which the toss of a stern cell is compensated by the multiplication of a neighbor, leading to neutral drift dynamics in which clones expand and contract at random until they either take over the crypt or they are lost. Combined with long-term clonal fate data, we show that the rate of stern cell replacement is comparable to the cell division rate, implying that neutral drift and symmetrical cell divisions are central to stem cell homeostasis. 10.1126/science.1196236

PepT1 is a di/tripeptide transporter highly expressed in the small intestine, but poorly or not expressed in the colon. However, during chronic inflammation, such as inflammatory bowel disease, PepT1 expression is induced in the colon. Commensal bacteria that colonize the human colon produce a large amount of di/tripeptides. To date, two bacterial peptides (N-formylmethionylleucyl-phenylalanine and muramyl dipeptide) have been identified as substrates of PepTl. We hypothesized that the proinflammatory tripeptide L-Ala-[gamma]-D-Glu-meso-DAP (Tri-DAP), a breakdown product of bacterial peptidoglycan, is transported into intestinal epithelial cells via PepT1. We found that uptake of glycine-sarcosine, a specific substrate of PepTl, in intestinal epithelial Caco2-BBE cells was inhibited by Tri-DAP in a dose-dependent manner. Tri-DAP induced activation of NF-[kappa]B and MAP kinases, consequently leading to production of the proinflammatory cytokine interleukin-8. Tri-DAP-induced inflammatory response in Caco2-BBE cells was significantly suppressed by silencing of PepT1 expression by using PepTl-shRNAs in a tetracycline-regulated expression (Tet-off) system. Colonic epithelial HT29-Cl.19A cells, which do not express PepT1 under basal condition, were mostly insensitive to Tri-DAP-induced inflammation. However, HT29-Cl.19A cells exhibited proinflammatory response to Tri-DAP upon stable transfection with a plasmid encoding PepTl. Accordingly, Tri-DAP significantly increased keratinocyte-derived chemokine production in colonic tissues from transgenic mice expressing PepT1 in intestinal epithelial cells. Finally, Tri-DAP induced a significant drop in intracellular pH in intestinal epithelial cells expressing PepT1, but not in cells that did not express PepT1. Our data collectively support the classification of Tri-DAP as a novel substrate of PepT1. Given that PepTl is highly expressed in the colon during inflammation, PepTl-mediated Tri-DAP transport may occur more effectively during such conditions, further contributing to intestinal inflammation. inflammation; inflammatory bowel diseases doi: 10.1152/ajpgi.00527.2009.

Gastrointestinal epithelium faces osmotic stress, both at physiological and pathophysiological conditions. JNK activation is an immediate cellular response to osmotic stress. We investigated the effect of osmotic stress on intestinal epithelial barrier function and delineated the role of JNK2 in osmotic stress-induced tight junction (TJ) regulation in Caco-2 cell monolayers and ileum of [Jnk.sup.-/-] and [Jnk2.sup.-/-] mice. The role of JNK activation in osmotic stress-induced TJ disruption was evaluated using JNK-specific inhibitor and antisense oligonucleotides. Furthermore, the effect of cold restraint stress in vivo on TJ integrity was determined in rats. Osmotic stress disrupted TJs and barrier function in Caco-2 cell monolayers without affecting cell viability. Osmotic stress activated JNK1 and JNK2 and the inhibition of JNK by SP600125 attenuated osmotic stress-induced TJ disruption. TJ disruption and barrier dysfunction by osmotic stress was associated with JNK-dependent remodeling of actin cytoskeleton. Knockdown of JNK2 accelerated TJ assembly and attenuated osmotic stress-induced TJ disruption in Caco-2 cell monolayers. In mouse ileum in vitro, osmotic stress increased paracellular permeability, which was attenuated by SP600125. Osmotic stress disrupted actin cytoskeleton and TJs and increased paracellular permeability in the ileum of wild-type and [JNK1.sup.-/-] mice, but not in [JNK2.sup.-/-] mouse ileum. Cold restraint stress activated JNK in rat ileum and caused JNK-dependent remodeling of actin cytoskeleton and redistribution of occludin and zona occluden-1 from the intercellular junctions. These results reveal the role of JNK2 in the mechanism of osmotic stress-induced TJ disruption in the intestinal epithelium. c-Jun N[H.sub.2]-terminal kinase; occludin; intestine; barrier function; stress doi: 10.1152/ajpgi.00265.2010.

Intestinal mucosal integrity is dependent on epithelial function and a regulated immune response to injury. Fucosyltransferase VII (Fuc-TVII) is an essential enzyme required for the expression of the functional ligand for E- and P-selectin. Trefoil factor 3 (TFF3) is involved in both protecting the intestinal epithelium against injury as well as aiding in wound repair following injury. The aim of the present study was to assess the interplay between barrier function and leukocyte recruitment in intestinal inflammation. More specifically, we aimed to examine how targeted disruption of Fuc-TVII either in wild-type or [TFF3.sup.-/-] mice would alter their susceptibility to colonic injury. TFF3 and Fuc-TVII double-knockout mice (TFF3/Fuc-[TVII.sup.-/-] mice) were generated by mating [TFF3.sup.-/-] and Fuc-[TVII.sup.-/-] mice. Colitis was induced by administration of dextran sodium sulfate (DSS) (2.5% wt/vol) in the drinking water. Changes in baseline body weight, diarrhea, and fecal blood were assessed daily. Upon euthanasia, extents of colonic inflammation were assessed macroscopically, microscopically, and through quantification of myeloperoxidase (MPO) activity. Colonic lymphocyte subpopulations were assessed at 6 days after administration of DSS by flow cytometry and immunohistochemistry. No baseline intestinal inflammation was found in TFF3/Fuc-[TVII.sup.-/-] [TFF3.sup.-/-], Fuc-[TVII.sup.-/-], or wild-type mice. Loss of Fuc-TVII resulted in a reduction in disease severity whereas [TFF3.sup.-/-] mice were markedly more susceptible to DSS-induced colitis. Remarkably, the loss of Fuc-TVII in [TFF3.sup.-/-] mice markedly decreased the severity of DSS-induced colitis as evidenced by reduced weight loss, diarrhea, decreased colonic MPO levels and improved survival. Furthermore, the loss of TFF3 resulted in increased severity of spontaneous colitis in IL-2/[beta-microglobulin-deficient mice. These studies highlight the importance of the interplay between factors involved in the innate immune response, mucosal barrier function, and genes involved in regulating leukocyte recruitment and other aspects of the immune response. trefoil factor family 3 doi: 10.1152/ajpgi.00228.2009.

Nitric oxide (NO) is associated with intestinal apoptosis in health and disease. This study aimed to investigate the role of intestinal NO in the regulation of apoptosis during fasting in rats. Male Wistar rats were divided into two groups and subcutaneously injected with saline (SA) or aminoguanidine (AG), followed by fasting for 24, 48, 60, and 72 h. At each time point, the jejunum was subjected to histological evaluation for enterocyte apoptosis by histomorphometric assessment and TUNEL analysis. We performed immunohistochemistry for inducible NO synthase (iNOS) expression in the jejunum and measured tissue nitrite levels using HPLC and 8-hydroxydeoxyguanosine adduct using ELISA, indicative of endogenous NO production and reactive oxygen species (ROS) production, respectively. Jejunal transcriptional levels of iNOS, neuronal NO synthase (nNOS), and interferon-[gamma], (IFN-[gamma]) were also determined by RT-PCR. Fasting caused significant jejunal mucosal atrophy due to attenuated cell proliferation and enhanced apoptosis with increase in iNOS transcription, its protein expression in intestinal epithelial cells (IEC), and jejunal nitrite levels. However, AG treatment histologically reduced apoptosis with inhibition of fasting-induced iNOS transcription, protein expression, and nitrite production. We also observed fasting-induced ROS production and subsequent IFN-[gamma] transcription, which were all inhibited by AG treatment. Furthermore, we observed reduced transcriptional levels of nNOS, known to suppress iNOS activation physiologically. These results suggest that fasting-induced iNOS activation in IEC may induce apoptosis mediators such as IFN-[gamma] via a ROS-mediated mechanism and also a possible role of nNOS in the regulation of iNOS activity in fasting-induced apoptosis. aminoguanidine; reactive oxygen species; intestinal epithelial cells; neuronal nitric oxide synthase doi: 10.1152/ajpgi.00429.2009.

The intestinal epithelium serves as a major protective barrier between the mammalian host and the external environment. Here we show that the transmembrane serine protease matriptase plays a pivotol role in the formation and integrity of the intestinal epithelial barrier. St14 hypomorphic mice, which have a 100-fold reduction in intestinal matriptase mRNA levels, display a 35% reduction in intestinal transepithelial electrical resistance (TEER). Matriptase is expressed during intestinal epithelial differentiation and colocalizes with E-cadherin to apical junctional complexes (AJC) in differentiated polarized Caco-2 monolayers. Inhibition of matriptase activity using a specific peptide inhibitor or by knockdown of matriptase by siRNA disrupts the development of TEER in barrier-forming Caco-2 monolayers and increases paracellular permeability to macromolecular FITC-dextran. Loss of matriptase was associated with enhanced expression and incorporation of the permeability-associated, 'leaky' tight junction protein claudin-2 at intercellular junctions. Knockdown of claudin-2 enhanced the development of TEER in matriptase-silenced Caco-2 monolayers, suggesting that the reduced barrier integrity was caused, at least in part, by an inability to regulate claudin-2 expression and incorporation into junctions. We find that matriptase enhances the rate of claudin-2 protein turnover, and that this is mediated indirectly through an atypical PKC[zeta]-dependent signaling pathway. These results support a key role for matriptase in regulating intestinal epithelial barrier competence, and suggest an intriguing link between pericellular serine protease activity and tight junction assembly in polarized epithelia. claudin-2 | intestinal barrier | St14 | type II transmembrane serine protease | tight junction doi/10.1073/pnas.0903923107

Fibroblast growth factor receptor 3 (FGFR-3) is expressed in the lower crypt epithelium, where stem cells of the intestine reside. The role of FGFR-3 signaling in regulating features of intestinal morphogenesis was examined in FGFR-3-null (FGFR-[3.sup.-/-]) mice. FGFR-[3.sup.-/-] mice had only about half the number of intestinal crypts and a marked decrease in the number of functional clonogenic stem cells, as assessed by an in vivo microcolony-forming assay, compared with wild-type littermates. A marked deficit in allocation of progenitor cells to Paneth cell differentiation was noted, although all the principal epithelial lineages were represented in FGFR-[3.sup.-/-] mice. The total cellular content and nuclear localization of [beta]-catenin protein were reduced in FGFR-[3.sup.-/-] mice, as was expression of cyclin D1 and matrix metalloproteinase-7, major downstream targets of [beta]-catenin/T cell factor-4 (Tcf-4) signaling. Activation of FGFR-3 in Caco-2 cells, an intestinal epithelial cell line, abrogated the fall in [beta]-catenin/Tcf-4 signaling activity that is normally observed in these cells as cultures become progressively more confluent. These findings are consistent with the hypothesis that, during intestinal development, FGFR-3 signaling regulates crypt epithelial stem cell expansion and crypt morphogenesis, as well as Paneth cell lineage specification, through [beta]-catenin/Tcf-4-dependent and -independent pathways. fibroblast growth factors; [beta]-catenin; T cell factor-4; intestinal morphogenesis

Members of the [K.sub.v]7 voltage-gated [K.sup.+] channel family are important determinants of cardiac and neuronal membrane excitability. Recently, we and others have shown that [K.sub.v]7 channels are also crucial regulators of smooth muscle activity. The aim of the present study was to assess the [K.sub.v]7 expression in different parts of the murine gastrointestinal (GI) tract and to assess their functional roles by use of pharmacological agents. Of KCNQ/[K.sub.v]7 members, both KCNQ4/[K.sub.v]7.4 and KCNQ5/[K.sub.v]7.5 genes and proteins were the most abundantly expressed [K.sub.v]7 channels in smooth muscles throughout the GI tract. Immunohistochemical staining also revealed that [K.sub.v]7.4 and [K.sub.v]7.5 but not [K.sub.v]7.1 were expressed in the circular muscle layer of the colon. In segments of distal colon circular muscle exhibiting spontaneous phasic contractions, the nonselective [K.sub.v]7 blockers XE991 and linopirdine increased the integral of tension. Increases in the integral of tension were also observed under conditions of neuronal blockade. Similar effects, although less marked, were observed in the proximal colon. As expected, the [K.sub.v]7.1-selective blocker chromanol 293B had no effect in either type of segment. These data show that [K.sub.v]7.X especially [K.sub.v]7.4 and [K.sub.v]7.5 are expressed in different regions of the murine gastrointestinal tract and blockers of [K.sub.v]7 channels augment inherent contractile activity. Drugs that selectively block [K.sub.v]7.4/7.5 might be promising therapeutics for the treatment of motility disorders such as constipation associated with irritable bowel syndrome. colon; voltage-gated [K.sup.+] channel; KCNE

For decades, enterocyte brush border microvilli have been viewed as passive cytoskeletal scaffolds that serve to increase apical membrane surface area. However, recent studies revealed that in the in vitro context of isolated brush borders, myosin-1 a (myol a) powers the sliding of microvillar membrane along core actin bundles. This activity also leads to the shedding of small vesicles from microvillar tips, suggesting that microvilli may function as vesicle-generating organelles in vivo. In this study, we present data in support of this hypothesis, showing that enterocyte microvilli release unilamellar vesicles into the intestinal lumen; these vesicles retain the right side out orientation of microvillar membrane, contain catalytically active brush border enzymes, and are specifically enriched in intestinal alkaline phosphatase. Moreover, myo 1 a knockout mice demonstrate striking perturbations in vesicle production, clearly implicating this motor in the in vivo regulation of this novel activity. In combination, these data show that microvilli function as vesicle-generating organelles, which enable enterocytes to deploy catalytic activities into the intestinal lumen.

A wireless drive and control method using rotational magnetic field on a capsule micro robot is proposed, and both outer and inner actuators are Nd-Fe-B magnet cylinders with multiple magnetic poles in tegular shape alternating with dissimilar radial magnetization, through magnetic coupling with rotational magnetic field generated by the rotating outer actuator, the capsule robot is rotated by an embedded inner actuator, and axial thrust is generated by fluid dynamic pressure distributing on outer surface of the robot with spiral blade, thus the screw motion of the micro robot in intestine is realized. Mathematical model on magnetic torque is established based on equivalent magnetic charge method, and the characteristics of magnetic torque with respect to structural parameters of actuators and swimming effect of the robot are studied theoretically and experimentally. Index Terms--Capsule micro robot, equivalent magnetic charge method, magnetic torque, spiral drive.

The integrity of the intestinal epithelial barrier depends on intercellular junctions that are highly regulated by numerous extracellular and intracellular factors. E-cadherin is found primarily at the adherens junctions in the intestinal mucosa and mediates strong cell-cell contacts that have a functional role in forming and regulating the epithelial barrier. Polyamines are necessary for E-cadherin expression, but the exact mechanism underlying polyamines remains elusive. The current study was performed to determine whether polyamines induce E-cadherin expression through the transcription factor c-Myc and whether polyamine-regulated E-cadherin plays a role in maintenance of the epithehal barrier integrity. Decreasing cellular polyamines reduced c-Myc and repressed E-cadherin transcription as indicated by a decrease in levels of E-cadherin promoter activity and its mRNA. Forced expression of the c-myc gene by infection with adenoviral vector containing c-Myc cDNA stimulated E-cadherin promoter activity and increased Ecadherin mRNA and protein levels in polyamine-deficient cells. Experiments using different E-cadherin promoter mutants revealed that induction of E-cadherin transcription by c-Myc was mediated through the E-Pal box located at the proximal region of the E-cadherin promoter. Decreased levels of E-cadherin in polyamine-deficient ceils marginally increased basal levels of paracellular permeability but, remarkably, potentiated [H.sub.2][O.sub.2]-induced epithelial barrier dysfunction. E-cadherin silencing by transfection with its specific small interfering RNA also increased vulnerability of the epithelial barrier to [H.sub.2][O.sub.2]. These results indicate that polyamines enhance E-cadherin transcription by activating c-Myc, thus promoting function of the epithelial barrier. paracellular permeability; adherens junctions; gene expression; ornithine decarboxylase; intestinal epithelial cells

Absorption of dietary iodide, presumably in the small intestine, is the first step in iodide ([I.sup.-]) utilization. From the bloodstream, [I.sup.-] is actively taken up via the [Na.sup.+]/[I.sup.-] symporter (NIS) in the thyroid for thyroid hormone biosynthesis and in such other tissues as lactating breast, which supplies [I.sup.-] to the newborn in the milk. The molecular basis for intestinal [I.sup.-] absorption is unknown. We sought to determine whether [I.sup.-] is actively accumulated by enterocytes and, if so, whether this process is mediated by NIS and regulated by [I.sup.-] itself. NIS expression was localized exclusively at the apical surface of rat and mouse enterocytes. In vivo intestine-to-blood transport of pertechnetate, a NIS substrate, was sensitive to the NIS inhibitor perchlorate. Brush border membrane vesicles accumulated [I.sup.-] in a sodium-dependent, perchlorate-sensitive manner with kinetic parameters similar to those of thyroid cells. NIS was expressed in intestinal epithelial cell line 6, and [I.sup.-] uptake in these cells was also kinetically similar to that in thyrocytes. [I.sup.-] downregulated NIS protein expression and its own NIS-mediated transport both in vitro and in vivo. We conclude that NIS is functionally expressed on the apical surface of enterocytes, where it mediates active [I.sup.-] accumulation. Therefore, NIS is a significant and possibly central component of the [I.sup.-] absorption system in the small intestine, a system of key importance for thyroid hormone biosynthesis and thus systemic intermediary metabolism. dietary iodide absorption; active iodide transport; sodium/iodide symporter; enterocyte brush border

Expansion of intestinal progenitors and putative stem cells (pISC) occurs early and transiently following ileo-cecal resection (ICR). The mechanism controlling this process is not defined. We hypothesized that glucagon-like peptide-2 (GLP-2) would augment jejunal pISC expansion only when administered to mice immediately after ICR. Since recent reports demonstrated increases in intestinal insulin-like growth factor (IGF)-I following GLP-2 administration, we further hypothesized that increased intestinal IGF-I expression would correlate with pISC expansion following ICR. To assess this, GLP-2 or vehicle was administered to mice either immediately after resection (early) or before tissue harvest 6 wk following ICR (late). Histological analysis quantified proliferation and intestinal morphometrics. Serum levels of GLP-2 were measured by ELISA and jejunal IGF-I mRNA by qRT-PCR. Expansion of jejunal pISC was assessed by fluorescent-activated cell sorting of side population cells, immunohistochemistry for phosphorylated [beta]-catenin at serine 552 (a pISC marker), percent of crypt fission, and total numbers of crypts per jejunal circumference. We found that early but not late GLP-2 treatment after ICR significantly augmented pISC expansion. Increases in jejunal IGF-I mRNA correlated temporally with early pISC expansion and effects of GLP-2. Early GLP-2 increased crypt fission and accelerated adaptive increases in crypt number and intestinal caliber. GLP-2 increased proliferation and intestinal morphometrics in all groups. This study shows that, in mice, GLP-2 promotes jejunal pISC expansion only in the period immediately following ICR. This is associated with increased IGF-I and accelerated adaptive increases in mucosal mass. These data provide clinical rationale relevant to the optimal timing of GLP-2 in patients with intestinal failure. intestinal adaptation

Human intestinal [CD3.sup.+][TCR[alpha][[beta].sup.+][CD8.sup.+] intraepithelial lymphocytes (IELs) are intimately associated with epithelial cells (ECs) through binding of CD103 to E-cadherin. How these two cell types functionally interact is largely unknown. IEL-EC cross talk was determined using HT-29 cells as the model EC and IL-8 as the readout. IL-8 was derived from both cell types and synergistically increased when the cells were combined. This synergistic effect required active transcription by both IELs and HT-29 cells. Cell contact was required as shown by the loss of the synergistic increase in IL-8 when the two cell types were separated by Transwells. Specifically, IL-8 release required the binding of CD2 on the IELs to CD58 on the HT-29 cells. The association of the CD3/TCR complex with major histocompatibility antigen class I antigens was not involved. Antibody neutralization of tumor necrosis factor-[alpha] (TNF-[alpha]), but not interferon-[gamma] (IFN-[gamma]), resulted in increased IL-8 production by the coculture. Although both TNF-[alpha] and IFN-[gamma] increased IL-8 synthesis and CD58 expression by the HT-29 cells, only IFN-[gamma] reduced IL-8 production by IELs. IL-8 production by either cell type involved phosphorylation of p38 and JNK. In summary, the synergistic synthesis of IL-8 occurs when IELs are stimulated through the CD2 pathway by CD58 on HT-29 cells, resulting in TNF-[alpha] release that, in turn, augments IL-8 synthesis and CD58 expression by the HT-29 cells. intraepithelial lymphocytes; epithelial cells; interleukin-8; tumor necrosis factor-[alpha]; interferon-[gamma]

Cellular retinol-binding protein type II (CRBPII) is abundantly expressed in the small intestinal enterocytes of many vertebrates and plays important physiological roles in intestinal absorption, transport, and metabolism of vitamin A. In the present study, we investigated regulation of human CRBPII gene expression using human intestinal Caco-2 BBe cells. We found that the human CRBPII gene contained a direct repeat 1 (DR-1-like nuclear receptor response element in the proximal promoter region and that endogenous hepatocyte nuclear factor-4[alpha] (HNF-4[alpha]) was a major transcription factor binding to the DR-1-like element. Cotransfection of HNF-4[alpha] expression vector transactivated the human CRBPII gene promoter activity, whereas mutation of the DR-1-like element abolished the promoter activity. Stably transfected Caco-2 BBe cells overexpressing HNF-4[alpha] significantly increased endogenous CRBPII gene expression and retinyl ester synthesis. Reduction of HNF-4[alpha] protein levels by HNF-4[alpha] small interference RNA decreased CRBPII gene expression. Caco-2 BBe cells treated with phorbol 12-myristate 13-acetate, a protein kinase C activator, decreased nuclear HNF-4[alpha] protein level and binding activity to the human CRBPII gene DR-1-like element, as well as CRBPII gene expression. Moreover, nuclear HNF-4[alpha] protein levels, HNF-4[alpha] protein binding to human CRBPII DR-1-like elements, and CRBPII gene expression level were coordinately increased during Caco-2 BBe cell differentiation. These results suggest that HNF-4[alpha] is an important transcriptional factor that regulates human CRBPII gene expression and provide the possibility for a novel function of HNF-4[alpha] in the regulation of human intestinal vitamin A absorption and metabolism. Caco-2 BBe cells; vitamin A; direct repeat-1; gene regulation

It has proved to be impossible to culture epithelial cells from the gastrointestinal tract of adult animals. Researchers have had to use either cell lines derived from newborn rat small intestine or colon carcinoma cell lines that have retained some of the properties of the gastrointestinal mucosa. We have described a method for establishing conditionally immortalized cell lines from the stomach, small intestine, colon, pancreas, and liver from tissue obtained from a transgenic mouse strain carrying a temperature-sensitive mutant of the SV40 large T gene (the 'Immortomouse'). This immortalizing gene has proved to be useful for establishing cell lines from a number of transgenic mice following crossbreeding of the Immortomouse with the transgenic mouse of interest. These cell lines are being used in numerous studies. In this review we describe the methods for developing such lines and list the range of cell lines that have been developed from colon, small intestine, stomach, liver, and pancreas of a number of transgenic mice. epithelium; intestine; mouse; tissue culture

The transport systems involved in intestinal methionine (Met) absorption are described as [Na.sup.+]-dependent and [Na.sup.+]-independent mechanisms. However, since recent studies have suggested the importance of the [H.sup.+] gradient as a driving force for intestinal nutrient absorption, the aim of the present work was to test whether Met transport across the apical membrane of Caco-2 cells is affected by extracellular pH. The results show that Land D-Met uptake was increased by lowering extracellular pH from 7.4 to 5.5, in both the presence and absence of [Na.sup.+]. Cis-inhibition experiments revealed that inhibition of L-Met transport by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) or L-lysine (L-Lys) was higher at a pH of 5.5. Moreover, the BCH-insensitive component was not affected by pH, whereas the L-Lys-insensitive component was increased by lowering extracellular pH, thus suggesting the participation of system L. The contribution of another mechanism, sensitive to both BCH and L-Lys, was also considered. The inhibition obtained with taurine (Tau) was also higher at a pH of 5.5, thus suggesting the involvement of system [B.sup.0,+] on pH-stimulated component. As for D-Met uptake, the results showed higher inhibition with L-Lys and Tau at a pH of 5.5 and no effect on the L-Lys- or Tau-insensitive component. In conclusion, Met transport across the apical membrane of Caco-2 cells is increased by low extracellular pH as the result of the stimulation of two transport systems functionally identified with systems L and [B.sup.0,+] for L-Met and with system [B.sup.0,+] for D-Met. intestine; amino acid transport; [H.sup.+] gradient

NHE8 transporter is a member of the sodium/hydrogen exchanger (NHE) family. This transporter protein is expressed at the apical membrane of epithelial cells of kidney and intestine and contributes to vectorial [Na.sup.+] transport in both tissues. Although NaCl absorption has been shown to be reduced in diarrhea associated with colitis and enteritis, little is known about the role of [Na.sup.+]/[H.sup.+] exchange and the involvement of NHE isoforms in the pathogenesis of inflammatory disorders and the mechanism of inflammation-associated diarrhea. This study investigated the role of NHE8 in the setting of inflammatory states. Jejunal mucosa was harvested from trinitrobenzene sulfonic acid (TNBS) colitis rats or lipopolysaccharide (LPS) rats for RNA extraction and brush-border membrane protein purification. The human NHE8 gene promoter was cloned from human genomic DNA and characterized in Caco-2 cells. The promoter was further used to study the mechanisms of TNF-[alpha]-mediated NHE8 expression down-regulation in Caco-2 cells. Results from Western blot and real-time PCR indicated that NHE8 protein and mRNA were significantly reduced in TNBS rats and LPS rats. In Caco-2 cells, TNF-[alpha] produces similar reduction levels in the endogenous NHE8 mRNA expression observed in our in vivo studies. The downregulation of NHE8 expression mediated by TNF-[alpha] could be blocked by transcription inhibitor actinomycin D, suggesting the involvement of transcriptional regulation. Further studies indicated that the human NHE8 gene transcription could be activated by Sp3 transcriptional factor, and TNF-[alpha] inhibits human NHE8 expression by reducing Sp3 interaction at the minimal promoter region of the human NHE8 gene. In conclusion, our studies suggest that TNF-[alpha] decreases NHE8 expression in inflammation induced by TNBS and LPS, which may contribute to the diarrhea associated with inflammation. trinitrobenzene sulfonic acid colitis; sodium/hydrogen exchanger 8; intestine

Enterocytes of the intestinal epithelium are continually regenerated. They arise from precursor cells in crypts, migrate along villi, and finally die, 3-4 days later, when they reach the villus apex. Their death is thought to occur by anoikis, a form of apoptosis induced by cell detachment, but the mechanism of this process remains poorly understood. We have previously shown that a key event in the onset of anoikis in normal enterocytes detached from the basal lamina is the disruption of adherens junctions mediated by E-cadherin (Fouquet S, Lugo-Martinez VH, Faussat AM, Renaud F, Cardot P, Chambaz J, Pincon-Raymond M, Thenet S. J Biol Chem 279: 43061-43069, 2004). Here we have further investigated the mechanisms underlying this disassembly of the adherens junctions. We show that disruption of the junctions occurs through endocytosis of E-cadherin and that this process depends on the tyrosine-kinase activity of the epidermal growth factor receptor (EGFR). Activation of EGFR was detected in detached enterocytes before E-cadherin disappearance. Specific inhibition of EGFR by tyrphostin AG-1478 maintained E-cadherin and its cytoplasmic partners [beta]- and [alpha]-catenin at cell-cell contacts and decreased anoikis. Finally, EGFR activation was evidenced in the intestinal epithelium in vivo, in rare individual cells, which were shown to lose their interactions with the basal lamina. We conclude that EGFR is activated as enterocytes become detached from the basal lamina, and that this mechanism contributes to the disruption of E-cadherin-dependent junctions leading to anoikis. This suggests that EGFR participates in the physiological elimination of the enterocytes. intestinal epithelial cells; cell-cell junctions; anoikis; epidermal growth factor receptor; E-cadherin

Monocarboxylate transporter (MCT1) plays an important role in the absorption of short-chain fatty acids (SCFA) such as butyrate in the human colon. Previous studies from our laboratory have demonstrated that phorbol ester, PMA (1 [micro]M, 24 h), upregulates butyrate transport and MCT1 protein expression in human intestinal Caco-2 cells. However, the molecular mechanisms involved in the transcriptional regulation of MCT1 gene expression by PMA in the intestine are not known. In the present study, we showed that PMA (0.1 [micro]M, 24 h) increased the MCT1 promoter activity (-871/+ 91) by approximately fourfold. A corresponding increase in MCT1 mRNA abundance in response to PMA was also observed. PMA-induced stimulation of MCT1 promoter activity was observed as early as 1 h and persisted until 24 h, suggesting that the effects of PMA are attributable to initial PKC activation. Kinase inhibitor and phosphorylation studies indicated that these effects may be mediated through activation of the atypical PKC-[xi] isoform. 5'-deletion studies demonstrated that the MCT1 core promoter region (-229/+91) is the PMA-responsive region. Site-directed mutagenesis studies showed the predominant involvement of potential activator protein 2 (AP2) binding site in the activation of MCT1 promoter activity by PMA. In addition, overexpression of AP2 in Caco-2 cells significantly increased MCT1 promoter activity in a dose-dependent manner. These findings showing the regulation of MCT1 promoter by PKC and AP2 are of significant importance for an understanding of the molecular regulation of SCFA absorption in the human intestine. short-chain fatty acid absorption; transcriptional regulation; human intestine; protein kinase C-[xi]; activator protein 2

The microcolony assay following gamma irradiation (IR) is a functional assay of intestinal stem cell fate. The cyclin-dependent kinase (CDK) inhibitor [p21.sup.Waf1/Cip1/Sdi1] (p21) regulates cell cycle arrest following DNA damage. To explore the role of p21 on stem cell fate, we examined the effects of p21 deletion on intestinal crypt survival following IR and expression of the stem/progenitor cell marker Musashi-1 (Msi-1) and the antiapoptotic gene survivin. Intestinal stem cell survival in adult wild-type (WT) and [p21.sup.-/-] mice was measured using the microcolony assay. Msi-1, p21, and survivin mRNA were measured using real-time PCR and immunohistochemical analysis. Laser capture microdissection (LCM) was used to isolate mRNA from the crypt stem cell zone. No differences in radiation-induced apoptosis were observed between WT and [p21.sup.-/-] mice. However, increased crypt survival (3.0-fold) was observed in [p21.sup.-/-] compared with WT mice 3.5 days after 13 Gy. Msi-1 and survivin mRNA were elevated 12-and 7.5-fold, respectively, in LCM-dissected crypts of [p21.sup.-/-] compared with WT mice. In conclusion, deletion of p21 results in protection of crypt stem/progenitor cells from IR-induced cell death. Furthermore, the increase in crypt survival is associated with increased numbers of Msi-1- and survivin-expressing cells in regenerative crypts. Musashi-1; gamma irradiation; survivin; crypt survival

Calcium absorption proceeds by transcellular and paracellular flux, with the latter accounting for most absorbed calcium when calcium intake is adequate. Vitamin D helps regulate transcellular calcium transport by increasing calcium uptake via a luminal calcium channel and by inducing the cytosolic calcium transporting protein, calbindin[D.sub.9k]. Recent studies utilizing knockout mice have challenged the functional importance of the channel and calbindin. To integrate the new findings with many previous studies, the function of the two molecules must be evaluated in the calcium transport and economy of mice. When calcium intake is high, transcellular calcium transport contributes little to total calcium absorption. Therefore, increasing calcium intake seems the most effective nutritional approach to ensure adequate absorption and prevent bone loss.

Humans cannot synthesize biotin and thus must obtain this vitamin from exogenous sources. The intestine is exposed to 2 sources of biotin: a dietary source and a bacterial source, which is normal microflora of the large intestine. Dietary protein-bound biotin is converted to free biotin prior to absorption. Absorption of free biotin in the small and large intestine involves a saturable and [Na.sup.+]-dependent carrier-mediated process that is shared with panthothenic acid and lipoate. For this reason, the involved transport system is referred to as the sodium-dependent multivitamin transporter (SMVT); in humans, it is designated as hSMVT. The hSMVT system has been cloned, demonstrated to be exclusively expressed at the apical membrane of enterocytes, and shown, by means of gene-specific short interfering RNA, to be the main biotin uptake system that operates in human intestinal epithelial cells. The 5'-regulatory region of the hSMVT gene has also been cloned and characterized both in vitro and in vivo. Further, the human intestinal biotin uptake process was adaptively up-regulated in biotin deficiency via a transcriptionally mediated mechanism(s) that involves Kruppel-like factor 4 sites. Studies on cell biology of hSMVT have shown a region in the cytoplasmic C-terminal domain of the polypeptide to be essential for its targeting to the apical membrane domain of epithelial cells. Intracellular trafficking of the hSMVT protein appears to involve distinct trafficking vesicles that require an intact microtubules network and the motor protein dynein for their mobility.

Intestine contributes to lipid homeostasis through the absorption of dietary lipids, which reach the apical pole of enterocytes as micelles. The present study aimed to identify the specific impact of these dietary lipid-containing micelles on gene expression in enterocytes. We analyzed, by microarray, the modulation of gene expression in Caco-2/TC7 ceils in response to different lipid supply conditions that reproduced either the permanent presence of albumin-bound lipids at the basal pole of enterocytes or the physiological delivery, at the apical pole, of lipid micelles, which differ in their composition during the interprandial (IPM) or the postprandial (PPM) state. These different conditions led to distinct gene expression profiles. We observed that, contrary to lipids supplied at the basal pole, apical lipid micelles modulated a large number of genes. Moreover, compared with the apical supply of IPM, PPM specifically impacted 46 genes from three major cell function categories: signal transduction, lipid metabolism, and cell adhesion/architecture. Results from this first large-scale analysis underline the importance of the mode and polarity of lipid delivery on enterocyte gene expression. They demonstrate specific and coordinated transcriptional effects of dietary lipid-containing micelles that could impact the structure and polarization of enterocytes and their functions in nutrient transfer. nutrients; alimentary lipids; mRNA; intestine

Tissue remodeling and mesenchymal cell accumulation accompanies chronic inflammatory disorders involving joints, lung, vasculature, and bowel. Chronic inflammation may alter DNA-mismatch repair (MMR) systems in mesenchymal cells, but is not defined in Crohn's disease (CD) and its associated intestinal remodeling and stricture formation. We determined whether DNA-MMR alteration plays a role in the pathogenesis of CD tissue remodeling. Control and CD bowel tissues were used to generate primary cultures of muscularis mucosa myofibroblasts, which were assessed directly or following stimulation with TNF-[alpha]/LPS or [H.sub.2][O.sub.2]. MutS homolog (MSH)2, MSH3, and MSH6 expression in tissues and myofibroblasts was determined. Immunohistochemical staining revealed an increased expression of MSH2 in CD muscularis mucosa and submucosal tissues compared with controls or uninvolved CD tissue, and MSH2 expression was increased in CD myofibroblasts compared with control cells. TNF-[alpha]/LPS and [H.sub.2][O.sub.2] further enhanced MSH2 expression in both control and CD cells, which were decreased by simvastatin. There were no significant changes in MSH3 and MSH6 expression. Proliferating cell nuclear antigen and Ki67 staining of CD tissue revealed increased proliferation in the muscularis mucosa and submucosa of chronically inflamed tissues, and enhanced proliferation was seen in CD myofibroblasts compared with controls. Simvastatin reversed the effects of inflammatory stress on the DNA-MMR and inhibited proliferation of control and CD myofibroblasts. Gene silencing with MSH2 siRNA selectively decreased CD myofibroblast proliferation. These data demonstrate a potential role for MSH2 in the pathogenesis of nonneoplastic mesenchymal cell accumulation and intestinal remodeling in CD chronic inflammation. ulcerative colitis; simvastatin; MSH2 siRNA

Segmentation in the guinea pig small intestine consists of a number of discrete motor patterns including rhythmic stationary contractions that occur episodically at specific locations along the intestine. The enteric nervous system regulates segmentation, but the exact circuit is unknown. Using simple computer models, we investigated possible circuits. Our computational model simulated the mean neuron firing rate in the feed-forward ascending and descending reflex pathways. A stimulus-evoked pacemaker was located in the afferent pathway or in a feedforward pathway. Output of the feedforward pathways was fed into a simple model to determine the response of the muscle. Predictions were verified in vitro by using guinea pig jejunum, in which segmentation was induced with luminal fatty acid. In the computational model, local stimuli produced an oral contraction and anal dilation, similar to in vitro responses to local distension, but did not produce segmentation. When the stimulus was distributed, representing a nutrient load, the result was either a tonic response or globally synchronized oscillations. However, when we introduced local variations in synaptic coupling, stationary contractions occurred around these locations. This predicts that severing the ascending and descending pathways will induce stationary contractions. An acute lesion in our in vitro model significantly increased the number of stationary contractions immediately oral and anal to the lesion. Our results suggest that spatially localized rhythmic contractions arise from a local imbalance between ascending excitatory and descending inhibitory muscle inputs and require a distributed stimulus and a rhythm generator in the afferent pathway. fed-state/postprandial motor pattern; computational modeling; enteric nervous system; myenteric plexus; enteric reflex circuits; feedforward networks

Differentiation of intestinal epithelial cells is accompanied by alterations in levels of expression of many genes, including those involved in nutrient uptake. Effects of differentiation of intestinal epithelial cells on the physiological and molecular parameters of the intestinal folate uptake process are not well characterized. To address this issue, we used two models, Caco-2 cells and native mouse intestine. Studies with Caco-2 cells showed a significant increase in the initial rate of carrier-mediated folic acid uptake during differentiation (i.e., as the cells transitioned from preconfluent to confluent and then to postconfluent stages). This increase was associated with an increase in the level of expression of the human reduced folate carrier (hRFC) and the human proton-coupled folate transporter (hPCFT) both at the protein and mRNA levels with differentiation; it was also associated with a significant increase in activity of the hRFC and hPCFT promoters. Studies with native mouse intestine showed a significantly higher folate uptake in villus compared with crypt cells, which was again associated with a significantly higher level of expression of the mouse RFC and PCFT at the protein and mRNA levels. Together, these studies demonstrate that the intestinal folate uptake process undergoes differentiation-dependent regulation and that this regulation is mediated via changes in the level of expression of both the RFC and PCFT. In addition, the studies suggest the possible involvement (at least in part) of a transcriptional mechanism(s) in this type of regulation of the intestinal folate uptake process. transport; epithelial transport; reduced folate carrier; proton-coupled folate transporter

In this study, the effect of feeding different types and amounts of dietary fiber (DF) on luminal environment and morphology in the small and large intestine of sows was studied. Three diets, a low-fiber diet (LF) and 2 high-fiber diets (high fiber 1, HF1, and high fiber 2, HF2) were used. Diet LF (DF, 17%; soluble DF 4.6%) was based on wheat and barley, whereas the 2 high-fiber diets (HF1: DF, 43%; soluble DF, 11.0%; and HF2: DF, 45%; soluble DF, 7.6%) were based on wheat and barley supplemented with different coproducts from the vegetable food and agroindustry (HF1 and HF2: sugar beet pulp, potato pulp, and pectin residue; HF2: brewers spent grain, seed residue, and pea hull). The diets were fed for a 4-wk period to 12 sows (4 receiving each diet). Thereafter, the sows were killed 4 h postfeeding, and digesta and tissue samples were collected from various parts of the small and large intestine. The carbohydrates in the LF diet were well digested in the small intestine, resulting in less digesta in all segments of the intestinal tract. The fermentation of nonstarch polysaccharides in the large intestine was affected by the chemical composition and physicochemical properties. The digesta from pigs fed the LF diet provided low levels of fermentable carbohydrates that were depleted in proximal colon, whereas for pigs fed the 2 high-DF diets, the digesta was depleted of fermentable carbohydrates at more distal locations of the colon. The consequence was an increased retention time, greater DM percentage, decreased amount of material, and a decreased tissue weight after feeding the LF diet compared with the HF diets. The concentration of short-chain fatty acids was consistent with the fermentability of carbohydrates in the large intestine, but there was no effect of the dietary composition on the molar short-chain fatty acid proportions. It was further shown that feeding the diet providing the greatest amount of fermentable carbohydrates (diet HF1, which was high in soluble DF) resulted in significant morphological changes in the colon compared with the LF diet. Key words: coproduct, carbohydrate, digestibility, luminal environment, morphology, sow

The intestine serves numerous purposes. In addition to digestion and absorption, the intestine functions as an organ that provides specific and nonspecific protection against pathological bacteria and noxious agents. At birth, and certainly when birth occurs prematurely, these functions are not yet fully developed. This article addresses the specific needs of the neonatal gut to perform these functions adequately and describes efforts to modulate intestinal barrier function by preterm formula supplemented with insulin-like growth factor 1.

Monoassociations of germ-free animals with colitogenic and probiotic bacterial strains trigger intestinal epithelial cell (IEC) activation and host-derived feedback mechanisms. To characterize the impact of a single nonpathogenic bacterial strain on the intestinal epithelium in the presence of an established microbiota, we inoculated reconstituted Lacotobacillus-free (RLF) mice at 8 wk of age with Lactobacillus reuteri 100-23. Primary IEC from the small intestine of L. reuteri-inoculated and control RLF mice were isolated 2, 6, and 21 d after inoculation followed by gene expression analysis (real-time PCR; Affymetrix microarrays) as well as 2-dimensional-gel electrophoreses (2D SDS-PAGE) and peptide mass fingerprinting via matrix-assisted laser desorption/ionization time of flight MS. At d 6, gene expression of proinflammatory cytokines and chemokines including interleukin (IL)-1[alpha], IL-6, interferon-[gamma],-inducible protein 10, and macrophage inflammatory protein 2 was transiently induced, whereas gene expression levels of regulatory proteins A20 and Toll-interacting protein decreased. In addition, 8 target proteins with changes in the steady-state protein expression levels were identified at d 2 and 6 of L. reuteri colonization. Consistent with the absence of histopathology, L. reuteri-induced activation of primary IEC returned to control levels by d 21 after inoculation of RLF mice. The capability of L. reuteri 100-23 to directly trigger epithelial cell activation was confirmed in small IEC cultures using the murine cell line Mode-K. These results clearly indicate that the intestinal epithelium is reactive toward environmental changes induced by the commensal bacterial strain L. reuteri even in the presence of an already-established microbiota. The induction of transient IEC activation may help to maintain mucosal homeostasis.

TNF is a pleiotropic cytokine that activates both anti- and proapoptotic signaling pathways, with cell fate determined by the balance between these two pathways. Activation of ErbB family members, including EGF receptor (EGFR/ErbB1), promotes cell survival and regulates several signals that overlap with those stimulated by TNF. This study was undertaken to determine the effects of TNF on EGFR and ErbB2 activation and intestinal epithelial cell survival. Mice, young adult mouse colon epithelial cells, and EGFR knockout mouse colon epithelial cells were treated with TNF. Activation of EGFR, ErbB2, Akt, Src, and apoptosis were determined in vivo and in vitro. TNF stimulated EGFR phosphorylation in young adult mouse colon epithelial cells, and loss of EGFR expression or inhibition of kinase activity increased TNF-induced apoptosis, which was prevented in WT but not by kinase-inactive EGFR expression. Similarly, TNF injection stimulated apoptosis in EGFR-kinase-defective mice ([EGFR.sup.wa2]) compared with WT mice. TNF also activated ErbB2, and loss of ErbB2 expression increased TNF-induced apoptosis. Furthermore, Src-kinase activity and the expression of both EGFR and ErbB2 were required for TNF-induced cell survival. Akt was shown to be a downstream target of TNF-activated EGFR and ErbB2. These findings demonstrate that EGFR and ErbB2 are critical mediators of TNF-regulated antiapoptotic signals in intestinal epithelial cells. Given evidence for TNF signaling in the development of colitis-associated carcinoma, this observation has significant implications for understanding the role of EGFR in maintaining intestinal epithelial cell homeostasis during cytokine-mediated inflammatory responses.

The very last member of the SLC2A gene family of facilitated hexose transporters to be cloned was SLC2A7 (hGLUT7). It has been assigned to the class II of the GLUT family on the basis of sequence similarity, and its closest family member is GLUT5, an intestinal fructose transporter. GLUT7 is primarily expressed in the small intestine and colon, although mRNA has been detected in the testis and prostate as well. The protein is expressed in the apical membrane of the small intestine and colon, and it has a high affinity ( glucose transporter proteins; SLC2A7; intestinal hexose absorption; structure-function

In a rabbit model of chronic intestinal inflammation, we previously demonstrated that the activity of Na-glucose cotransporter (SGLT1), SLC5A1, is inhibited. This inhibition is secondary to a decrease in the number of cotransporters, indicating that the regulation of SGLT1 during chronic inflammation is at the level of transcription. However, the regulation of SGLT1 expression and the transcription factors involved in the regulation are not yet known. In this report, we describe the cloning and characterization of rabbit SGLT1 promoter and the identification of transcription factors affected in villus cells during chronic intestinal inflammation. The promoter sequence for SGLT1 gene was identified by using the publicly available rabbit genomic sequence. Even though rabbit SGLT1 promoter did not have considerable overall homology with other mammalian SGLT1 promoters, two specificity protein 1 (Sp1) and a hepatocyte nuclear factor 1 (HNF1) binding sites were highly conserved among the species. Rabbit SGLT1 cDNA was encoded by 15 exons. Minimal promoter region determination showed that 196 nucleotides upstream of the transcription start site were sufficient for optimal promoter activity. This region encompassed two transcription factor binding sites, Sp1 and HNF1. For maximal SGLT1 promoter activity, these two transcription factor binding sites were essential, and their effect was synergistic, indicating that two separate regulatory pathways might be involved in their regulation. Using mobility shift assays, we further demonstrated that the binding of both Sp1 and HNF1 transcription factors to SGLT1 promoter regions were affected during chronic intestinal inflammation. Thus this report demonstrates that Sp1 and HNF1 transcription factors act in concert to regulate SGLT1 transcription in the chronically inflamed intestine. Na-glucose cotransporter 1; rabbit SGLT1 promoter; transcriptional regulation

Alterations in EGF receptor (EGFR) signaling occur in intestinal disorders associated with dysregulated epithelial transport. In the present study, we investigated a role for the EGFR in the chronic regulation of intestinal epithelial secretory function. Epithelial [Cl.sup.-] secretion was measured as changes in short-circuit current ([I.sup.sc]) across voltage-clamped monolayers of T84 cells in Ussing chambers. Acute treatment of T84 cells with EGF (100 ng/ml, 15 min) chronically enhanced [I.sub.sc] responses to a broad range of secretagogues. This effect was apparent within 3 h, maximal by 6 h, and sustained for 24 h after treatment with EGF. The [Na.sup.+]/[K.sup.+]/2[Cl.sup.-] cotransporter (NKCCl) inhibitor bumetanide (100 [micro]M) abolished the effect of EGF, indicating increased responses are due to potentiated [Cl.sup.-] secretion. Neither basal nor agonist-stimulated levels of intracellular [Ca.sup.2+] or PKA activity were altered by EGF, implying that the effects of the growth factor are not due to chronic alterations in levels of second messengers. EGF increased the expression of NKCC1 with a time course similar to that of its effects on [Cl.sup.-] secretion. This effect of EGF was maximal after 6 h, at which time NKCC1 expression in EGF-treated cells was 199.9 [+ or -] 21.9% of that in control cells (n = 21, P < 0.005). EGF-induced NKCCl expression was abolished by actinomycin D, and RT-PCR analysis demonstrated EGF increased expression of NKCCl mRNA. These data increase our understanding of mechanisms regulating intestinal fluid and electrolyte transport and reveal a novel role for the EGFR in the chronic regulation of epithelial secretory capacity through upregulation of NKCCl expression. epidermal growth factor; epithelium; chloride secretion; ion transport

Repetitive deformation due to villous motility or peristalsis may support the intestinal mucosa, stimulating intestinal epithelial proliferation under normal circumstances and restitution in injured and inflamed mucosa rich in tissue fibronectin. Cyclic strain enhances Caco-2 and IEC-6 intestinal epithelial cell migration across fibronectin via ERK. However, the upstream mediators of ERK activation are unknown. We investigated whether Src and FAK mediate strain-induced ERK phosphorylation and migration in human Caco-2 intestinal epithelial cells on fibronectin. Monolayers on tissue fibronectin-precoated membranes were subjected to an average 10% repetitive deformation at 10 cycles/min. Phosphorylation of Src-[Tyr.sup.418], FAK-[Tyr.sup.397]-[Tyr.sup.576]-[Tyr.sup.925], and ERK were significantly increased by deformation. The stimulation of wound closure by strain was prevented by Src blockade with PP2 (10 [micro]mol/l) or specific short interfering (si)RNA. Src inhibition also prevented strain-induced FAK phosphorylation at [Tyr.sup.397] and [Tyr.sup.576] but not FAK-[Tyr.sup.925] or ERK phosphorylation. Reducing FAK by siRNA inhibited strain-induced ERK phosphorylation. Transfection of N[H.sub.2]-terminal tyrosine phosphorylation-deficient FAK mutants Y397F, Y576F-Y577F, and Y397F-Y576F-Y577F did not prevent the activation of ERK2 by cyclic strain, but a FAK mutant at the COOH terminal (Y925F) prevented the strain-induced activation of ERK2. Although the Y397F-Y576F-Y577F FAK construct exhibited less basal FAK[Tyr.sup.925] phosphorylation under static conditions, it nevertheless exhibited increased FAK-[Tyr.sup.925] phosphorylation in response to strain. These results suggest that repetitive deformation stimulates intestinal epithelial motility across fibronectin in a manner that requires both Src activation and a novel Src-independent FAK-[Tyr.sup.925]-dependent pathway that activates ERK. This pathway may be an important target for interventions to promote mucosal healing in settings of intestinal ileus or fasting. migration; cyclic strain; intestine; mechanotransduction; signaling

Long distance transportation may affect the health of pigs; thus, adding a rest stop (lairage) during long journeys may improve their well-being. The objective of this study was to determine whether a mid-journey lairage influenced swine innate immunity and intestinal microbial populations after a 16-h transport. Four replications were conducted, 1 in each of 4 seasons. Eighteen-kilogram pigs were housed in 16 pens (13 to 16 pigs/pen) with 8 pens/treatment. Lairage pigs were transported for 8 h, given a rest with food and water for 8 h, then transported for an additional 8 h. Continuous pigs were continuously transported for 16 h. Jugular blood samples and intestinal tissue and contents were collected from 16 pigs (8/treatment) on d 1, 3, 7, and 14 posttransport. Hematocrit and white blood cell counts were determined and neutrophil cell functions, including phagocytosis/oxidative burst and phagocytosis of latex beads and leukocyte phenotypic cell markers (CD14 and CD18), were analyzed using flow cytometry. Expression of toll-like receptors 2, 4, and 5; IL-8 (a cytokine that is a chemoattractant for neutrophils); CCL20 (a chemokine that is a chemoattractant for dendritic cells); and the antimicrobial peptide PR39 were determined from ileal and jejunal total RNA. Denaturing gradient gel electrophoresis was used to determine shifts in intestinal microbial populations. Total white blood cell and granulocyte counts in continuous pigs were greater (P < 0.01) on d 1 than in lairage pigs. Phagocytosis of microbeads was greater in continuous (P < 0.05) than in lairage pigs on d 7. Expression of IL-8 in jejunum was greater (P < 0.05) for continuous than for lairage pigs on d 1. Expression of CCL20 in the ileum was greater (P < 0.05) on d 14 for the continuous pigs. Expression of PR39 was greatest (P < 0.05) in the jejunum of lairage pigs on d 3. Lairage pigs had a greater (P < 0.05) variation in microbial populations (lower similarity coefficient) in the jejunum contents on d i and 7, in the cecum contents and tissue on d 3, and in the jejunum contents and tissue on d 14. However, continuous pigs had greater (P < 0.05) variation in the ileal tissues on d 14. This study indicates that adding a lairage to an extended transport alters immune functions, receptor, cytokine and chemokine expression, and gut microbiota compared with pigs transported for 16 h without lairage. Key words: commensal bacteria, innate immunity, lairage, Salmonella, swine, transport

The effects of feeding different types of fiber to weanling pigs on growth performance, intestinal and liver cytokine expression, circulating acute-phase proteins, and IGF-I were evaluated. Intestinal tissue abundance of DNA, protein, and phosphorylated S6 kinase were also determined. Pigs (n = 120; initially 5.2 kg and 24 d of age) were randomly assigned to diets containing 1 of 4 fiber sources: 1) control diets containing no added fiber source, 2) diets containing 7.5% distillers dried grains with solubles (DDGS), 3) diets containing 7.5% soybean hulls, or 4) diets containing 7.5% citrus pulp. The experimental diets were fed for 4 wk in 2 phases (phase 1, wk 1 and 2; phase 2, wk 3 and 4). Intestinal tissue samples, liver samples, and blood samples were collected from a subset (n = 24; 6 pigs/treatment) of the pigs on d 7, and blood samples were collected from another subset (n = 24; 6 pigs/ treatment) of pigs on d 28 of the experiment. Dietary treatment had no effect on ADG, ADFI, or G:F throughout the experiment. Likewise, pig BW variability (CV), plasma IGF-I, or the plasma concentration of the acute phase proteins, [[alpha].sub.1]-acid glycoprotein, C-reactive protein, and haptoglobin, were not affected by dietary treatment. Real-time RT-PCR analysis revealed that on d 7, pigs fed DDGS had a greater (P < 0.05) relative abundance of the mRNA encoding IL-6, IL-1[beta], and IL-10 in ileum tissue than pigs fed all other diets. Diets containing DDGS had no effect on the relative abundance of tumor necrosis factor a or interferon-[gamma] mRNA in ileum tissue on d 7. The d-7 mRNA expression of cytokines was not altered in jejunum, colon, or liver tissue by dietary treatment. Intestinal tissue protein content or jejunum and ileum DNA concentrations were not affected by diet. Western blot analysis found no effect of dietary treatment on the activation of S6 kinase in jejunum, ileum, or colon tissue on d 7. These results indicate that feeding 7.5% of a fiber source as DDGS, soybean hulls, or citrus pulp does not affect growth performance or circulating markers of inflammation in weanling pigs and that feeding DDGS increases the expression of both proinflammatory and antiinflammatory cytokines in intestinal tissue. Key words: cytokine, fiber, nursery diet, pig

Under conditions of starvation and disease, the gut barrier becomes impaired, and trophic feeding to prevent gut mucosal atrophy has become a standard treatment of critically ill patients. However, the mechanisms responsible for the beneficial effects of enteral nutrition have remained a mystery. Using in vitro and in vivo models, we demonstrate that the brush-border enzyme, intestinal alkaline phosphatase (IAP), has the ability to detoxify lipopolysaccharide and prevent bacterial invasion across the gut mucosal barrier, IAP expression and function are lost with starvation and maintained by enteral feeding. It is likely that the lAP silencing that occurs during starvation is a key component of the gut mucosal barrier dysfunction seen in critically ill patients. gut barrier | lipopolysaccharide | trophic feeding | bacterial translocation | [NF.sub.[kappa]B

Although preterm birth and formula feeding increase the risk of necrotizing enterocolitis (NEC), the influences of cesarean section (CS) and vaginal delivery (VD) are unknown. Therefore, gut characteristics and NEC incidence and severity were evaluated in preterm pigs (92% gestation) delivered by CS or VD. An initial study showed that newborn CS pigs (n = 6) had decreased gastric acid secretion, absorption of intact proteins, activity of brush-border enzymes and pancreatic hydrolases, plasma cortisol, rectal temperature, and changes in blood chemistry, indicating impaired respiratory function, compared with VD littermates (n = 6). In a second experiment, preterm CS (n = 16) and VD (n = 16) pigs were given total parenteral nutrition (36 h) then fed porcine colostrum (VD-COL, n = 6; CS-COL, n = 6) or infant milk formula (VD-FORM, n = 10; CS-FORM, n = 10) for 2 days. Across delivery, FORM pigs showed significantly higher NEC incidence, tissue proinflammatory cytokines (IFN-7 and IL-6), Clostridium colonization, and impaired intestinal function, compared with COL pigs. NEC incidence was equal for CS (6/16) and VD (6/16) pigs, CS pigs had decreased bacterial diversity and density, higher villus heights, and increased brush-border enzyme activities (lactase, aminopeptidases) compared with VD pigs. In particular, VD-FORM pigs showed reduced mucosal proportions, reduced lactase and aminopeptidases, and increased proinflammatory cytokine IL-6 compared with CS-FORM (P < 0.06). Despite the initial improvement of intestinal and metabolic functions following VD, gut function, and inflammation were similar, or more negatively affected in VD neonates than CS neonates. Both delivery modes exhibited positive and negative influences on the preterm gut, which may explain the similar NEC incidence. vaginal; cesarean section; bacteria; birth; intestine

Multiple studies in animal models have shown that the commonly used food additive carrageenan (CGN) induces inflammation and intestinal neoplasia. We performed the first studies to determine the effects of CGN exposure on human intestinal epithelial cells (IEC)in tissue culture and tested the effect of very low concentrations (1-10 mg/L) of undegraded, high-molecular weight CGN. These concentrations of CGN are less than the anticipated exposure of the human colon to CGN from the average Western diet. In the human colonic epithelial cell line NCM460 and in primary human colonic epithelial cells that were exposed to CGN for 1-8 d, we found increased cell death, reduced cell proliferation, and cell cycle arrest compared with unexposed control cells. After 6-8 d of CGN exposure, the percentage of cells reentering G0-G1 significantly decreased and the percentages of cells in S and G2-M phases significantly increased. Increases in activated p53, p21, and p15 followed CGN exposure, consistent with CGN-induced cell cycle arrest. Additional data, including DNA ladder, poly ADP ribose polymerase Western blot, nuclear DNA staining, and activities of caspases 3 and 7, indicated no evidence of increased apoptosis following CGN exposure and were consistent with CGN-induced necrotic cell death. These data document for the first time, to our knowledge, marked adverse effects of low concentrations of CGN on survival of normal human IEC and suggest that CGN exposure may have a role in development of human intestinal pathology.

Previously we have demonstrated the use of [sup.1]H magic angle spinning (MAS) NMR spectroscopy for the topographical variations in functional metabolic signatures of intact human intestinal biopsy samples. Here we have analyzed a series of MAS [sup.1]H NMR spectra (spin--echo, one-dimensional, and diffusion-edited) and [sup.31]P--{[sup.1]H} spectra and focused on analyzing the enhancement of information recovery by use of the statistical total correlation spectroscopy (STOCSY) method. We have applied a heterospectroscopic cross-examination performed on the same samples and between [sup.1]H and [sup.31]P--{[sup.1]H} spectra (heteronuclear STOCSY) to recover latent metabolic information. We show that heterospectroscopic correlation can give new information on the molecular compartmentation of metabolites in intact tissues, including the statistical 'isolation' of a phosphlipid/triglyceride vesicle pool in intact tissue. The application of [sup.31]P--[sup.1]H HETSTOCSY allowed the cross-assignment of major [sup.31]P signals to their equivalent [sup.1]H NMR spectra, e.g., for phosphorylcholine and phosphorylethanolamine. We also show pathway correlations, e.g., the ascorbate--glutathione pathway, in the STOCSY analysis of intact tissue spectra. These [sup.31]P--[sup.1] HET-STOCSY spectra also showed different topographical regions, particular for minor signals in different tissue microenvironments. This approach could be extended to allow the detection of altered distributions within metabolic subcompartments as well as conventional metabonomics concentration-based diagnostics.

Glutamine is the most abundant amino acid in the body and is extensively taken up in gut and liver in healthy humans. To determine whether glucocorticosteroids alter splanchnic glutamine metabolism, the effect of prednisone was assessed in healthy volunteers using isotope tracer methods. Two groups of healthy adults received 5-h intravenous infusions of L-[1-[sup.14]C]leucine and L-[[sup.2][H.sub.5]]glutamine, along with q. 20 min oral sips of tracer doses of L-[[1-.sup.13]C]glutamine in the fasting state, either 1) at baseline (control group; n = 6) or 2) after a 6-day course of 0.8 mg*[kg.sup.-1]*[day.sup.-1] prednisone (prednisone group; n = 8). Leucine and glutamine appearance rates (Ra) were determined from plasma [[1-.sup.14]C]ketoisocaproate and [[sup.2][H.sub.5]]glutamine, respectively, and leucine and glutamine oxidation from breath [sup.14]C[O.sub.2] and [sup.13]C[O.sub.2], respectively. Splanchnic glutamine extraction was estimated by the fraction of orally administered [[sup.13]C]glutamine that failed to appear into systemic blood. Prednisone treatment 1) did not affect leucine Ra or leucine oxidation; 2) increased plasma glutamine Ra, mostly owing to enhanced glutamine de novo synthesis (medians [+ or -] interquartiles, 412 [+ or -] 61 vs. 280 [+ or -] 190 [micro]mol*[kg.sup.-1]*[h.sup.-1], P = 0.003); and 3)increased the fraction of orally administered glutamine undergoing extraction in the splanchnic territory (means [+ or -] SE 64 [+ or -] 6 vs. 42 [+ or -] 12%, P < 0.05), without any change in the fraction of glutamine oxidized (means [+ or -] SE, 75 [+ or -] 4 vs. 77 [+ or -] 4%, not significant). We conclude that high-dose glucocorticosteroids increase in splanchnic bed the glutamine requirements. The role of such changes in patients receiving chronic corticoid treatment for inflammatory diseases or suffering from severe illness remains to be determined. nutrition; protein metabolism; stable isotopes; stress; gut

Normal cellular models able to efficiently recapitulate intestinal epithelial cell differentiation in culture are not yet available. The aim of this work was to establish and genetically characterize a mesenchymal-epithelial coculture system to identify transcriptional regulators involved in this process. The deposition of rat intestinal epithelial cells on human intestinal mesenchymal cells led to the formation of clustered structures that expanded shortly after seeding. These structures were composed of polarized epithelial cells with brush borders and cell junction complexes. A rat GeneChip statistical analysis performed at different time points during this process identified hepatocyte nuclear factor-4[alpha] (HNF-4[alpha]) and hepatocyte nuclear factor-1[alpha] (HNF-1[alpha]) as being induced coincidently with the apparition of polarized epithelial structures. Stable introduction of HNF-4[alpha] in undifferentiated epithelial cells alone led to the rapid induction of HNF-1[alpha] and several intestinal-specific markers and metabolism-related genes for which mRNA was identified to be upregulated during epithelial differentiation. HNF-4[alpha] was capable to transactivate the calbindin 3 gene promoter, a process that was synergistically increased in the presence of HNF-l[alpha]. When HNF-4[alpha]-expressing cells were plated on mesenchymal cells, an epithelial monolayer formed rapidly with the apparition of dome structures that are characteristics of vectorial ion transport. Forced expression of HNF-1[alpha] alone did not result in dome structures formation. In sum, this novel coculture system functionally identified for the first time HNF-4[alpha] as an important modulator of intestinal epithelial differentiation and offers an innovative opportunity to investigate molecular mechanisms involved in this process. intestinal epithelial cells; mesenchymal cells; HNF-4[alpha]; HNF-1[alpha]; differentiation

The high rate of turnover of the intestinal epithelium is maintained by a group of stem cells that reside at the base of the crypts of Lieberkuhn. Whereas the existence of these intestinal epithelial stem cells has been well established, their study has been limited due to the inability to isolate them. Previous work has utilized side population (SP) sorting of the murine small intestine to isolate a viable fraction of cells enriched for putative intestinal epithelial stem cells. In the present study, we have used gene expression profiling techniques to characterize the molecular features of this potential stem cell population. Further in situ hybridization studies reveal that transcripts enriched in the SP tend to localize to the intestinal crypt base/progenitor cell zone, while de-enriched transcripts localize outside of this region. From a functional standpoint, gene ontology and pathway mapping analyses demonstrate that immune, mesenchymal, and differentiated epithelial cells are depleted in the SP fraction, while putative progenitor cells are enriched in this cell population. Furthermore, the significance of the maturity onset diabetes of the young pathway in these cells suggests that enteroendocrine progenitors are enriched in this cell fraction as well. In conclusion, SP sorting of mouse small intestinal mucosa does appear to isolate cells with progenitor characteristics. These findings provide the foundation for membrane protein-based sorting procedures that can be used to further fractionate these cells for transplantation experiments in the future. stem cells; epithelium; progenitor; microarray; functional genomics

The impact of an altered bacterial colonization on gut development has not been thoroughly studied, despite the increased risk of certain diseases with a disturbed microbiota after birth. This study was conducted to determine the effect of microbial manipulation, i.e., antibiotic treatment or Escherichia coli exposure, of the dam on bacterial colonization and gut development in the offspring. Pregnant rats were administered either broad-spectrum antibiotics 3 days before parturition or live nonpathogenic E. coli Culture Collection of University of G6teborg, Sweden type strain (CCUG [29300.sup.T]) 1 wk before parturition and up to 14 days of lactation in the drinking water. Cecal bacterial levels, gut growth, intestinal permeability, digestive enzyme levels, and intestinal inflammation were studied in 2-wk-old rats. Pups from dams that were antibiotic-treated had higher densities of Enterobacteriaceae, which correlated with a decreased stomach growth and function, lower pancreatic protein levels, higher intestinal permeability, and increased plasma levels of the acute phase protein, haptoglobin, compared with pups from untreated mothers. Exposure of pregnant/lactating mothers to E. coli CCUG [29300.sup.T], also resulting in increased Enterobacteriaceae levels, gave in the offspring similar results on the stomach and an increased small intestinal growth compared with the control pups. Furthermore, E. coli pups showed increased mucosal disaccharidase activities, increased liver, spleen, and adrenal weights, as well as increased plasma concentrations of haptoglobin. These findings indicate that disturbing the normal bacterial colonization after birth, by increasing the densities of cecal Enterobacteriaceae, appears to have lasting effects on the postnatal microflora, which affects gut growth and function. bacteria; gut permeability; Enterobacteriaceae; neonatal; intestine

Defecation in the nematode worm Caenorhabditis elegans is a highly rhythmic behavior that is regulated by a [Ca.sup.2+] wave generated in the 20 epithelial cells of the intestine, in part through activation of the inositol 1,4,5-trisphosphate receptor. Execution of the defecation motor program (DMP) can be modified by external cues such as nutrient availability or mechanical stimulation. To address the likelihood that environmental regulation of the DMP requires integrating distinct cellular and organismal processes, we have developed a method for studying coordinate [Ca.sup.2+] oscillations and defecation behavior in intact, freely behaving animals. We tested this technique by examining how mutations in genes known to alter [Ca.sup.2+] handling [including egl-8/phospholipase C (PLC)-[beta], kqt-3/KCNQ1, sca-1/sarco (endo)plasmic reticulum [Ca.sup.2+] ATPase, and unc-43/[Ca.sup.2+]-CaMKII] contribute to shaping the [Ca.sup.2+] wave and asked how [Ca.sup.2+] wave dynamics in the mutant backgrounds altered execution of the DMP. Notably, we find that [Ca.sup.2+] waves in the absence of PLC[beta] initiate ectopically, often traveling in reverse, and fail to trigger a complete DMP. These results suggest that the normal supremacy of the posterior intestinal cells is not obligatory for [Ca.sup.2+] wave occurrence but instead helps to coordinate the DMP. Furthermore, we present evidence suggesting that an underlying pacemaker appears to oscillate at a faster frequency than the defecation cycle and that arrhythmia may result from uncoupling the pacemaker from the DMP rather than from disrupting the pacemaker itself. We also show that chronic elevations in [Ca.sup.2+] have limited influence on the defecation period but instead alter the interval between successive steps of the DMP. Finally, our results demonstrate that it is possible to assess [Ca.sup.2+] dynamics and muscular contractions in a completely unrestrained model organism. calcium; oscillation; Caenorhabditis elegans; biosensor

We previously showed that phosphorylation of p70 $6 kinase (p[70.sup.S6k]) in the intestine is increased during viral enteritis. In this study, we hypothesized that during rotavirus infection, oral Arg, which stimulates p[70.sup.S6k] activation, will further stimulate intestinal protein synthesis and mucosal recovery, whereas the p70s6k inhibitor rapamycin (Rapa) will inhibit mucosal recovery. Newborn piglets were fed a standard milk replacer diet supplemented with Arg (0.4 g * [kg.sup.-1] * [d.sup.-1], twice daily by gavage), Rapa (2 mg * [m.sup.-2]. [d.sup.-1]), Arg + Rapa, or saline (controls). They were infected on d 6 of life with porcine rotavirus. Three days postinoculation, we measured the piglets' body weight, fecal rotavirus excretion, villus-crypt morphology, epithelial electrical resistance in Ussing chambers, and p[70.sup.S6k] activation by Western blotting and immunohistochemistry. We previously showed a 2-fold increase in jejunal protein synthesis during rotavirus diarrhea. In this experiment, Arg stimulated jejunal protein synthesis 1.3-fold above standard medium, and the Arg stimulation was partially inhibited by Rapa. Small bowel stimulation of p[70.sup.S6k] phosphorylation and p[70.sup.S6k] levels were inhibited >80% by Rapa. Immunohistochemistry revealed a major increase of p[70.sup.S6k] and ribosomal protein S6 phosphorylation in the crypt and lower villus of the infected piglets. However, in Arg-treated piglets, p[70.sup.S6k] activation occurred over the entire villus. Jejunal villi of the Rapa-treated group showed inactivation of p[70.sub.s6k] and a decrease in mucosal resistance (reflecting increased permeability), the latter of which was reversed by Arg. We conclude that, early in rotavirus enteritis, Arg has no impact on diarrhea but augments intestinal protein synthesis in part by p70s6k stimulation, while improving intestinal permeability via a mammalian target of rapamycin/p[70.sup.S6k]-independent mechanism.

Abrupt transfer of rainbow trout from freshwater to 65% seawater caused transient disturbances in extracellular fluid ionic composition, but homeostasis was reestablished 48 h posttransfer. Intestinal fluid chemistry revealed early onset of drinking and slightly delayed intestinal water absorption that coincided with initiation of NaC1 absorption and HC[O.sup.-.sub.3] secretion. Suggestive of involvement in osmoregulation, relative mRNA levels for vacuolar [H.sup.+]-ATPase (V-ATPase), [Na.sup.+]-[K.sup.+]-ATPase, [Na.sup.+]/[H.sup.+] exchanger 3 (NHE3), [Na.sup.+]-HC[O.sup.-.sub.3] cotransporter 1, and two carbonic anhydrase (CA) isoforms [a general cytosolic isoform trout cytoplasmic CA (tCAc) and an extracellular isoform trout membrane-bound CA type IV (tCAIV)], were increased transiently in the intestine following exposure to 65% seawater. Both tCAc and tCAIV proteins were localized to apical regions of the intestinal epithelium and exhibited elevated enzymatic activity after acclimation to 65% seawater. The V-ATPase was localized to both basolateral and apical regions and exhibited a 10-fold increase in enzymatic activity in fish acclimated to 65% seawater, suggesting a role in marine osmoregulation. The intestinal epithelium of rainbow trout acclimated to 65% seawater appears to be capable of both basolateral and apical [H.sup.+] extrusion, likely depending on osmoregulatory status and intestinal fluid chemistry. water absorption; HC[O.sup.-.sub.3] secretion; intestinal [H.sup.+] transport; tCAIV; tCAc

To better understand the role of human equilibrative (hENTs) and concentrative (hCNTs) nucleoside transporters in physiology and pharmacology, we investigated the regional, cellular, and spatial distribution of two hCNTs (hCNT1 and hCNT2) and two hENTs (hENT1 and hENT2) in four human tissues. Using in situ hybridization and immunohistochemical techniques, we found that the duodenum expressed hCNT1 and hCNT2 mRNAs in enterocytes and hENT1 and hENT2 mRNAs in crypt cells. In these cells, the hCNT and hENT proteins were predominantly localized in the apical and lateral membrane, respectively. Hepatocytes expressed higher levels of mRNAs of hENT1, hCNT1, and hENT2 than of hCNT2 and expressed all these proteins at hepatocyte cell borders and in the cytoplasm. While the kidney expressed hCNT1 and hCNT2 mRNAs in the proximal tubules, hENT1 and hENT2 mRNAs were present in the distal tubules, glomeruli, endothelial cells, and vascular smooth muscle cells. Proximal tubules adjacent to corticomedullary junctions expressed hENT1, hCNT1, and hCNT2 mRNA. Immunolocalization studies revealed predominant localization of hCNTs in the brush-border membrane of the proximal tubular epithelial cells and hENTs in the basolateral membrane of the distal tubular epithelial cells. Chorionic villi sections of human term placenta expressed mRNAs and proteins for hENT1 and hENT2 but only mRNA for hCNT2. Immunolocalization studies showed presence of hENT1 in the brush-border membrane of the syncytiotrophoblasts. These data are critical for a better understanding of the role of nucleoside transporters in the physiological and pharmacological effects of nucleosides and nucleoside drugs, respectively. expression; localization; human tissue