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Congenital diarrheal disorders (CDDs) are a collection of rare, heterogeneous enteropathies with early onset and often severe outcomes. Here, we report a family of Ashkenazi Jewish descent, with 2 out of 3 children affected by CDD. Both affected children presented 3 days after birth with severe, intractable diarrhea. One child died from complications at age 17 months. The second child showed marked improvement, with resolution of most symptoms at 10 to 12 months of age. Using exome sequencing, we identified a rare splice site mutation in the DGAT1 gene and found that both affected children were homozygous carriers. Molecular analysis of the mutant allele indicated a total loss of function, with no detectable DGAT1 protein or activity produced. The precise cause of diarrhea is unknown, but we speculate that it relates to abnormal fat absorption and buildup of DGAT substrates in the intestinal mucosa. Our results identify DGAT1 loss-of-function mutations as a rare cause of CDDs. These findings prompt concern for DGAT1 inhibition in humans, which is being assessed for treating metabolic and other diseases., Introduction Congenital diarrheal disorders (CDDs) are rare and heterogeneous enteropathies, often with severe clinical manifestations (1), (2). Those whose cause has been identified typically result from autosomal recessive mutations. Affected [...]

Intestinal enteroendocrine cells are critical to central regulation of caloric consumption, since they activate hypothalamic circuits that decrease appetite and thereby restrict meal size by secreting hormones in response to nutrients in the gut. Although guanylyl cyclase and downstream cGMP are essential regulators of centrally regulated feeding behavior in invertebrates, the role of this primordial signaling mechanism in mammalian appetite regulation has eluded definition. In intestinal epithelial cells, guanylyl cyclase 2C (GUCY2C) is a transmembrane receptor that makes cGMP in response to the paracrine hormones guanylin and uroguanylin, which regulate epithelial cell dynamics along the crypt-villus axis. Here, we show that silencing of GUCY2C in mice disrupts satiation, resulting in hyperphagia and subsequent obesity and metabolic syndrome. This defined an appetite-regulating uroguanylin-GUCY2C endocrine axis, which we confirmed by showing that nutrient intake induces intestinal prouroguanylin secretion into the circulation. The prohormone signal is selectively decoded in the hypothalamus by proteolytic liberation of uroguanylin, inducing GUCY2C signaling and consequent activation of downstream anorexigenic pathways. Thus, evolutionary diversification of primi-tive guanylyl cyclase signaling pathways allows GUCY2C to coordinate endocrine regulation of central food acquisition pathways with paracrine control of intestinal homeostasis. Moreover, the uroguanylin-GUCY2C endocrine axis may provide a therapeutic target to control appetite, obesity, and metabolic syndrome., Introduction Body mass reflects central regulation of caloric consumption by enteroendocrine cells that sense nutrients in the gastrointestinal tract and secrete hormones that activate hypothalamic circuits limiting meal size (1). [...]

Studies in embryonic development have guided successful efforts to direct the differentiation of human embryonic and induced pluripotent stem cells (PSCs) into specific organ cell types in vitro (1,2). For example, human PSCs have been differentiated into monolayer cultures of liver hepatocytes and pancreatic endocrine cells (3-6) that have therapeutic efficacy in animal models of liver disease (7,8) and diabetes (9), respectively. However, the generation of complex three-dimensional organ tissues in vitro remains a major challenge for translational studies. Here we establish a robust and efficient process to direct the differentiation of human PSCs into intestinal tissue in vitro using a temporal series of growth factor manipulations to mimic embryonic intestinal development (10). This involved activin-induced definitive endoderm formation (11), FGF/Wnt-induced posterior endoderm pattering, hindgut specification and morphogenesis (12-14), and a pro-intestinal culture system (15,16) to promote intestinal growth, morphogenesis and cytodifferentiation. The resulting three-dimensional intestinal 'organoids' consisted of a polarized, columnar epithelium that was patterned into villus-like structures and crypt-like proliferative zones that expressed intestinal stem cell markers (17). The epithelium contained functional enterocytes, as well as goblet, Paneth and enteroendocrine cells. Using this culture system as a model to study human intestinal development, we identified that the combined activity of WNT3A and FGF4 is required for hindgut specification whereas FGF4 alone is sufficient to promote hindgut morphogenesis. Our data indicate that human intestinal stem cells form denovoduring development. We also determined that NEUROG3, a pro-endocrine transcription factor that is mutated in enteric anendocrinosis (18), is both necessary and sufficient for human enteroendocrine cell development invitro. PSC-derived human intestinal tissue should allow for unprecedented studies of human intestinal development and disease., The epithelium of the intestine is derived from a simple sheet of cells called the definitive endoderm (17). As a first step to generating intestinal tissue from PSCs (summarized in [...]

Homeostasis of self-renewing small intestinal crypts results from neutral competition between Lgr5 stem cells, which are small cycling cells located at crypt bottoms (1,2). Lgr5 stem cells are interspersed between terminally differentiated Paneth cells that are known to produce bactericidal products such as lysozyme and cryptdins/defensins (3). Single Lgr5-expressing stem cells can be cultured to form long-lived, self-organizing crypt-villus organoids in the absence of non-epithelial niche cells (4). Here we find a close physical association of Lgr5 stem cells with Paneth cells in mice, both in vivo and in vitro. [CD24.sup.+] Paneth cells express EGF, TGF-α, Wnt3 and the Notch ligand Dll4, all essential signals for stem-cell maintenance in culture. Co-culturing of sorted stem cells with Paneth cells markedly improves organoid formation. This Paneth cell requirement can be substituted by a pulse of exogenous Wnt. Genetic removal of Paneth cells in vivo results in the concomitant loss of Lgr5 stem cells. In colon crypts, [CD24.sup.+] cells residing between Lgr5 stem cells may represent the Paneth cell equivalents. We conclude that Lgr5 stem cells compete for essential niche signals provided by a specialized daughter cell, the Paneth cell., In a Matrigel-based culture system containing EGF, the Wnt agonist R-spondin 1 and the BMP inhibitor noggin (4), single Lgr5 stem cells autonomously grow into crypt-like structures with de novo [...]

Intestinal stem cells (ISCs) in the Drosophila adult midgut are essential for maintaining tissue homeostasis and replenishing lost cells in response to tissue damage. Here we demonstrate that the Hippo (Hpo) signaling pathway, an evolutionarily conserved pathway implicated in organ size control and tumorigenesis, plays an essential role in regulating ISC proliferation. Loss of Hpo signaling in either midgut precursor cells or epithelial cells stimulates ISC proliferation. We provide evidence that loss of Hpo signaling in epithelial cells increases the production of cytokines of the Upd family and multiple EGFR ligands that activate JAK-STAT and EGFR signaling pathways in ISCs to stimulate their proliferation, thus revealing a unique non--cell-autonomous role of Hpo signaling in blocking ISC proliferation. Finally, we show that the Hpo pathway mediator Yorkie (Yki) is also required in precursor cells for injury-induced ISC proliferation in response to tissue-damaging reagent DSS. Yap | wts | regeneration doi/ 10.1073/pnas.1012759107

Structural components of the cell surface have an impact on some of the beneficial attributes of probiotic bacteria. In silico analysis of the L. acidophilus NCFM genome sequence revealed the presence of a putative cell surface protein that was predicted to be a myosin cross-reactive antigen (MCRA). As MCRAs are conserved among many probiotic bacteria, we used the upp-based counterselective gene replacement system, designed recently for use in L. acidophilus, to determine the functional role of this gene (LBA649) in L. acidophilus NCFM. Phenotypic assays were undertaken with the parent strain (NCK1909) and deletion mutant (NCK2015) to assign a function for this gene. The growth of NCK2015 (ALBA649) was reduced in the presence of lactate, acetate, porcine bile and salt. Adhesion of NCK2015 to Caco-2 cells was substantially reduced for both stationary-phase (~45% reduction) and exponential-phase cells (~50% reduction). Analysis of NCK2015 by scanning electron microscopy revealed a longer cell morphology after growth in MRS broth compared to NCK1909. These results indicate a role for LBA649 in stress tolerance, cell wall division and adherence to Caco-2 cells. DOI 10.1099/mic.0.043158-0

Studies have demonstrated that angiotensin II (Ang II) can regulate intestinal fluid and electrolyte transport and control intestinal wall muscular activity. Ang II is also a proinflammatory mediator that participates in inflammatory responses such as apoptosis, angiogenesis, and vascular remodeling; accumulating evidence suggests that this hormone may be involved in gastrointestinal (GI) inflammation and carcinogenesis. Ang II binds to two distinct G protein-coupled receptor subtypes, the AT1R and AT2R, which are widely expressed in the GI system. Together these studies suggest that Ang II-AT1R/-AT2R actions may play an important role in GI tract physiology and pathophysiology. Currently it is not known whether miRNAs can regulate the expression of the human AT1R (hAT1R) in the GI system. PCR and in situ hybridization experiments demonstrated that miR-802 was abundantly expressed in human colon and intestine. Luciferase reporter assays demonstrated that miR-802 could directly interact with the bioinformatics-predicted target site harbored within the 3'-untranslated region of the hAT1R mRNA. To validate that the levels of miR-802 were physiologically relevant in the GI system, we demonstrated that miR-802 'loss-offunction' experiments resulted in augmented hAT1R levels and enhanced Ang II-induced signaling in a human intestinal epithelial cell line. These results suggest that miR-802 can modulate the expression of the hATeR in the GI tract and ultimately play a role in regulating the biological efficacy of Ang II in this system. gastrointestinal system; miRNAs doi: 10.1152/ajpgi.00120.2010.

Early epithelial restitution is an important repair modality in the gut mucosa and occurs as a consequence of epithelial cell migration. Canonical transient receptor potential-1 (TRPC1) functions as a store-operated [Ca.sup.2+] channel (SOCs) in intestinal epithelial cells (IECs) and regulates intestinal restitution, but the exact upstream signals initiating TRPC1 activation after mucosal injury remain elusive. Stromal interaction molecule 1 (STIM1) is a single membrane-spanning protein and is recently identified as essential components of SOC activation. The current study was performed to determine whether STIM1 plays a role in the regulation of intestinal epithelial restitution by activating TRPC1 channels. STIM1 translocation to the plasma membrane increased after wounding, which was followed by an increase in IEC migration to reseal wounds. Increased STIM1 levels at the plasma membrane by overexpressing EF-hand mutant STIM1 enhanced [Ca.sup.2+] influx through SOCs and stimulated IEC migration after wounding. STIM1 interacted with TRPC1 and formed STIM1/TRPC1 complex, whereas inactivation of STIM1 by STIM1 silencing decreased SOC-mediated [Ca.sup.2+] influx and inhibited epithelial restitution. In cells overexpressing EF-hand mutant STIM1, TRPC1 silencing also decreased STIM1/ TRPC1 complex, reduced SOC-mediated [Ca.sup.2+] influx, and repressed cell migration after wounding. Our findings demonstrate that induced STIM1 translocation to the plasma membrane promotes IEC migration after wounding by enhancing TRPC1-mediated [Ca.sup.2+] signaling and provide new insight into the mechanism of intestinal epithelial restitution. mucosal injury; early rapid repair; cell migration; capacitative [Ca.sup.2+] entry; intestinal epithelial cells; surface biotinylation doi: 10.1152/ajpcell.00066.2010.

Surprisingly few pathways signal between cells, raising questions about mechanisms for tissue-specific responses. In particular, Wnt ligands signal in many mammalian tissues, including the intestinal epithelium, where constitutive signaling causes cancer. Genomewide analysis of DNA cis-regulatory regions bound by the intestine-restricted transcription factor CDX2 in colonic cells uncovered highly significant overrepresentation of sequences that bind TCF4, a transcriptional effector of intestinal Wnt signaling. Chromatin immunoprecipitation confirmed TCF4 occupancy at most such sites and cooccupancy of CDX2 and TCF4 across short distances. A region spanning the single nucleotide polymorphism rs6983267, which lies within a MYC enhancer and confers colorectal cancer risk in humans, represented one of many co-occupied sites. Co-occupancy correlated with intestine-specific gene expression and CDX2 loss reduced TCF4 binding. These results implicate CDX2 in directing TCF4 binding in intestinal cells. Co-occupancy of regulatory regions by signal-effector and tissue-restricted transcription factors may represent a general mechanism for ubiquitous signaling pathways to achieve tissue-specific outcomes. context specificity of signaling | genome-wide chromatin immunoprecipitation | signaling | Wnt signaling in intestine | tissue-specific gene regulation doi/ 10.1073/pnas.1003822107

Hyperplasia of smooth muscle contributes to the thickening of the intestinal wall that is characteristic of inflammation, but the mechanisms of growth control are unknown. Nitric oxide (NO) from enteric neurons expressing neuronal NO synthase (nNOS) might normally inhibit intestinal smooth muscle cell (ISMC) growth, and this was tested in vitro. In ISMC from the circular smooth muscle of the adult rat colon, chemical NO donors inhibited [[sup.3]H]thymidine uptake in response to FCS, reducing this to baseline without toxicity. This effect was inhibited by the guanylyl cyclase inhibitor ODQ and potentiated by the phosphodiesterase-5 inhibitor zaprinast. Inhibition was mimicked by 8-bromo (8-Br)-cGMP, and ELISA measurements showed increased levels of cGMP but not cAMP in response to sodium nitroprusside. However, 8-Br-cAMP and cilostamide also showed inhibitory actions, suggesting an additional role for cAMP. Via a coculture model of ISMC and myenteric neurons, immunocytochemistry and image analysis showed that innervation reduced bromodeoxyuridine uptake by ISMC. Specific blockers of nNOS (7-NI, NAAN) significantly increased [[sup.3]H]thymidine uptake in response to a standard stimulus, showing that nNOS activity normally inhibits ISMC growth. In vivo, nNOS axon number was reduced threefold by day 1 of trinitrobenzene sulfonic acid-induced rat colitis, preceding the hyperplasia of ISMC described earlier in this model. We conclude that NO can inhibit ISMC growth primarily via a cGMP-dependent mechanism. Functional evidence that NO derived from nNOS causes inhibition of ISMC growth in vitro predicts that the loss of nNOS expression in colitis contributes to ISMC hyperplasia in vivo. colitis; growth inhibition; intestine nitric oxide; neuronal nitric oxide synthase; cGMP; cAMP doi: 10.1152/ajpgi.00259.2009.

Intestinal epithelium is a rapidly self-renewing tissue in the body, and its homeostasis is tightly regulated by numerous factors including polyamines. Decreased levels of cellular polyamines increase activating transcription factor (ATF)-2, but the exact role and mechanism of induced ATF-2 in the regulation of intestinal epithelial cell (IEC) growth remain elusive. Cyclin-dependent kinase (CDK) 4 is necessary for the G1-to-S phase transition during the cell cycle, and its expression is predominantly controlled at the transcription level. Here, we reported that induced ATF-2 following polyamine depletion repressed CDK4 gene transcription in IECs by increasing formation of the ATF-2/JunD heterodimers. ATF-2 formed complexes with JunD as measured by immunoprecipitation using the ATF-2 and JunD antibodies and by glutathione S-transferase (GST) pull-down assays using GST-ATF-2 fusion proteins. Studies using various mutants of GST-ATF-2 revealed that formation of the ATF-2/JunD dimers depended on the COOH-terminal basic region-leucine zipper domain of ATF-2. Polyamine depletion increased ATF-2/JunD complex and inhibited CDK4 transcription as indicated by a decrease in the levels of CDK4-promoter activity and its mRNA. ATF-2 silencing not only prevented inhibition of CDK4 transcription in polyamine-deficient cells but also abolished repression of CDK4 expression induced by ectopic JunD overexpression. ATF-2 silencing also promoted IEC growth in polyamine-depleted cells. These results indicate that induced ATF-2/JunD association following polyamine depletion represses CDK4 transcription, thus contributing to the inhibition of IEC growth. activating protein-1; gene expression; cell proliferation; growth arrest; ornithine decarboxylase; [alpha]-difluoromethylornithine doi: 10.1152/ajpcell.00021.2010.

Mutations in the Nod2 gene are among the strongest genetic risk factors in the pathogenesis of ileal Crohn's disease, but the exact contributions of Nod2 to intestinal mucosal homeostasis are not understood. Here we show that Nod2 plays an essential role in controlling commensal bacterial flora in the intestine. Analysis of intestinal bacteria from the terminal ilea of Nod2-deficient mice showed that they harbor an increased load of commensal resident bacteria. Furthermore, Nod2-deficient mice had a diminished ability to prevent intestinal colonization of pathogenic bacteria. In vitro, intestinal crypts isolated from terminal ilea of Nod2-deficient mice were unable to kill bacteria effectively, suggesting an important role of Nod2 signaling in crypt function. Interestingly, the expression of Nod2 is dependent on the presence of commensal bacteria, because mice re-derived into germ-free conditions expressed significantly less Nod2 in their terminal ilea, and complementation of commensal bacteria into germ-free mice induced Nod2 expression. Therefore, Nod2 and intestinal commensal bacterial flora maintain a balance by regulating each other through a feedback mechanism. Dysfunction of Nod2 results in a break-down of this homeostasis. commensal bacteria | Crohn's disease | mouse | NLR

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

Several studies are conducted for the characterization of endocrine-cerebro-osteodysplasia (ECO), a multiplex human syndrome. The syndrome is shown to implicate a very important role for the intestinal cell kinase in the development of the central nervous, skeletal and the endocrine systems.

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 enteric serotonin transporter (SERT) plays a critical role in modulating serotonin availability and thus has been implicated in the pathogenesis of various intestinal disorders. To date, SERT expression and function in the human intestine have not been investigated. Current studies were designed to characterize the function, expression, distribution, and membrane localization of SERT in the native human intestine. Real-time PCR studies showed relatively higher SERT mRNA expression in the human small intestine compared with colon (ileum [much greater than] duodenum [much greater than] jejunum). Northern blot analysis revealed three mRNA hybridizing species encoding SERT (3.0, 4.9, and 6.8 kb) in the hurhan ileum. Consistent with SERT mRNA expression, SERT immunostaining was mainly detected in the epithelial cells of human duodenal and ileal resected tissues. Notably, SERT expression was localized predominantly to the apical and intracellular compartments and was distributed throughout the crypt-villus axis. Immunoblotting studies detected a prominent protein band (~70 kDa) in the ileal apical plasma membrane vesicles (AMVs) isolated from mucosa obtained from organ-donor intestine. Functional studies showed that uptake of [[sup.3]H]serotonin (150 nM) in human ileal AMVs was 1) significantly increased in the presence of both [Na.sup.+] and C[I.sup.-]; 2) inhibited (~50%) by the neuronal SERT inhibitor, fluoxetine (10 [micro]M) and by unlabeled 5-HT; and 3) exhibited saturation kinetics indicating the presence of a carrier-mediated process. Our studies demonstrated differential expression of SERT across various regions of the human intestine and provide evidence for the existence of a functional SERT capable of removing intraluminal serotonin in human ileal epithelial cells. serotonin uptake; serotonin transporter expression

Changes in diet greatly affect the mucosal immune system, particularly in diseases such as Crohn's disease and necrotizing enterocolitis. This article examines the hypothesis that alterations in the luminal environment of the intestine regulate the expression of genes in the enterocyte responsible for signaling to immune cells. Genes expressed by the epithelium orchestrate leukocytes in the lamina propria. For example, chemokine expression in the mouse intestinal epithelium, through transgenic means, induced the recruitment of neutrophils and lymphocytes into intestinal tissues. Diet alters the expression of the genes responsible for signaling by a variety of pathways. The introduction of a normal diet to a weanling mouse up-regulates MHC class II expression through a particular isoform of the class II transactivator, a protein that acts in the nucleus. SCFA concentrations in the intestinal lumen vary markedly with diet. SCFAs increase IL-8 and insulin-like growth factor binding protein-2 expression by inhibiting histone deacetylase activity in the enterocyte. Down-regulation of gene expression by butyrate can act through acetylation of the inhibitory transcription factor Sp3. The review therefore describes a number of molecular pathways, explaining how changes in diet may alter leukocyte recruitment by regulating enterocyte gene expression. Myofibroblasts enhance enterocyte chemotactic activity by cleaving inactive precursors; and myofibroblast genes also are regulated by SCFA. It is likely that other similar regulatory mechanisms remain to be discovered.

Nucleotide oligomerization domain (NOD) 2 functions as a mammalian cytosolic pathogen recognition molecule, and mutant forms have been genetically linked to Crohn's disease (CD). NOD2 associates with the caspase activation and recruitment domain of RIP-like interacting caspase-like apoptosis regulatory protein kinase (RICK)/RIP2 and activates nuclear factor (NF)-[kappa]B in epithelial cells and macrophages, whereas NOD2 mutant 3020insC, which is associated with CD, shows an impaired ability to activate NF-[kappa]B. To gain insight into the molecular mechanisms of NOD2 function, we performed a functional analysis of deletion and substitution NOD2 mutants. NOD2, but not NOD2 3020insC mutant, associated with cell surface membranes of intestinal epithelial cells. Membrane targeting and subsequent NF-[kappa]B activation are mediated by two leucine residues and a tryptophan-containing motif in the COOH-terminal domain of NOD2. The membrane targeting of NOD2 is required for NF-[kappa]B activation after the recognition of bacterial muramyl dipeptide in intestinal epithelial cells.

Inactivation of Cdx2 by homologous recombination results in the development of forestomach epithelium at ectopic sites in pericaecal areas of the midgut of heterozygote mice. Local factors subsequently result in the secondary induction of tissues exhibiting an orderly sequence of tissue types between the ectopic forestomach tissue and the surrounding colon. Clonal analysis of this secondarily generated tissue using Y chromosome painting in chimaeric mice indicates that once differentiated to express Cdx2, host colonic epithelium can only form small intestinal-type epithelium, while Cdx2 mutant cells give rise to a succession of gastric-type tissue but never to a small intestine morphology. Our results indicate a difference in potency between forestomach and midgut precursor endodermal cells. Keywords: Cdx2; Homeobox; Intercalation; Gut development

Clinical expression of Hirschsprung disease (HSCR) requires the interaction of multiple susceptibility genes. Molecular genetic analyses have revealed that interactions between mutations in the genes encoding the RET receptor tyrosine kinase and the endothelin receptor type B (EDNRB) are central to the genesis of HSCR. We have established two locus noncomplementation assays in mice, using allelic series at Ednrb in the context of Ret kinase-null heterozygotes, to understand the clinical presentation, incomplete penetrance, variation in length of aganglionic segment, and sex bias observed in human HSCR patients. Titration of Ednrb in the presence of half the genetic dose of Ret determines the presentation of an enteric phenotype in these strains, revealing or abrogating a sex bias in disease expression depending on the genotype at Ednrb. RET and EDNRB signaling pathways are also critical for the normal development of other tissues, including the kidneys and neural crest-derived melanocytes. Our data demonstrate that interaction between these genes is restricted to the enteric nervous system and does not affect renal, coat color, and retinal choroid development.

Proliferation and carcinogenesis of the large intestinal epithelial cells (IEC) cells is significantly increased in transgenic mice that overexpress the precursor progastrin (PG) peptide. It is not known if the in vivo growth effects of PG on IEC cells are mediated directly or indirectly. Full-length recombinant human PG (rhP[G.sub.1-80]) was generated to examine possible direct effects of PG on IEC cells. Surprisingly, rhPG (0.1-1.0 nM) was more effective than the completely processed gastrin 17 (G17) peptide as a growth factor. Even though IEC cells did not express CC[K.sub.1] and CC[K.sub.2] receptors (-R), fluorescently labeled G17 and Gly-extended G17 (G-Gly) were specifically bound to the cells, suggesting the presence of binding proteins other than CC[K.sub.1]-R and CC[K.sub.2]-R on IEC cells. High-affinity ([K.sub.d] = 0.5-1.0 nM) binding sites for [sup.I]-rhPG were discovered on IEC cells that demonstrated relative binding affinity for gastrinlike peptides in the order PG [greater than or equal to] COOH-terminally extended G17 [greater than or equal to] G-Gly > G17 > *CCK-8 (* significant difference; P < 0.05). In conclusion, our studies demonstrate for the first time direct growth effects of the full-length precursor peptide on IEC cells in vitro that are apparently mediated by the high-affinity PG binding sites that were discovered on these cells. progastrin-preferring receptors; confocal microscopy; in vitro growth effects; Gly-gastrin

Descriptive studies of phoronid development have concluded that the mesoderm of these animals originates from the endoderm during gastrulation. This interpretation has been tested by labeling one blastomere of 4- through 16-cell embryos and examining the position and germ layers occupied by the labeled clones of cells in the larva. No 2 injections gave rise to identical clones of cells, suggesting that the cleavage program does not generate cells of unique identity and that cell fates are established at later developmental time points. In many cases, a relatively large sector composed of ectodermal cells was labeled. When these labeled cells were adjacent to the mouth or anus of the larva, muscle and mesenchyme cells originated from the labeled clones. Under these circumstances, nerve cells also originated from these labeled sectors. These labeling studies also showed that endodermal cells can give rise to mesodermal and neural cells. These results suggest that nerve and muscle cells are induced to form at ectodermal-endodermal boundaries from both germ layers. These marking experiments also confirmed the observation that nerve cells originate both from the apical organ and the trunk region and show for the first time that the intestine originates by ingression of posterior ectoderm. Key Words: actinotroch larva; cell labeling; origin of mesoderm; origin of neural cells; origin of intestine; interactions at germ layer boundaries.

Iron deficiency is the most common nutritional disorder worldwide, whereas pathologic elevations of body iron stores can occur under certain circumstances due to genetic abnormalities or in association with other diseases. The intestine is the exclusive locus of homeostatic regulation of body iron stores, which is accomplished by changes in iron absorption efficiency by largely unknown molecular mechanisms in response to alterations in body iron stores. Recently, a number of novel genes involved in iron metabolism, such as the iron uptake transporter DMT1/DCT1/Nramp2 and the iron export transporter IREG1/ferroportin1/MTP1, have been identified, providing important insights about molecular aspects of intestinal iron absorption and its regulation. The aim of this study was to investigate the effects of iron treatment on DMT1 and IREG1 mRNA expression in Caco-2 cells, a human intestinal cell line. Exposure of the cells to iron (200 [micro]mol/L ferric nitrilotriacetic acid for 72 h) significantly decreased transferrin receptor mRNA (80%), DMT1 mRNA (57%) and IREG1 mRNA (52%). These observations are consistent with the notion of parallel regulation of these iron-responsive genes in vivo to protect the enterocyte from iron toxicity and mediate a decreased efficiency of intestinal iron absorption to prevent iron overload. J. Nutr. 132: 693-696, 2002. KEY WORDS: * iron absorption * ferroportin1 * DCT1 * Nramp2 * iron responsive protein * MTP1

Mammalian nutritional status affects the homeostatic balance of multiple physiological processes and their associated gene expression. Although DNA array analysis can monitor large numbers of genes, there are no reports of expression profiling of a micronutrient deficiency in an intact animal system. In this report, we have tested the feasibility of using cDNA arrays to compare the global changes in expression of genes of known function that occur in the early stages of rodent zinc deficiency. The gene-modulating effects of this deficiency were demonstrated by real-time quantitative PCR measurements of altered mRNA levels for metallothionein 1, zinc transporter 2, and uroguanylin, all of which have been previously documented as zinc-regulated genes. As a result of the low level of inherent noise within this model system and application of a recently reported statistical tool for statistical analysis of microarrays [Tusher, V.G., Tibshirani, R. & Chu, G. (2001) Proc. Natl. Acad. Sci. USA 98, 5116-5121], we demonstrate the ability to reproducibly identify the modest changes in mRNA abundance produced by this single micronutrient deficiency. Among the genes identified by this array profile are intestinal genes that influence signaling pathways, growth, transcription, redox, and energy utilization. Additionally, the influence of dietary zinc supply on the expression of some of these genes was confirmed by real-time quantitative PCR. Overall, these data support the effectiveness of cDNA array expression profiling to investigate the pleiotropic effects of specific nutrients and may provide an approach to establishing markers for assessment of nutritional status. nutrition | microarray | micronutrient

We have previously shown that the Caenorhabditis elegans gut-specific esterase gene (Ce-ges-1) has the unusual ability to be expressed in different modules of the embryonic digestive tract (anterior pharynx, posterior pharynx, and rectum) depending on sequence elements within the Ce-ges-1 promoter. In the present paper, we analyze the expression of the ges-1 homolog (Cb-ges-1) from the related nematode Caenorhabditis briggsae and show that Cb-ges-1 also has the ability to switch expression between gut and pharynx + rectum. The control of this expression switch centres on a tandem pair of WGATAR sites in the Cb-ges-1 5'-flanking region, just as it does in Ce-ges-1. We use sequence alignments and subsequent deletions to identify a region at the 3'-end of both Ce-ges-1 and Ce-ges-1 that acts as the ges-1 cryptic pharynx enhancer whose activity is revealed by removal of the 5' WGATAR sites. This region contains a conserved binding site for PHA-4 (the C. elegans ortholog of forkhead/HNF3 [alpha], [beta], [gamma] factors), which is expressed in all cells of the developing pharynx and a subset of cells of the developing rectum. We propose a model in which the normal expression of ges-1 is controlled by the gut-specific GATA factor ELT-2. We propose that, in the pharynx (and rectum), PHA-4 is normally bound to the ges-1 3'-enhancer sequence but that the activation function of PHA-4 is kept repressed by a (presently unknown) factor binding in the vicinity of the 5' WGATAR sites. We suggest that this control circuitry is maintained in Caenorhabditis because pharyngeal expression of ges-1 is advantageous only under certain developmental or environmental conditions. Key Words: Caenorhabditis elegans; Caenorhabditis briggsae; ges-1; gene expression; intestine; pharynx; elt-2; pha-4.

The RET tyrosine kinase receptor isoforms and differential activities in mammalian embryogenesis are discussed. Targeted mutagenesis was used to generate mice that express RET9 or RET51. Some RET9 transgenes can rescue kidney agenesis in RET-deficient mice or can cause kidney hypodysplasia in wild-type animals. Mice without RET9 lack enteric ganglia from the colon. Human mutations of c-RET bring on Hirschsprung's disease.

The molecular mechanisms of intestinal alkaline phosphatase gene transcription were investigated using sodium butyrate-treated HT-29 cells as an in vitro model system. Transient transfection assays that utilize human intestinal alkaline phosphatase-CAT reporter genes together with DNase I footprinting were employed in the localization of a critical cis element corresponding to the sequence 5'-GACTGGGCGGGGTCAAGATGGA-3'. A significant reduction in reporter gene activity was observed after deletion of the cis element, with the element functioning in the context of a heterologous promoter in a cell-type specific manner.

We evaluated the effects of the probiotic Escherichia coli Nissle 1917 (EcN) and the antibiotic Ciprofloxacin (Cipro) on mRNA expression of intestinal epithelial cells (IEC) in gnotobiotic (Gn) piglets colonized with a defined commensal microflora (DMF) and inoculated with human rotavirus (HRV) that infects IECs. We analyzed mRNA levels of IEC genes for enteroendocrine cells [chromogranin A (CgA)], goblet cells [mucin 2 (MUC2)], transient amplifying progenitor cell [proliferating cell nuclear antigen (PCNA)], intestinal epithelial stem cell (SOX9) and enterocytes (villin). Cipro treatment enhanced HRV diarrhea and decreased the mRNA levels of MUC2 and villin but increased PCNA. These results suggest that Cipro alters the epithelial barrier, potentially decreasing the numbers of mature enterocytes (villin) and goblet cells secreting protective mucin (MUC2). These alterations may induce increased IEC proliferation (PCNA expression) to restore the integrity of the epithelial layer. Coincidental with decreased diarrhea severity in EcN treated groups, the expression of CgA and villin was increased, while SOX9 expression was decreased representing higher epithelial integrity indicative of inhibition of cellular proliferation. Thus, EcN protects the intestinal epithelium from damage by increasing the gene expression of enterocytes and enteroendocrine cells, maintaining the absorptive function and, consequently, decreasing the severity of diarrhea in HRV infection. Keywords: Probiotic, Antibiotic, Commensal microflora, Gnotobiotic piglets, Human rotavirus, Intestinal epithelial cell-specific genes, Author(s): Francine C. Paim[sup.1] , Stephanie N. Langel[sup.1] , David D. Fischer[sup.1] , Sukumar Kandasamy[sup.1] , Lulu Shao[sup.1,2] , Moyasar A. Alhamo[sup.1] , Huang-Chi Huang[sup.1] , Anand Kumar[sup.1,3] , Gireesh [...]

The Caenorhabditis elegans elt-2 gene encodes a single-finger GATA factor, previously cloned by virtue of its binding to a tandem pair of GATA sites that control the gut-specific ges-1 esterase gene. In the present paper, we show that elt-2 expression is completely gut specific, beginning when the embryonic gut has only two cells (one cell cycle prior to ges-1 expression) and continuing in every cell of the gut throughout the life of the worm. When elt-2 is expressed ectopically using a transgenic heat-shock construct, the endogenous ges-1 gene is now expressed in most if not all cells of the embryo; several other gut markers (including a transgenic elt-2-promoter::1acZ reporter construct designed to test for elt-2 autoregulation) are also expressed ectopically in the same experiment. These effects are specific in that two other C. elegans GATA factors (elt-1 and elt-3) do not cause ectopic gut gene expression. An imprecise transposon excision was identified that removes the entire elt-2 coding region. Homozygous elt-2 null mutants die at the L1 larval stage with an apparent malformation or degeneration of gut cells. Although the loss of elt-2 function has major consequences for later gut morphogenesis and function, mutant embryos still express ges-1. We suggest that elt-2 is part of a redundant network of genes that controls embryonic gut development; other factors may be able to compensate for elt-2 loss in the earlier stages of gut development but not in later stages. We discuss whether elements of this regulatory network may be conserved in all metazoa. Key Words: C. elegans; GATA factor; elt-2; gut development.

Immunohistochemical and immunogold labeling electron microscopy was utilized to determine the distribution and function of cysteine-rich intestinal protein (CRIP) in rat IEC-6 intestinal epithelial cell lines. Analysis of rats IEC-6 indicated the presence of CRIP in the cytoplasmic granules of Paneth cells and epithelium of the villus tip. Furthermore, CRIP was also detected in murine RAW 264.7 mononuclear macrophage cell lines indicating the role of the protein in mediating host defense and tissue differentiation.

The secretagogue-stimulated Cl- transport activity of T84 colonocytes are enhanced by the interleukin-1alpha (IL-1alpha)-induced stimulation of the subepithelial myofibroblasts (SEMF) colonic and jejunal cell lines. Enhanced cyclooxygenase gene expression initiate the action of IL-1alpha-activated neurotransmitter and inflammatory mediators. Furthermore, IL-1alpha utilizes Ca2+ to activate the mediators that sensitizes Cl- secretion in enterocytes and stimulate the production of prostaglandin E2.

The expression of hepatocyte nuclear factors 1-alpha (HNF) and HNF-1-beta mRNA during growth and along the length of the intestine was studied. This study was aimed at determining the role of the two HNF-1 factors in the characterization of various intestinal epithelial cell lineages in channelizing alpha-fetoprotein (AFP) to enteroendocrine cells. The HNF factors were found to be expressed along the vertical axis of the intestine, gradiently.

Introduction The intestine is one of the most diverse and complex bacterial habitats of the human body, harboring ~1,000 bacterial phylotypes [1]. Recent studies have associated the human intestinal microbiome [...]

Characterization of the homeoprotein-containing regulatory complexes, when it comes, will be helpful to elucidation of molecular mechanisms that underlie the control of morphological diversity by Hox genes in animal development. Effects of hexapeptide mutations in vivo on targets that are well defined will make the investigation of the HOX proteins possible in the absence of cooperativity. Mutations in homeodomains that get rid of DNA binding may give some insight into composition of the complex on in vivo targets.

The quantification of the mucin-degrading bacterium Akkermansia muciniphila found in the human intestinal tract is demonstrated. The bacterium colonizes in early life, develops in a year in adults and decreases in the elderly.

Using monolayers of human intestinal (Caco-2) cells, we found that oxidants and ethanol damage the cytoskeleton and disrupt barrier integrity; epidermal growth factor (EGF) prevents damage by enhancement of protein kinase C (PKC) activity and translocation of the PKC-[Beta]1 isoform. To see if PKC-[Beta]1 mediates EGF protection, cells were transfected to stably over- or underexpress PKC-[Beta]1. Transfected monolayers were preincubated with low or high doses of EGF (1 or 10 ng/ml) or 1-oleoyl-2-acetyl-sn-glycerol [OAG; a PKC activator (0.01 or 50 [micro]M)] before treatment with oxidant (0.5 mM [H.sub.2][O.sub.2]). Only in monolayers overexpressing PKC-[Beta]1 (3.1-fold) did low doses of EGF or OAG initiate protection, increase tubulin polymerization (assessed by quantitative immunoblotting) and microtubule architectural integrity (laser scanning confocal microscopy), maintain normal barrier permeability (fluorescein sulfonic acid clearance), and cause redistribution of PKC-[Beta]1 from cytosolic pools into membrane and/or cytoskeletal fractions (assessed by immunoblotting), thus indicating PKC-[Beta]1 activation. Antisense inhibition of PKC-[Beta]1 expression (-90%) prevented these changes and abolished EGF protection. We conclude that EGF protection against oxidants requires PKC-[Beta]1 isoform activation. This mechanism may be useful for development of novel therapies for the treatment of inflammatory gastrointestinal disorders including inflammatory bowel disease. tubulin; cytoskeleton; growth factors; paracellular permeability; fluorescein sulfonic acid clearance; Caco-2 cells; protein kinase C transfection, epidermal growth factor

Differential activation of intestinal gene promoters: functional interactions between GATA-5 and HNF-1[Alpha]. Am J Physiol Gastrointest Liver Physiol 281: G69-G84, 2001.--The effects of GATA-4, -5, and -6, hepatocyte nuclear factor-1[Alpha] (HNF-1[Alpha] and -[Beta], and Cdx-2 on the rat and human lactase-phlorizin hydrolase (LPH) and human sucrase-isomaltase (SI) promoters were studied using transient cotransfection assays in Caco-2 cells. GATA factors and HNF-1[Alpha] were strong activators of the LPH promoters, whereas HNF-1[Alpha] and Cdx-2 were strong activators of the SI promoter, although GATA factors were also necessary for maximal activation of the SI gene. Cotransfection of GATA-5 and HNF-1[Alpha] together resulted in a higher activation of all three promoters than the sum of the activation by either factor alone, demonstrating functional cooperativity. In the human LPH promoter, an intact HNF-1 binding site was required for functional synergy. This study is the first to demonstrate 1) differential activation of the LPH and SI promoters by multiple transcription factors cotransfected singly and in combination and 2) that GATA and HNF-1 transcription factors cooperatively activate intestinal gene promoters. Synergistic activation is a mechanism by which higher levels of tissue-specific expression might be attained by overlapping expression of specific transcription factors. lactase-phlorizin hydrolase; sucrase-isomaltase; Cdx-2; hepatocyte nuclear factor-1

We aim to study the development of endoderm and the differentiation of the intestinal epithelium in mice, using embryonic gut culture, ES cell differentiation, and mouse models. We have established an innovative mouse embryonic gut culture system (caternary culture) in our laboratory that maintains the three-dimensional architecture of the gut. This provides an opportunity to directly study the function of genes in a system that closely mimics the development and differentiation of the gut in vivo. The development of the epithelial cell layer in caternary cultures has been studied in detail using light and electron microscopy and immunohistochemistry for molecular markers of gut development. We have also demonstrated that plasmids encoding genes can be introduced into the epithelial cell layer using a low-voltage electroporation technique. The effects of expressing mutant versions of components of the APC, Ras, and TGF[Beta] signaling pathways on intestinal cell biology will be examined using this system. Along side this work, we also aim to identify novel genes that can promote intestinal differentiation using a unique screening strategy in embryonic stem (ES) cells. This is based on a unique ES cell line generated in our laboratory that contains a LacZ marker gene under the control of the A33 promoter (a definitive marker of intestinal epithelium). The ultimate objective of this project is to screen an expression library for genes that can promote intestinal differentiation.

To investigate whether human intestinal epithelial cell survival involves distinct control mechanisms depending on the state of differentiation, we analyzed the in vitro effects of insulin, pharmacological inhibitors of Fak, MEK/Erk, and PI3-K/Akt, and integrin ([Beta]1, [Beta]4)-blocking antibodies on the survival of the well-established human Caco-2 enterocyte-like and HIEC-6 cryptlike cell models. In addition, relative expression levels of six Bcl-2 homologs (Bcl-2, Bcl-[X.sub.L], Mcl-1, Bax, Bak, and Bad) and activation levels of Fak, Erk-2, and Akt were analyzed. Herein, we report that 1) the enterocytic differentiation process results in the establishment of distinct profiles of Bcl-2 homolog expression levels, as well as [p125.sup.Fak], [p42.sup.Erk-2], and [p57.sup.Akt] activated levels; 2) the inhibition of Fak, of the MEK/ Erk pathway, or of PI3-K, have distinct impacts on enterocytic cell survival in undifferentiated (subconfluent Caco-2, confluent HIEC-6) and differentiated (30 days postconfluent Caco-2) cells; 3) exposure to insulin and the inhibition of Fak, MEK, and PI3-K resulted in differentiation state-distinct modulations in the expression of each Bcl-2 homolog analyzed; and 4) Fak, [Beta]1 and [Beta]4 integrins, as well as the MEK/ Erk and PI3-K/Akt pathways, are distinctively involved in cell survival depending on the state of cell differentiation. Taken together, these data indicate that human intestinal epithelial cell survival is regulated according to differentiation state-specific control mechanisms. anoikis; apoptosis; gut; intestine; signal transduction

Abstract Background and aims--Inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by nitric oxide (NO) in intestinal preconditioning could modify the rate of formation of glycolytic intermediates. Fructose-1,6-biphosphate (F16BP) is a glycolytic intermediate [...]

GATA family transcription factors activate lactase gene promoter in intestinal Caco-2 cells. Am J Physiol Gastrointest Liver Physiol 280: G58-G67, 2001.--The GATA family of transcription factors regulate tissue-specific patterns of gene expression during development. We have characterized the interaction between GATA proteins and the lactase gene promoter. Nuclear protein bound to the lactase gene GATA region cis element (-97 to -73) was analyzed by electrophoretic mobility shift assays (EMSA) and supershift assays with GATA antibodies. Lactase promoter activities were assayed in Caco-2 cells transfected with wild-type and mutated luciferase promoter-reporter constructs and GATA-4/5/6 expression constructs. EMSA with the GATA region probe yields a specific DNA-protein complex that requires the GATA factor binding site WGATAR. The complex is recognized by GATA-4- and GATA-6-specific antibodies. GATA-4/ 5/6 expression constructs are able to activate transcription driven by the wild-type promoter, but not by a promoter in which the GATA binding site is mutated, in Caco-2 and nonintestinal QT6 cells. GATA factor binding to the lactase cis element correlates with functional promoter activation. We conclude that each of the GATA family zinc finger proteins expressed in the intestine, GATA-4, -5, and -6, can interact with the lactase promoter GATA element and can function to activate the promoter in Caco-2 cells. GATA-4; GATA-5; GATA-6; enterocyte

This study was conducted to investigate the influence of dietary zinc on intestinal apoB mRNA editing in hamsters. Apolipoprotein B-48 (apoB-48) is synthesized from the same gene as apoB-100 by a post-transcriptional, site-specific cytidine deamination, a process known as apoB mRNA editing, A cDNA encoding the hamster apoB mRNA editing enzyme was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) and the deduced amino acid sequence was found to possess high amino acid sequence identity to apoB mRNA editing enzymes from several other species. Editing activity was detected in the small intestine and colon but, like humans, none was detected in the liver. Analysis by RT-PCR indicated that the small intestine possessed the highest expression of editing enzyme mRNA abundance, whereas both liver and small intestine expressed relatively high levels of apoB mRNA. The influence of dietary zinc on intestinal apoB mRNA editing levels was examined in Golden Syrian hamsters (7 wk old) assigned to one of the following three dietary treatments: Zn-adequate (ZA, 30 mg Zn/kg diet), Zn-deficient (ZD, [is less than] 0.5 mg Zn/kg diet), or Zn-replenished (ZDA, ZD hamsters receiving ZA diet for last 2 d) for 7 wk. Hamsters consuming the ZD diet had modestly but significantly lower intestinal editing activity than ZA hamsters. Intestinal editing activity in the ZDA group, was not different from that of ZA hamsters. Data derived from these studies contribute to the understanding of lipoprotein metabolism in hamsters, a suitable model for the study of atherosclerosis. J. Nutr. 130: 2166-2173, 2000. KEY WORDS: * hamsters * dietary zinc * cardiovascular disease * cholesterol * low density lipoprotein

Chatterjee, Nabendu S., Chandira K. Kumar, Alvaro Ortiz, Stanley A. Rubin, and Hamid M. Said. Molecular mechanism of the intestinal biotin transport process. Am. J. Physiol. 277 (Cell Physiol. 46): C605-C613, 1999.--Previous studies have characterized different aspects of the cellular/ membrane mechanism and regulation of the intestinal uptake process of the water-soluble vitamin biotin. Little, however, is known about the molecular mechanisms of the uptake process. In this study, we have identified a cDNA from rat small intestine that appears to be involved in biotin transport. The open reading frame of this cloned cDNA consisted of 1,905 bases and was identical to that identified for the vitamin transporter in placental tissue. Significant heterogeneity, however, was found in the 5' untranslated region of this clone, with three distinct variants (II, III, IV) being identified in the small intestine; the placental variant (variant I), however, was not present in the small gut. Variant II was found to be the predominant form expressed in the rat small and large intestines. Functional identity of the cloned intestinal cDNA was confirmed by stable expression in COS-7 cells, which showed a four- to fivefold increase in biotin uptake in transfected COS-7 cells compared with controls. The induced biotin uptake in transfected COS-7 cells was found to be 1) [Na.sup.+] dependent, 2) saturable as a function of concentration with an apparent [K.sub.m] of 8.77 [Micro]M and a [V.sub.max] of 779.7 pmol.mg [protein.sup.-1] [multiplied by] 3 [min.sup.-1], and 3) inhibited by unlabeled biotin and pantothenic acid and their structural analogs. The distribution of complementary mRNA transcripts of the cloned cDNA along the vertical and longitudinal axes of the intestinal tract was also determined. Results of this study describe the molecular characteristics of the intestinal biotin absorption process and report the identification of a cDNA that encodes a [Na.sup.+]-dependent biotin uptake carrier that appears to exist in the form of multiple variants. water-soluble vitamin; molecular transport mechanism; 5' untranslated region; reverse transcriptase-polymerase chain reaction