Oxytocin is expressed throughout the human gastrointestinal tract

BACKGROUND/AIM: Several studies have described that oxytocin exerts stimulatory or inhibitory effects on gut functions. Recently, mRNA for oxytocin and its receptor was found throughout the entire human gastrointestinal (GI) tract. The aim of this study was to examine the cellular localization and distribution of the corresponding proteins. MATERIAL AND METHODS: Full-thickness biopsies from 24 patients, covering the entire GI tract, were collected during operations at the Department of Surgery in Malm? and Lund. The biopsies were taken from non_affected margins. The biopsies were fixed by immersion, rinsed in buffered sucrose, and kept frozen at 70 degrees C. Indirect immunofluorescence with primary antibodies to oxytocin and its receptor was used. RESULTS: Oxytocin was expressed in nerve cell bodies and nerve fibres in the myenteric and submucous ganglia all along the GI tract. Immunoreactive nerve cell bodies in myenteric ganglia predominated in the proximal (antrum and duodenum) and distal gut, while those in the submucous ganglia were more numerous in the ileum and colon. The oxytocin receptor was not detectable by two different antibodies in any tissue in the GI tract. CONCLUSION: Oxytocin is expressed in the myenteric and submucous ganglia and nerve fibres along the entire human GI tract. The role for oxytocin in the physiology and pathophysiology of the bowel remains to be settled.     

Comparative expression of hexose transporters (SGLT1, GLUT1, GLUT2 and GLUT5) throughout the mouse gastrointestinal tract

Hexose transporters play a pivotal role in the absorption of food-derived monosaccharides in the gastrointestinal tract. Although a basic knowledge of the hexose transporters has already been gained, their detailed distribution and comparative intensities of expression throughout the gastrointestinal tract have not been fully elucidated. In this study, we quantitatively evaluated the expression of SGLT1, GLUT1, GLUT2, and GLUT5 by in situ hybridization and real-time PCR techniques using a total of 28 segments from the gastrointestinal tract of 9-week-old mice. GLUT2 and GLUT5 mRNA expressions were detected predominantly from the proximal to middle parts of the small intestine, showing identical expression profiles, while SGLT1 mRNA was expressed not only in the small intestine but also in the large intestine. Notably, GLUT1 mRNA was expressed at a considerable level in both the stomach and large intestine but was negligible in the small intestine. Immunohistochemistry demonstrated the polarized localization of hexose transporters in the large intestine: SGLT1 on the luminal surface and GLUT1 on the basal side of epithelial cells. The present study provided more elaborate information concerning the localization of hexose transporters in the small intestine. Furthermore, this study revealed the significant expression of glucose transporters in the large intestine, suggesting the existence of the physiological uptake of glucose in that location in mice.     

Constitutive expression and induction of CYP1B1 mRNA in the mouse.

Previous studies appeared to indicate that CYP1B1 was not constitutively expressed in mouse liver. In our laboratory, we demonstrated using aromatic hydrocarbon-responsive receptor knock-out (AHR-(-)/-) mice that both piperonyl butoxide (PBO) and acenaphtyhlene (ACN) are AHR-independent inducers of murine CYP1A2 and CYP1B1 mRNA. In the current study, we demonstrate both constitutive levels and induction of CYP1B1 in mouse liver. The induction of CYP1B1 mRNA by PBO or ACN was higher in DBA/2 (Ahrd) than in C57BL/6 (Ahrb-1) mice, while 3-methylcholanthrene induced CYP1B1 more in C57BL/6 than in DBA/2 mice. These results suggest that CYP1B1 may also be induced by more than one mechanism. In addition, constitutive expression of CYP1B1 was detected in liver, kidney, and lung of untreated C57BL/6 mice. There was no gender difference in CYP1B1 expression; however, in C57BL/6 mice, the kidney contained less CYP1B1 than either liver or lung.     

Two alternatively spliced mouse urokinase receptor mRNAs with different histological localization in the gastrointestinal tract

Two mouse urokinase-type plasminogen activator receptor (muPAR) cDNAs were isolated: muPAR1 is homologous to the human urokinase-type plasminogen activator receptor while muPAR2 codes for a 199 residue protein sharing the first 133 residues with muPAR1. Mouse genomic DNA sequencing indicates that the two different mRNAs arise by alternative splicing. In situ hybridization showed differential expression of the two mRNAs in mouse gastric mucosa. muPAR1 mRNA is located in luminal epithelial cells situated close to urokinase-type plasminogen activator-producing connective tissue cells of the lamina propria, pointing to plasmin generation controlled by the cooperation of different cells that may play a role in the release of gastric epithelial cells. muPAR2 mRNA is expressed in the basal epithelial cells, and the deduced protein sequence includes the receptor ligand binding domain, but omits the region involved in glycolipid-mediated membrane anchoring, suggesting that muPAR2 may code for a secreted uPA binding protein.     

The protein chaperone Ssa1 affects mRNA localization to the mitochondria

Many nuclear-transcribed mRNAs encoding mitochondrial proteins are localized near the mitochondrial outer membrane. A yet unresolved question is whether protein synthesis is important for transport of these mRNAs to their destination. Herein we present a connection between mRNA localization in yeast and the protein chaperone Ssa1. Ssa1 depletion lowered mRNA association with mitochondria while its overexpression increased it. A genome-wide analysis revealed that Ssa proteins preferentially affect mRNAs encoding hydrophobic proteins, which are expected targets for these protein chaperones. Importantly, deletion of the mitochondrial receptor Tom70 abolished the impact of Ssa1 overexpression on mRNAs encoding Tom70 targets. Taken together, our results suggest a role for Ssa1 in mediating localization of nascent peptide-ribosome-mRNA complexes to the mitochondria, consistent with a co-translational transport process.     

Identification and localization of soluble sulfotransferases in the human gastrointestinal tract

Soluble SULTs (sulfotransferases) are important in the regulation of messenger molecules and the elimination of xenobiotics. However, sulfo-conjugation of various substrates can also lead to the formation of reactive metabolites that may induce cancer and cause other damage. The aim of the present study was to identify the SULT forms expressed in the human gastrointestinal tract, especially the colon and rectum (common sites for cancer), and to determine their cellular localization. Normal colonic or rectal tissue, resected with tumours, was obtained from 39 subjects. For comparison, we additionally studied one to four samples from stomach, jejunum, ileum, cecum and liver. SULTs were detected by immunoblotting, immunohistochemistry and measurement of enzyme activities. SULT1A1, 1A3 and 1B1 were found in all parts of the gastrointestinal tract, often exceeding levels in liver (where these forms were present at high, undetectable and low levels respectively). They were predominantly localized in differentiated enterocytes. SULT1E1 and 2A1 were only detected in liver, jejunum, ileum and cecum. SULT1C1 was readily found in stomach, but was negligible elsewhere. SULT1A2 was present at low levels in individual samples. The remaining forms were not detected with the limitation that only high levels could be recognized with the antisera used. In conclusion, SULTs are abundant in the gastrointestinal tract of man. We suspect that they are involved in the presystemic elimination of bioactive food-borne components, including aglycones released by gut microbiota, as well as the bioactivation of some procarcinogens.     

The yeast Cbk1 kinase regulates mRNA localization via the mRNA-binding protein Ssd1

The mRNA-binding protein Ssd1 is a substrate for the Saccharomyces cerevisiae LATS/NDR orthologue Cbk1, which controls polarized growth, cell separation, and cell integrity. We discovered that most Ssd1 localizes diffusely within the cytoplasm, but some transiently accumulates at sites of polarized growth. Cbk1 inhibition and cellular stress cause Ssd1 to redistribute to mRNA processing bodies (P-bodies) and stress granules, which are known to repress translation. Ssd1 recruitment to P-bodies is independent of mRNA binding and is promoted by the removal of Cbk1 phosphorylation sites. SSD1 deletion severely impairs the asymmetric localization of the Ssd1-associated mRNA, SRL1. Expression of phosphomimetic Ssd1 promotes polarized localization of SRL1 mRNA, whereas phosphorylation-deficient Ssd1 causes constitutive localization of SRL1 mRNA to P-bodies and causes cellular lysis. These data support the model that Cbk1-mediated phosphorylation of Ssd1 promotes the cortical localization of Ssd1-mRNA complexes, whereas Cbk1 inhibition, cellular stress, and Ssd1 dephosphorylation promote Ssd1-mRNA interactions with P-bodies and stress granules, leading to translational repression.     

The ultrastructural localization and quantitation of cholinergic receptors at the mouse motor endplate

Summary The snake venom toxin, -bungarotoxin, is known to bind specifically to the acetylcholine receptor at skeletal muscle endplates. In this study, tritiated -bungarotoxin has been used in conjunction with electron-microscope autoradiography to visualize and enumerate acetylcholine receptor sites at the neuromuscular junctions of the mouse diaphragm. From an analysis of the grain distribution, the receptor sites appear to be located specifically on the postjunctional membrane. The density there is about 8,500/² of membrane surface. For comparison purposes, cholinesterases and related active centers were labeled using [³H] diisopropylfluorophosphate; they were shown to be at this same concentration over the synaptic membranes (or along the cleft). The 11 relationship of the receptors to the cholinesterase type of site, found previously to hold in studies on whole endplates, is also true at the ultrastructural level in this case. In fact, this 11 relationship is believed to be a characteristic of the postsynaptic membranes of endplates in other muscles and other vertebrates.Based on the constant density value thus arrived at, the total surface areas of postsynaptic and of presynaptic membranes are at once obtained from the known total numbers of these sites per endplate, available from previous studies in this laboratory. Examples of such synaptic surface area values are given. These values are only reliable for a given muscle type if the approximate fiber size is defined.     

Persistence of colicinogenic Escherichia coli in the mouse gastrointestinal tract

Background  

The ability of a bacterial strain to competitively exclude or displace other strains can be attributed to the production of narrow spectrum antimicrobials, the bacteriocins. In an attempt to evaluate the importance of bacteriocin production for Escherichia coli strain residence in the gastrointestinal tract, a murine model experimental evolution study was undertaken.     

CYP4A mRNA, protein, and product in rat lungs: novel localization in vascular endothelium.

The vasodilatory effect of 20-hydroxyeicosatetraenoic acid (20-HETE) on lung arteries is opposite to the constrictor effect seen in cerebral and renal vessels. These observations raise questions about the cellular localization of 20-HETE-forming isoforms in pulmonary arteries and other tissues. Using in situ hybridization, we demonstrate for the first time CYP4A (a family of cytochrome P-450 enzymes catalyzing formation of 20-HETE from the substrate arachidonic acid) mRNA in pulmonary arterial endothelial and smooth muscle cells, bronchial smooth muscle and bronchial epithelial cells, type I epithelial cells, and macrophages in adult male rat lungs. Moreover, we detect CYP4A protein in rat pulmonary arteries and bronchi as well as cultured endothelial cells. Finally, we identify endogenously formed 20-HETE by using fluorescent HPLC techniques, as well as the capacity to convert arachidonic acid into 20-HETE in pulmonary arteries, bronchi, and endothelium. These data show that 20-HETE is an endogenous product of several pulmonary cell types and is localized to tissues that optimally position it to modulate physiological functions such as smooth muscle tone or electrolyte flux.     

Cellular distribution of prostanoid EP receptors mRNA in the rat gastrointestinal tract

The inhibition of PGE(2) synthesis resulting from sustained NSAIDs therapy has been linked to gastrointestinal irritations and ulceration. The multiple physiological effects of PGE(2) in the gut are mediated through the activation of four receptors termed EP(1-4). The aim of the study was to determine the precise distribution of the four prostaglandin E(2) receptors in the rat stomach, small intestine, and colon. We used non-radioactive in situ hybridization techniques on paraffin-embedded tissue. Mucous cells of the stomach and goblet cells of the small intestine and colon were found to express mRNA for all four EP subtypes. A positive hybridization signal for EP(1), EP(3), and EP(4) was detected in the parietal cells of the stomach whereas the chief cells expressed low levels of EP(1) and EP(3). The EP(1) and EP(3) receptor mRNA could also be detected in the muscularis mucosa, longitudinal muscle and enteric ganglias of the stomach and small intestine. However, close examination of the enteric ganglias indicated that most of the positive labeling was localized to the glial cells, although some neurons did express EP(3). In conclusion, we have detailed the distribution of prostanoid EP receptors in the gut at the cellular level, giving new insights to the role of prostaglandins in gastrointestinal functions.     

Intracellular localization and protein interactions of the gene 1 protein p28 during mouse hepatitis virus replication

Coronaviruses encode the largest replicase polyprotein of any known positive-strand RNA virus. Replicase protein precursors and mature products are thought to mediate the formation and function of viral replication complexes on the surfaces of intracellular double-membrane vesicles. However, the functions of only a few of these proteins are known. For the coronavirus mouse hepatitis virus (MHV), the first proteolytic processing event of the replicase polyprotein liberates an amino-terminal 28-kDa product (p28). While previous biochemical studies have suggested that p28 is associated with viral replication complexes, the intracellular localization and interactions of p28 with other proteins during the course of MHV replication have not been defined. We used immunofluorescence confocal microscopy to show that p28 localizes to viral replication complexes in the cytoplasm during early times postinfection. However, at late times postinfection, p28 localizes to sites of M accumulation distinct from the replication complex. Furthermore, by yeast two-hybrid and coimmunoprecipitation analyses, we demonstrate that p28 specifically binds to p10 and p15, two coronavirus replicase proteins of unknown function. Deletion mutagenesis experiments determined that the carboxy terminus of p28 is not required for its interactions with p10 and p15. These results suggest that p28 may play a part at the replication complex by interacting with p10 and p15. Moreover, our findings highlight a potential role for p28 at virion assembly sites.     

Immunohistochemical localization of LIM mineralization protein 1 during mouse molar development

LIM mineralization protein 1 (LMP-1) is an essential positive regulator of osteoblast differentiation, maturation and bone formation. Our previous investigations on the distribution of LMP-1 in mature human teeth indicated that LMP-1 might play a role in the odontoblast differentiation and dentin matrix mineralization. The aim of the present study was to use immunohistochemistry to determine the expression of LMP-1 during tooth development in mouse molars. In embryonic and postnatal Kunming mice, LMP-1 protein was expressed during molar development, but the expression levels and patterns differed at various developmental stages. At embryonic day 13.5 (E13.5), LMP-1 was found in the enamel organ. At E14.5, LMP-1 was detected in the entire enamel organ and in the underlying mesenchyme. At E16.5, LMP-1 was observed in the inner and outer enamel epithelium and the stratum intermedium. The expression also converged at the cusps in the dental papilla. At E18.5 and postnatal day 2.5 (P2.5), LMP-1 was restricted to the stratum intermedium, in differentiating dental papilla cells at cusps, while it disappeared in terminal differentiated ameloblasts and odontoblasts. At P13.5, no positive staining was detected in the odontoblasts or in the dental pulp cells. Therefore, LMP-1 showed spatiotemporal expression patterns during molar development and might participate in molar crown morphogenesis and odontoblast differentiation at late molar development.     

ASH1 mRNA localization in yeast involves multiple secondary structural elements and Ash1 protein translation

Localization of ASH1 mRNA to the distal cortex of daughter but not mother cells at the end of anaphase is responsible for the two cells' differential mating-type switching during the subsequent cell cycle. This localization depends on actin filaments and a type V myosin (She1/Myo4). The 3' untranslated region (3' UTR) of ASH1 mRNA is reportedly capable of directing heterologous RNAs to a mother cell's bud [1] [2]. Surprisingly, however, its replacement has little or no effect on the localisation of ASH1 mRNA. We show here that, unlike all other known localization sequences that have been found in 3' UTRs, all the elements involved in ASH1 mRNA localization are located at least partly within its coding region. A 77 nucleotide region stretching from 7 nucleotides 5' to 67 nucleotides 3' of the stop codon of ASH1 mRNA is sufficient to localize mRNAs to buds; the secondary structure of this region, in particular two stems, is important for its localizing activity. Two regions entirely within coding sequences, both sufficient to localize green fluorescent protein (GFP) mRNA to growing buds, are necessary for ASH1 mRNA localization during anaphase. These three regions can anchor GFP mRNA to the distal cortex of daughter cells only inefficiently. The tight anchoring of ASH1 mRNA to the cortex of the daughter cell depends on translation of the carboxy-terminal sequences of Ash1 protein.     

Assessment of mouse strain on bacterial translocation from the gastrointestinal tract

The translocation of indigenous bacteria from the gastrointestinal tract to the mesenteric lymphnodes was compared in ten strains of mice. Indigenous Escherichia coli were cultured from the mesenteric lymphnodes of only two of the six mouse strains examined. Thus, spontaneous translocation of indigenous enteric bacteria across the intestinal barrier did not occur to any significant extent in any of the mouse strains examined. Since bacterial overgrowth in the gastrointestinal tract promotes bacterial translocation, bacterial translocation was tested in ten mouse strains including B10 series after antibiotic-decontaminated and subsequent colonization with streptomycin-resistant E. coli C25. E. coli C25 populated the ceca of the mice at levels of 10(8) to 10(9) per gram and translocated to 90-100% of the mesenteric lymphnodes with mean of 10(1.13) to 10(1.86) per mesenteric lymphnode. However, there were no significant differences between mouse strains as to the translocation incidence or the numbers of viable E. coli C25 per mesenteric lymphnode. Thus, genetic differences between mouse strains did not influence bacterial translocation from the gastrointestinal tract to the mesenteric lymphnodes.