Biosynthesis of intestinal mucins. Effect of puromycin on mucoprotein biosynthesis by sheep colonic mucosal tissue.
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ABSTRACT:
SUBMITTER: Allen A
PROVIDER: S-EPMC1198499 | biostudies-other | 1968 Jan
REPOSITORIES: biostudies-other
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Project description:1. Incubation of sheep colonic mucosal scrapings in Krebs-Ringer buffer for 2(1/2)hr. in the presence of salicylate (15mm) resulted in decreased incorporation of radioactivity into the epithelial glycoprotein from the following labelled precursors: 16.6mum-d-[2-(14)C]glucose (83.9% inhibition), 20mum-l-[U-(14)C]threonine (82%) and (35)SO(4) (2-)(79%). Oxygen uptake measured simultaneously was diminished to 41% of the control value. 2. At lower concentrations of salicylate (e.g. 3.75mm), incorporation of 20mum-l-[U-(14)C]threonine was little affected (3-6% inhibition), whereas utilization of 4mum-d-[U-(14)C]glucose and (35)SO(4) (2-) was inhibited (41-48% and 40-59% of the control values respectively). 3. Analysis of the papain-digested glycoprotein from tissue incubations with 16.6mum-d-[2-(14)C]glucose in the presence of salicylate (3.75mm) showed large decreases in labelling of N-acetylneuraminic acid and N-glycollylneuraminic acid residues (57% and 34% of the control values respectively) and of hexosamine constituents (glucosamine, 55% inhibition; galactosamine, 33% inhibition). Labelling of neutral sugars (galactose and fucose) was relatively little affected (9 and 11% inhibition respectively). 4. Glucose 6-phosphate transaminase and glucosamine 6-phosphate acetylase in particle-free enzyme preparations of the sheep tissue were unaffected by salicylate at the above concentrations. Acetyl-CoA synthetase was markedly inhibited. 5. Human gastric mucosa (from operation), on incubation as above, had in one experiment an oxygen consumption of 9.9mul./hr./mg. dry wt. of tissue and incorporated 5mum-d-[U-(14)C]glucose (15.8% of the total radioactivity added) into bound hexosamine (20.6% of the total radioactivity incorporated), hexoses (glucose and galactose, 5.7%) and fucose (14.2%). The presence of salicylate (15mm) decreased the incorporation of 5mum-d-[U-(14)C]glucose into the glycoprotein by 74%, all sugar constituents being affected, without influence on the rate of oxygen consumption. 6. The results suggest an inhibitory effect of salicylate on glycoprotein biosynthesis at the level of the amino sugar intermediates.
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Project description:1. Sheep colonic mucin contains three types of sialic acids, separable from the macrostructure by mild acidic hydrolysis. These are composed chiefly of N-acetyl-and N-glycollyl-neuraminic acid in ratios between 1:1.2 and 1:3.5 for different preparations of the mucin. The third sialic acid appears to be a diacetylated neuraminic acid. 2. A particle-free enzyme preparation, obtained from sheep colonic mucosa by gentle homogenization and high-speed centrifugation, catalyses a series of reactions involving N-acylamino sugars and leading to the formation of sialic acids in vitro: (i) phosphorylation by ATP of d-glucosamine, N-acetyl-and N-glycollyl-d-glucosamine; (ii) conversion of N-acetylglucosamine 6-phosphate into N-acetyl-d-glucosamine 1-phosphate; (iii) formation of sialic acids from phosphoenolpyruvate and N-acetyl- or N-glycollyl-d-glucosamine; (iv) formation of N-acetylneuraminic acid from uridine diphospho-N-acetylglucosamine or from N-acetylmannosamine; (v) incorporation of l-[U-(14)C]serine into the mucin by whole mucosal preparations.
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Project description:Throughout the gastrointestinal (GI) tract, a distinct mucus layer composed of highly glycosylated proteins called mucins plays an essential role in providing lubrication for the passage of food, participating in cell signaling pathways and protecting the host epithelium from commensal microorganisms and invading pathogens, as well as toxins and other environmental irritants. These mucins can be broadly classified into either secreted gel-forming mucins, those that provide the structural backbone for the mucus barrier, or transmembrane mucins, those that form the glycocalyx layer covering the underlying epithelial cells. Goblet cells dispersed among the intestinal epithelial cells are chiefly responsible for the synthesis and secretion of mucins within the gut and are heavily influenced by interactions with the immune system. Evidence from both clinical and animal studies have indicated that several GI conditions, including inflammatory bowel disease (IBD), colorectal cancer, and numerous enteric infections are accompanied by considerable changes in mucin quality and quantity. These changes include, but are not limited to, impaired goblet cell function, synthesis dysregulation, and altered post-translational modifications. The current review aims to highlight the structural and functional features as well as the production and immunological regulation of mucins and the impact these key elements have within the context of barrier function and host defense in intestinal inflammation.
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Project description:Pig colonic mucins isolated from the adherent mucus gel in the presence of proteinase inhibitors were solubilized by homogenization and the component mucins fractionated by CsC1 density-gradient centrifugation. Polymeric and reduced pig colonic mucin were both largely excluded on Sepharose CL-2B, papain-digested colonic mucin was included. The M(r) values of polymeric, reduced and digested mucins were 5.5 x 10(6), 2.1 x 10(6) and 0.6 x 10(6) respectively. This suggests that pig colonic mucin is comprised of 2-3 subunits, each subunit containing 3-4 glycosylated regions. The intrinsic viscosities of polymeric, reduced and digested mucin were 240 ml.g-1, 100 ml.g-1 and 20 ml.g-1 respectively. Polymeric pig colonic mucin comprised 16% protein per mg of glycoprotein and was rich in serine, threonine and proline (43% of total amino acids). There were approx. 150 disulphide bridges and 53 free thiol groups per mucin polymer. A seventh of the protein content was lost on reduction. This protein was particularly rich in proline and the hydrophobic amino acids. Papain-digested pig colonic mucin contained 11% protein per mg of glycoprotein and was rich in serine, threonine, glutamate and aspartate. All types of amino acids with the exception of aspartate were lost on digestion. The amino acid analysis of the proteolytically digested regions of pig colonic mucin are markedly different to the tandem repeat regions of the human mucin genes shown to be expressed in the colon.
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Project description:BACKGROUND:Canine inflammatory bowel disease (IBD) is a group of chronic gastrointestinal (GI) disorders of still largely unknown etiology. Canine IBD diagnosis is time-consuming and costly as other diseases with similar signs should be initially excluded. In human IBD microRNA (miR) expression changes have been reported in GI mucosa and blood. Thus, there is a possibility that miRs may provide insight into disease pathogenesis, diagnosis and even treatment of canine IBD. The aim of this study was to determine the colonic mucosal and serum relative expression of a miRs panel in dogs with large intestinal IBD and healthy control dogs. RESULTS:Compared to healthy control dogs, dogs with large intestinal IBD showed significantly increased relative expression of miR-16, miR-21, miR-122 and miR-147 in the colonic mucosa and serum, while the relative expression of miR-185, miR-192 and miR-223 was significantly decreased. Relative expression of miR-146a was significantly increased only in the serum of dogs with large intestinal IBD. Furthermore, serum miR-192 and miR-223 relative expression correlated to disease activity and endoscopic score, respectively. CONCLUSION:Our data suggest the existence of dysregulated miRs expression patterns in canine IBD and support the potential future use of serum miRs as useful noninvasive biomarkers.
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Project description:Sialic acid occupies terminal positions on O-glycans of cervical mucins, where they contribute to the increased viscosity of mucin thereby regulating sperm transport. This study characterized the sialylated cervical mucins from follicular phase mucus of six European ewe breeds with known differences in pregnancy rates following cervical artificial insemination (AI) using frozen-thawed semen at both synchronized and natural estrus cycles. These were Suffolk (low fertility) and Belclare (medium fertility) in Ireland, Ile de France and Romanov (both with medium fertility) in France, and Norwegian White Sheep (NWS) and Fur (both with high fertility) in Norway. Expression of mucin and sialic acid related genes was quantified using RNA-sequencing in cervical tissue from Suffolk, Belclare, Fur, and NWS only. Cervical tissue was also assessed for the percentage of cervical epithelial populated by mucin secreting goblet cells in the same four ewe breeds. Biochemical analysis showed that there was an effect of ewe breed on sialic acid species, which was represented by Suffolk having higher levels of Neu5,9Ac2 compared with NWS (P < 0.05). Suffolk ewes had a lower percentage of goblet cells than Fur and NWS (P < 0.05). Gene expression analysis identified higher expression of MUC5AC, MUC5B, ST6GAL1, and ST6GAL2 and lower expression of ST3GAL3, ST3GAL4, and SIGLEC10 in Suffolk compared with high fertility ewe breeds (P < 0.05). Our results indicate that specific alterations in sialylated mucin composition may be related to impaired cervical sperm transport.
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Project description:Precancerous or cancerous lesions of the gastrointestinal tract often require surgical resection via endomucosal resection. Although excision of the colonic mucosa is an effective cancer treatment, removal of large lesions is associated with high morbidity and complications including bleeding, perforation, fistula formation, and/or stricture, contributing to high clinical and economic costs and negatively impacting patient quality of life. The present study investigates the use of a biologic scaffold derived from extracellular matrix (ECM) to promote restoration of the colonic mucosa following short segment mucosal resection. Six healthy dogs were assigned to ECM-treated (tubular ECM scaffold) and mucosectomy only control groups following transanal full circumferential mucosal resection (4 cm in length). The temporal remodeling response was monitored using colonoscopy and biopsy collection. Animals were sacrificed at 6 and 10 wk, and explants were stained with hematoxylin and eosin (H&E), Alcian blue, and proliferating cell nuclear antigen (PCNA) to determine the temporal remodeling response. Both control animals developed stricture and bowel obstruction with no signs of neomucosal coverage after resection. ECM-treated animals showed an early mononuclear cell infiltrate (2 weeks post-surgery) which progressed to columnar epithelium and complex crypt structures nearly indistinguishable from normal colonic architecture by 6 weeks after surgery. ECM scaffold treatment restored colonic mucosa with appropriately located PCNA+ cells and goblet cells. The study shows that ECM scaffolds may represent a viable clinical option to prevent complications associated with endomucosal resection of cancerous lesions in the colon.
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Project description:BACKGROUND: Polyamines are essential for cell growth. Dietary and probably gut bacterial derived polyamines contribute significantly to the polyamine body pool. AIMS: To evaluate the influence of dietary, luminal polyamines on growth and development of different gastrointestinal organs in normally growing rats. METHODS: Male suckling Wistar rats were randomly allocated to four treatment groups: polyamine deficient diet (PDD); PDD plus antibiotics (neomycin 2 g/kg and metronidazole 34 mg/kg); PDD plus polyamine supplementation at normal concentrations; or normal standard laboratory chow. After a six month feeding period 7-10 animals/group were sacrificed. RESULTS: No differences in body weight gain, food consumption, or general behaviour could be observed between the four groups of animals. Feeding of PDD alone or PDD plus antibiotics resulted in a highly significant decrease in organ weight, protein content, and DNA content in small intestinal and colonic mucosa whereas no alterations were found in the liver. CONCLUSIONS: Long term feeding of polyamine deficient diets resulted in a significant hypoplasia of small intestinal and colonic mucosa. Dietary, luminal polyamines are important local factors for growth and the development of small intestinal and colonic mucosa.
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Project description:The mechanisms behind acute mucosal injury responses in humans are poorly investigated.We thus examined the acute responses to mucosal barrier injury in humans with and without UC. A standardized mucosal wound extending to the muscularis mucosa layer was inflicted in the sigmoid colon of 6 patients with quiescent UC and 6 control subjects. Macroscopic, microscopic and molecular injury responses were assessed at 24 and 48 hours by repeated high resolution imaging and collection of biopsies across the wounds which were assessed by RNA sequencing.
2021-10-06 | GSE164918 | GEO