THE ISOLATION AND CHARACTERIZATION OF SUBCELLULAR COMPONENTS OF THE EPITHELIAL CELLS OF RABBIT SMALL INTESTINE.
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ABSTRACT:
SUBMITTER: PORTEOUS JW
PROVIDER: S-EPMC1206917 | biostudies-other | 1965 Jul
REPOSITORIES: biostudies-other
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Project description:1. Only a single phosphofructokinase isoenzyme is present in the mucosa of rat small intestine. 2. Mucosal phosphofructokinase was purified to yield a homogeneous preparation of specific activity 175 units/mg of protein. 3. The native enzyme is a tetramer, with monomer Mr 84 500 +/- 5000. 4. The native enzyme may be degraded by the action of endogenous proteinases to give two products with the same specific activity as the native enzyme: degradation occurs in the order native enzyme leads to proteolytic product 1 leads to proteolytic product 2. 5. Proteolytic product 1 has a greater mobility in cellulose acetate electrophoresis at pH8 and binds more strongly to DEAE-cellulose than does native enzyme; the converse is true for proteolytic product 2. 6. Proteolytic product 1 is a tetramer with a monomer Mr about 74 300; proteolytic product 2 is also a tetramer. 7. Native enzyme can only be prepared in the presence of proteinase inhibitors; partial purifications based on simple fractionation of crude mucosal extracts in the absence of proteinases inhibitors contain proteolytic product 2 as the main component and proteolytic product 1 together with little native enzyme. 8. Purified native mucosal phosphofructokinase displayed little co-operativity with respect to fructose 6-phosphate at pH 7.0 and was only weakly inhibited by ATP.
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Project description:Human noroviruses are a major cause of diarrheal illness, but pathogenesis is poorly understood. Here, we investigate the cellular tropism of norovirus in specimens from four immunocompromised patients. Abundant norovirus antigen and RNA are detected throughout the small intestinal tract in jejunal and ileal tissue from one pediatric intestinal transplant recipient with severe gastroenteritis. Negative-sense viral RNA, a marker of active viral replication, is found predominantly in intestinal epithelial cells, with chromogranin A-positive enteroendocrine cells (EECs) identified as a permissive cell type in this patient. These findings are consistent with the detection of norovirus-positive EECs in the other three immunocompromised patients. Investigation of the signaling pathways induced in EECs that mediate communication between the gut and brain may clarify mechanisms of pathogenesis and lead to the development of in vitro model systems in which to evaluate norovirus vaccines and treatment.
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Project description:Rabbit small intestine 16S RNAc Genome sequencing
| PRJNA896483 | ENA
Project description:Rabbit small intestine 16S RNAc Genome sequencing
| PRJNA896711 | ENA
Project description:1. The preparation of gram quantities of isolated epithelial-cell ;ghosts' from mucosal scrapings of rat small intestine is described. The method involves dispersing the tissue by gentle homogenization in 6% dextran in Krebs-Ringer phosphate, pH7.4, followed by filtration through nylon cloth and sedimentation by low-speed centrifuging. 2. The isolated epithelial-cell ;ghosts' contained all of the DNA, but only 52% of the protein and 53-57% of the RNA of the original homogenate. They contained most of the activity of the following enzymes found in the homogenate: aminopeptidase (71%); alkaline beta-glycerophosphatase (82%); invertase (92%); adenosine triphosphatase (93-116%); acid beta-glycerophosphatase (83%); nonspecific esterase (76%); succinate dehydrogenase (96%). Only small proportions of the total lactate-dehydrogenase (10%) and phosphoglucose-isomerase (2%) activities found in the homogenate were recovered in the isolated cell ;ghosts'. 3. The epithelial-cell ;ghost' preparation did not respire unless cofactors and substrates were added, and did not consume glucose or produce lactic acid from glucose. 4. The effect of varying the composition of the homogenization medium was studied. Concentrations of dextran (mol.wt. 15x10(4)) from 1 to 12%, solutions of dextrans (all at 6%) with mol.wt. varying between 3.6x10(4) and 2x10(6), and a solution of 8% polyethylene glycol (mol.wt. 4000) served equally well for the production of epithelial-cell ;ghosts'. Two of these solutions, however, 12% dextran (mol.wt.15x10(4)) and 6% dextran (mol.wt. 2x10(6)), were too viscous to allow the complete sedimentation of the cell ;ghosts' at low relative centrifugal forces. Omission of either Krebs-Ringer phosphate or dextran from the medium resulted in almost complete cell breakage during the homogenization. 5. The isolated cell ;ghosts' were used as a starting material for subcellular fractionation of rat intestinal mucosa by differential centrifugation. The distributions of protein and succinate-dehydrogenase activity among the fractions were compared with corresponding values in fractions isolated by differential centrifugation of mucosa homogenized in 0.3m-sucrose-5mm-EDTA, pH7.4. The method in which cell ;ghosts' were used as starting material gave a better separation and cleaner fractions than the method in which untreated mucosal scrapings were used.
| S-EPMC1207073 | biostudies-other
Project description:Phosphofructokinase from the epithelial cells of rat small intestine was characterized with respect to isoenzyme type in a comparison of its properties with those of the skeletal-muscle, brain and major liver isoenzymes by using five different techniques, namely electrophoresis on cellulose acetate and in polyacrylamide gels, chromatography on DEAE-cellulose, (NH4)2SO4 precipitation and immunotitration. When precautions were taken to inhibit the formation of active proteolytic artifacts by the action of endogenous proteinases, each technique revealed that rat intestinal mucosa contains only a single form of phosphofructokinase. The mucosal isoenzyme was found to be very similar to, although not identical with, the major liver isoenzyme and to be quite distinct from the skeletal-muscle isoenzyme when studied by the techniques of cellulose acetate electrophoresis, chromatography on DEAE-cellulose and immunotitration, whereas the converse was true when studied by the techniques of (NH4)2SO4 precipitation and polyacrylamide-gel electrophoresis. The mucosal isoenzyme was distinct from the brain isoenzyme when studied by each of the five techniques. Tsai & Kemp [(1973) J. Biol. Chem. 248, 785-792] reported that animal tissues contain three principal isoenzymes of phosphofructokinase, type A found as the sole isoenzyme in skeletal muscle, type B found as the major isoenzyme in liver and type C found as a significant isoenzyme in brain. Phosphofructokinase from mucosa is distinct from each of these isoenzymes. Following the nomenclature of Tsai & Kemp (1973), the isoenzyme from the mucosa of rat intestinal epithelial cells is designated phosphofructokinase D. The mucosal and liver isoenzymes behave so similarly with respect to their charge and immunological characteristics, on which the typing of isoenzymes is conventionally based, that it is likely that some tissues reported to contain the liver isoenzyme contain instead the mucosal isoenzyme.
| S-EPMC1152401 | biostudies-other
Project description:BackgroundEnterocytes exert an absorptive and protective function in the intestine, and they encounter many different challenging factors such as feed, bacteria, and parasites. An intestinal epithelial in vitro model can help to understand how enterocytes are affected by these factors and contribute to the development of strategies against pathogens.ResultsThe present study describes a novel method to culture and maintain primary chicken enterocytes and their characterization by immunofluorescence and biomolecular approaches. Starting from 19-day-old chicken embryos it was possible to isolate viable intestinal cell aggregates that can expand and produce a self-maintaining intestinal epithelial cell population that survives until 12 days in culture. These cells resulted positive in immunofluorescence to Cytokeratin 18, Zonula occludens 1, Villin, and Occludin that are common intestinal epithelial markers, and negative to Vimentin that is expressed by endothelial cells. Cells were cultured also on Transwell® permeable supports and trans-epithelial electrical resistance, was measured. This value gradually increased reaching 64 Ω*cm2 7 days after seeding and it remained stable until day 12.ConclusionsBased on these results it was confirmed that it is possible to isolate and maintain chicken intestinal epithelial cells in culture and that they can be suitable as in vitro intestinal model for further studies.
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Project description:The early steps of embryogenesis are controlled exclusively by the quality of oocyte that linked closely to its mechanical properties. The mechanical properties of an oocyte were commonly characterized by assuming it was homogeneous such that the result deviated significantly from the true fact that it was composed of subcellular components. In this work, we accessed and characterized the subcellular components of the oocytes and developed a layered high-fidelity finite element model for describing the viscoelastic responses of an oocyte under loading. The zona pellucida (ZP) and cytoplasm were isolated from an oocyte using an in-house robotic micromanipulation platform and placed on AFM to separately characterizing their mechanical profiling by analyzing the creep behavior with the force clamping technique. The spring and damping parameters of a Kelvin-Voigt model were derived by fitting the creeping curve to the model, which were used to define the shear relaxation modulus and relaxation time of ZP or cytoplasm in the ZP and cytoplasm model. In the micropipette aspiration experiment, the model was accurate sufficiently to deliver the time-varying aspiration depth of the oocytes under the step negative pressure of a micropipette. In the micropipette microinjection experiment, the model accurately described the intracellular strain introduced by the penetration. The developed oocyte FEM model has implications for further investigating the viscoelastic responses of the oocytes under different loading settings.
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Project description:Phosphodiesterase II activity was determined by using a synthetic substrate, the 2,4-dinitrophenyl ester of thymidine 3'-phosphate. The enzyme activity was determined in fractions obtained by differential centrifugation of homogenates of epithelial cells from the small intestinal mucosa of guinea pigs and rats. In guinea-pig preparations phosphodiesterase II occurred with highest specific activity in those fractions rich in succinate dehydrogenase and acid phosphatase. A lysosomal location for the guinea-pig enzyme was indicated by its structure-linked latency and by its association with particles that under-went a characteristic decrease in equilibrium density when Triton WR-1339 was injected into the animals. With rat preparations a much greater proportion of the phosphodiesterase II activity was found in the soluble fraction after ultracentrifugation. The rat enzyme exhibited a lower degree of latency and administration of Triton WR-1339 had no effect. The rat enzyme further differed from that of the guinea pig in other respects; it was more labile at 60 degrees C, it exhibited a lower pH optimum and it had a higher molecular weight as determined by gel-filtration chromatography.
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Project description:The kinetic properties of a maltase-glucoamylase complex with a neutral pH optimum, purified to homogeneity from the brush borders of the rabbit small intestine, are described. It has a broad range of substrate specificity, hydrolysing di- and poly-saccharides with alpha-1,4 and alpha-1,6 linkages. The Km and Vmax, values of the enzyme for the various substrates were determined. Starch and maltose were its best substrates. The kinetics of hydrolysis of two synthetic linear maltosaccharides, namely maltotriose and maltopentaose, were studied. Mixed-substrate incubation studies revealed the presence of at least two interacting sites on the enzyme, and the data were further analysed by the use of a number of non-substrate inhibitors.
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