The isolation and properties of epithelial-cell "ghosts" from rat small intestine.
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ABSTRACT:
SUBMITTER: Clark B
PROVIDER: S-EPMC1207073 | biostudies-other | 1965 Aug
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:Understanding regional distribution and specialization of small intestinal epithelial cells is crucial for developing methods to control appetite, stress, and nutrient uptake in swine. To establish a better understanding of specific epithelial cells found across different regions of the small intestine in pigs, we utilized single-cell RNA sequencing (scRNA-seq) to recover and analyze epithelial cells from duodenum, jejunum, and ileum. Cells identified included crypt cells, enterocytes, BEST4 enterocytes, goblet cells, and enteroendocrine (EE) cells. EE cells were divided into two subsets based on the level of expression of the EE lineage commitment gene, NEUROD1. NEUROD1hi EE cells had minimal expression of hormone-encoding genes and were dissimilar to EE cells in humans and mice, indicating a subset of EE cells unique to pigs. Recently discovered BEST4 enterocytes were detected in both crypts and villi throughout the small intestine via in situ staining, unlike in humans, where BEST4 enterocytes are found only in small intestinal villi. Proximal-to-distal gradients of expression were noted for hormone-encoding genes in EE cells and nutrient transport genes in enterocytes via scRNA-seq, demonstrating regional specialization. Regional gene expression in EE cells and enterocytes was validated via quantitative PCR (qPCR) analysis of RNA isolated from epithelial cells of different small intestinal locations. Though many genes had similar patterns of regional expression when assessed by qPCR of total epithelial cells, some regional expression was only detected via scRNA-seq, highlighting advantages of scRNA-seq to deconvolute cell type-specific regional gene expression when compared to analysis of bulk samples. Overall, results provide new information on regional localization and transcriptional profiles of epithelial cells in the pig small intestine.
| S-EPMC9831138 | biostudies-literature
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.
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Project description:The functional roles of transient receptor potential (TRP) channels in the gastrointestinal tract have garnered considerable attention in recent years. We previously reported that daikenchuto (TU-100), a traditional Japanese herbal medicine, increased intestinal blood flow (IBF) via adrenomedullin (ADM) release from intestinal epithelial (IE) cells (Kono T et al. J Crohns Colitis 4: 161-170, 2010). TU-100 contains multiple TRP activators. In the present study, therefore, we examined the involvement of TRP channels in the ADM-mediated vasodilatatory effect of TU-100. Rats were treated intraduodenally with the TRP vanilloid type 1 (TRPV1) agonist capsaicin (CAP), the TRP ankyrin 1 (TRPA1) agonist allyl-isothiocyanate (AITC), or TU-100, and jejunum IBF was evaluated using laser-Doppler blood flowmetry. All three compounds resulted in vasodilatation, and the vasodilatory effect of TU-100 was abolished by a TRPA1 antagonist but not by a TRPV1 antagonist. Vasodilatation induced by AITC and TU-100 was abrogated by anti-ADM antibody treatment. RT-PCR and flow cytometry revealed that an IEC-6 cell line originated from the small intestine and purified IE cells expressed ADM and TRPA1 but not TRPV1. AITC increased ADM release in IEC cells remarkably, while CAP had no effect. TU-100 and its ingredient 6-shogaol (6SG) increased ADM release dose-dependently, and the effects were abrogated by a TRPA1 antagonist. 6SG showed similar TRPA1-dependent vasodilatation in vivo. These results indicate that TRPA1 in IE cells may play an important role in controlling bowel microcirculation via ADM release. Epithelial TRPA1 appears to be a promising target for the development of novel strategies for the treatment of various gastrointestinal disorders.
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Project description:Endothelial cells (ECs) from the small intestine, colon, liver, and heart have distinct phenotypes and functional adaptations that are dependent on their physiological environment. Gut ECs adapt to low oxygen, heart ECs to contractile forces, and liver ECs to low flow rates. Isolating high-purity ECs in sufficient quantities is crucial to study their functions. Here, we describe protocols combining magnetic and fluorescent activated cell sorting for rapid and reproducible EC purification from four adult murine tissues. For complete details on the use and execution of these protocols, please refer to Kalucka et al. (2020).
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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.
| S-EPMC1168318 | biostudies-other
Project description:Epidermal growth factor (EGF) regulates the proliferation of cells of a rat intestinal epithelial-cell line (RIE-1) in culture. Confluent RIE-1 cells were stimulated to proliferate by EGF with a half-maximal effect at 1-2 ng/ml. In contrast, the growth of sparse RIE-1 cells was inhibited by the growth factor. Binding studies at 4 degrees C with 125I-EGF identified two classes of binding sites for EGF on RIE-1 cells, one of high affinity (KD = 1.8 X 10(-10)M; 1.8 X 10(4) receptors/cell) and one of lower affinity (KD = 5.2 X 10(-9)M; 6.3 X 10(4) receptors/cell). After binding to the cells at 37 degrees C, 125I-EGF was rapidly internalized and subsequently degraded. Degradation products were released into the medium after a lag of 15-30 min. The degradation of 125I-EGF did not occur at 4 degrees C and was inhibited at 37 degrees C by chloroquine, methylamine or NH4Cl, but not by colchicine. Exposure of RIE-1 cells to EGF caused a time- and dose-dependent loss of EGF receptors from the cell surface. The recovery of receptors after the removal of EGF was retarded in the absence of serum and prevented by the presence of cycloheximide or actinomycin D. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis separation of the 125I-EGF-receptor complex from RIE-1 cells after covalently cross-linking with disuccinimidyl suberate indicated a receptor of Mr congruent to 160 000. The demonstration of functional EGF receptors in this cell line provides further evidence that EGF may regulate intestinal-epithelial-cell physiology.
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Project description:1. Homogenization of the epithelial cells of rabbit small intestine in 0.3m-sucrose-5mm-EDTA, pH7.4, maintains intact the microvillus sheets that form the lumenal surface of the cells, the nuclei, the mitochondria and the vesicles (microsomes) formed from the endoplasmic reticulum. 2. These particulate components of the cell, and the cell-sap fraction, have been isolated by differential centrifuging of cell homogenates. 3. The nuclei and microvillus sheets sediment together and it has been impossible to separate these subcellular components by centrifugal methods. 4. The isolated subcellular fractions have been identified by a combination of light-microscopic examination, electron-microscopic examination, chemical analysis and assay for selected enzyme activities.
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Project description:Proximal mouse small intestine from mice bearing the Lgr5 GFP/+ and Mex3a Tom/+ alleles were used to obtain single cell preparations. Cells were selected for GFP expression and different levels of tdTomato were defined. Sorted cells were lysed and processed for transcriptomic analysis
2017-06-01 | GSE90629 | GEO
Project description:Experiment description 1. Experimental design 1) Authors, laboratory, and contact: Xue-Lin Cui1, Patricia Soteropoulos2, Peter Tolias2, Ronaldo P. Ferraris1: 1Dept. of Pharmacology and Physiology, UMDNJ-New Jersey Medical School, Newark, NJ 07103-2714, 2Center for Applied Genomics, PHRI, 225 Warren Street Newark, NJ 07103-3506. Contact to Dr. Ronaldo P. Ferraris. 2) Type of the experiment: treated vs. untreated comparison. 3) Experimental factors: Twenty to twenty-two old SD rats were use in the experiment. The intestines of paired pups were perfused with 100 mM fructose (HF) and 100 mM glucose (HG) for 4-h or 20-min, respectively. 4) Totally 14 experiments were done. 5) A common reference was not used for all the hybridizations. 6) Quality control steps ( 14) Miller RA, Galecki A, Shmookler-Reis RJ. Interpretation, design, and analysis of gene array expression experiments. J Gerontol A Biol Sci Med Sci. 2001 Feb;56(2):B52-7. (15) Tseng GC, Oh MK, Rohlin L, Liao JC, Wong WH. Issues in cDNA microarray analysis: quality filtering, channel normalization, models of variations and assessment of gene effects. Nucleic Acids Res. 2001 Jun 15;29(12):2549-57. ( 16) Yang YH, Speed T. Design issues for cDNA microarray experiments. Nat Rev Genet. 2002 Aug;3(8):579-88. (17) Yang YH, Dudoit S, Luu P, Lin DM, Peng V, Ngai J, Speed TP. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res. 2002 Feb 15;30(4):e15.): a) In the 4 h perfusion experiment, the intestines of the paired pups were perfused with 100 mM fructose (HF) and 100 mM glucose (HG) for 4-h, respectively. Totally five replicates were conducted. The RNA samples were extracted from HF- and HG-perfused intestines (n=5 pairs) then labelled with Cy 5 (HF1, HF2, HF3, HF4, and HF5) and Cy 3 (HG1, HG2, HG3, HG4, and HG5), respectively. At the same time, one dye swap experiment, in which the samples extracted from HF- and HG-perfused intestines were labelled with Cy 3 (HF1) and Cy 5 (HG1), respectively, was done and used in the data analysis. b) In the 20 min perfusion experiment, the intestines of of the paired pups were perfused with 100 mM fructose (HF) and 100 mM glucose (HG) for 20-min, respectively. Totally four replicates were conducted. In two of the four-replicate experiments, the samples extracted from HF- and HG-perfused intestines were labelled with Cy 5 (HF1a and HF3a) and Cy 3 (HG1a and HG3a), respectively. In the other two of the four-replicate experiments, the dye was swapped, that is, the samples extracted from HF- and HG-perfused intestines were labelled with Cy 3 (HF2a and HF4a) and Cy 5 (HG2a and HG4a), respectively. c) Three self-self experiments using different samples (HF1, HG1, and HF2) and one capture primer alone experiment were conducted to eliminate the false positive genes and aid to analyze the data. 7) The goal of the experiment: Fructose induced the expression of GLUT5 mRNA in the small intestinal epithelial cells. But it is not clear about the mechanisms involved in the phenomenal. In this experiment, we hypothesize: the expression of some genes must be changed after fructose perfusion for 4-h, these genes may regulate GLUT5 gene transcription; the expression of some genes must be changed even after 20-min fructose perfusion, these genes may trigger the GLUT5 gene expression as early response gene. Our goal in this study is to find out these potencial gene. 8) Links (URL), citations 2. Samples used, extract preparation, labeling and hybridization 1) Bio-source properties a) Sprague Dawley rats (NCBI taxonomy); b) The rat was anesthetized using urathene. The rat body was kept at 37 oC under light. c) Descriptors relevant to the particular sample: the jejunum of the 20-d old rat pups (unknown sex) was perused. 2) Biomaterial manipulations: the jejunum was in vivo perfused with 100 mM HF or HG for 4-h or 20-min. At the end of each experiment, the jejunum was immediately cut out and snap frozen in liqued nitrogen, and then stored the tissue in -80 oC freezer. 3) RNA isolation: A RNeasy Midi Kit (QIAGEN Inc., Valencia, CA) was used to extract total RNA from the jejunum. The brief procesure is described in the Total RNA Extraction for RT-PCR and Microarray above. 4) Sample assignment to slides for labeling and hybridization: S1: HF1(Cy 5) vs HG1 (Cy 3); S2: HF2 (Cy 5) vs HG2 (Cy 3); S3: HF3 (Cy 5) vs HG3 (Cy 3); S4: HF4 (Cy 5) vs HG4 (Cy 3); S5: HF5 (Cy 5) vs HG5 (Cy 3); S6: HF2 (Cy 3) vs HG2 (Cy 5); S7: HF1a (Cy 5) vs HG1a (Cy 3); S8: HF3a (Cy 5) vs HG3a (Cy 3); S9: HF2a (Cy 3) vs HG2a (Cy 5); S10: HF4a (Cy 3) vs HG4a (Cy 5); S11: HG1 (Cy 5) vs HG1 (Cy 3); S12: HG3 (Cy 5) vs HG3 (Cy 3); S13: HF3 (Cy 5) vs HF3 (Cy 3); S14: Cy 3 vs Cy 5 capture primer. 5) Labeling and hybridization procedures and parameters: The 3DNA Submicro Oligo Expression Array Detection Kit (Genisphere Inc., Hatfield, PA) was employed to synthesize and label the complimentary DNA (cDNA) with fluorescent dye: Cy3 or Cy5. The reason why this indirect labeling method was chosen is based on a previous study which demonstrated that the method was more sensitive and consistent than direct labeling method or other indirect techniques like aminoallyl labeling method and MICROMAX tyramide signal amplification labeling method. (18)Yu J, Othman MI, Farjo R, et al: Molecular Vision 2002; 8:130-137). In the primary experiments, three, four, and five g of total RNA were tested for labeling and hybridization conditions. In the official experiments, four g of total RNA isolated from each sample were used for labeling and hybridization, because the signal to noise ratio is high at this concentration. In addition, at this concentration, all most of the low expressed genes are detectable and the highly expressed genes are not fully saturated. The procedure was conducted following the manufacturer's protocol. Briefly, the first strand cDNA was synthesized using a specific poly T Cy3 RT primer or a specific poly T Cy5 RT primer in different tubes for paired samples. Combine the Cy3 and Cy5 reactions in one tube and precipitate the sample in ethanol. After briefly drying the pellet, resuspend the pellet with 40 l of hybridization buffer. Subsequently, apply the hybridization mixture onto the array slide and hybridize overnight at 55 oC in a humidified chamber. Following the hybridization, the slides were washed by placing them into 50 ml conical tubes containing 2 x SSC, 0.2% SDS for 15 min, 2 x SSC for 10 min, 0.2 x SSC for 10 min, and 95% ethanol for 1 min all at room temperature, except for the first wash, in which the temperature was 42 oC. After drying the slides, the Cy3 and Cy5 capture (connected to dye molecues) reagents mixed in hybridization buffer was pipeted onto the slides, and then hybridize at 65 oC for 3 h following slides wash as described above. 3. Measurement data and spesifications of data processing 1) Raw data description Slides were scanned for Cy3 and Cy5 fluorescence using an Axon GenePix4000 microarray reader (GenePix 4000A, Axon instruments, Foster City, CA) and quantitated using GenePix software. Scan parameters: Scan power: Cy 3 : Cy 5 = 100:100; Laser power: Cy 3 : Cy 5 = 1.15:1.76; Spatial resolution: ???; Pixel size: 10 or 100 ???; Pixel number: F pixels: 50-100; B pixels: 400-500 ???; PMT voltage: 600-750 for Cy 5 channel, 650-850 for Cy 3 channel. 2) Image analysis and quantitation For the image analysis, the density of the spots was detected using Gene Spring (Silicon Genetics, Redwood City, CA) software (GeniPix 4.0) (19) Axon Instruments Inc. (1999) GenePix 4000A User's Guide. Axon Instruments, Union City, CA). It is commercially available. Description of the algorithm and all the parameters used: R = red channel signal = F635; G = green channel signal = B 532; M = log2(R/G); A = 1/2xlog2(RxG); User-difined parameter f = 40%. For the complete image analysis out put of each image, see the NCBI's GEO database (www.ncbi.nlm.nih.gov/geo). 3) Normalized and summarized data-gene expression data matrix For the normalization, the following two steps were taken. a) When measuring the density of the spots using Gene Spring software, the ratio of Cy 3 and Cy 5 dyes was adjusted as 1 as a globle normalization for the whole slide. b) For the gene expression analysis, the results (midian of the density of the pixel in one spot was used) were normalized by a LOWESS (locally weighed scatter smoother) software provided by the Center for Applied Genomics, so that the results can be compared among the experiments performed at different times. (20)Yang YH, Dudoit S, Luu P, Lin DM, Peng V, Ngai J, Speed TP. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res. 2002 Feb 15;30(4):e15.). c) The following rules were made to decide the genes that were significantly changed: 1) Eliminate the genes with high density (over than 2X of background) in the 3DNA capture probe experiments; 2) Eliminate the genes with density lower than 2X of background ( 21) Lee HM, Greeley GH Jr, Englander EW. Age-associated changes in gene expression patterns in the duodenum and colon of rats. Mech Ageing Dev. 2001 Apr 15;122(4):355-71.); 3) Eliminate the genes with change over than 1.5X in dye flip-over experiment; 4) Eliminate the genes with opposite results in one experiments between dye flip-over comparisons; 5) Only the genes significantly changed over 3 in 5 comparisons in 4 h perfusion experiment and over 2 in 4 comparisons in 20 min perfusion experiment were considered as reliable and biologically important. d) The final results discribed in this paper were presented as Means + SE and the frequency of gene changed in the HF-perfused intestine. About the image, raw, and normalized data, and other detailed parameters, please see the gene expression data tables derived from the experiments in the NCBI's GEO database. The design for the dye labeling in this experiments is as the follows: 1) 5 comparisons were conducted in the 4 h-perfused HG vs HF experiment, in which in 3 comparisons, the cDNA samples synthesized from HG- and HF-perfused intestines were labeled with Cy3 and Cy5, respectively; and in 2 comparisons, the cDNA samples synthesized from HG- and HF-perfused intestines were labeled with Cy5 and Cy3, respectively. 2) 4 comparisons were conducted in the 20 min-perfused HG vs HF experiment, in which in 2 comparisons, the cDNA samples synthesized from HG- and HF-perfused intestines were labeled with Cy3 and Cy5, respectively; and in 2 comparisons, the cDNA samples synthesized from HG- and HF-perfused intestines were labeled with Cy5 and Cy3, respectively. 3) 3 dye flip-over experiments using 1 RNA sample extracted from HF-perfused intestine and 2 samples extracted from HG-perfused intestines were performed to eliminate the false positive results induced by the dye labeling technique or other unsolved problems. 4) 2 experiments only applying 3DNA fluorescent probe, which is connected to complementary capture sequence, were performed to eliminate the false positive genes that were low expressed in the tissue. The procedures for synthesizing the first strand cDNA, labeling the cDNA, hybridizaion, and detecting the signals were performed following the manufacturer's protocols. Keywords: other
2004-01-28 | GSE996 | GEO