The development of arylsulphatase in the small intestine of the rat.
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ABSTRACT:
SUBMITTER: Danovith SH
PROVIDER: S-EPMC1184861 | biostudies-other | 1969 Sep
REPOSITORIES: biostudies-other
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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
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.
2010-06-09 | E-GEOD-996 | biostudies-arrayexpress
Project description:Acid lipase was identified in the rat small intestine by using esters of 4-methylumbelliferone as substrates. Maximum activity towards the oleate ester was found at pH 4.0. In adult animals, the activity of acid lipase exhibited both latency and sedimentability, indicating a lyosomal localization. The activity of acid lipase was practically the same along the height of the villus, thus paralleling the distribution of acid beta-galactosidase. In adult rats, the activity of acid lipase in proximal (jejunum) and middle (mid-jejunum) sections of the small intestine was practically the same and exceeded the activity in the distal (ileum) section by a factor of 2. In suckling rats, the activity of the enzyme in the mid-jejunum exceeded that in the jejunum and ileum by 2.5- and 1.5-fold respectively. During postnatal development, the acid lipase activity of the mid-jejunum showed a peak between days 10 and 15, at which time it exceeded the adult mid-jejunum activity by 5--6-fold.
| S-EPMC1165014 | biostudies-other
Project description:Total pyruvate dehydrogenase activities in hamster intestine increase from 40 nmol/min (munits) per g of intestine in the foetal animals to 460 munits/g in the adult, whereas the fraction of the enzyme in the active form increases from 34 to 42% of the total activity over the same period. However, a complete conversion of the enzyme into the active form is observed in the neonatal animal immediately after birth. Results from experiments in vitro suggested that the activation of pyruvate dehydrogenase is controlled, in part, by the [NAD+]/[NADH] ratio. This proposal was tested in vivo by examining the proportion of the enzyme in the active form during conditions when the [NAD+]/[NADH] ratio was markedly altered, and the data show a direct relationship between the mitochondrial redox state and activity of the active form.
| S-EPMC1164848 | biostudies-other
Project description:Microsomal fractions from rat small intestine contain a fatty-acid chain-elongation activity. Cofactor requirements are similar to those of the liver microsomal system, but substrate specificity is different. The polyunsaturated arachidonic and timnodonic acids were elongated at very low rates. These results suggest that the relative contents of specific chain-elongation enzymes are different in liver and small intestine.
| S-EPMC1146981 | biostudies-other
Project description:The major glutathione transferases in the rat small-intestine cytosol were isolated and characterized. The enzymes active with 1-chloro-2,4-dinitrobenzene as second substrate were almost quantitatively recovered after affinity chromatography on immobilized S-hexylglutathione. The different basic forms of glutathione transferase, which account for 90% of the activity, were resolved by chromatofocusing. Fractions containing enzymes with lower isoelectric points were not further resolved. The isolated fractions were characterized by their elution position in chromatofocusing, apparent subunit Mr, reactions with specific antibodies, substrate specificities and inhibition characteristics. The major basic forms identified were glutathione transferases 1-1, 4-4 and 7-7. In addition, evidence for the presence of a variant form of subunit 1, as well as trace amounts of subunits 2 and 3, was obtained. A significant amount of transferase 8-8 in the fraction of acidic enzyme forms was demonstrated by immunoblot and Ouchterlony double-diffusion analysis. In the comparison of the occurrence of the different forms of glutathione transferase in liver, lung, kidney and small intestine, it was found that the small intestine is the richest source of glutathione transferase 7-7.
| S-EPMC1149368 | biostudies-other
Project description:To investigate the effects of the somatostatin analogue, octreotide, on maltose and sucrase activities and expression of glucose transporter type 2 (GLUT2) in obese rat intestinal mucosa.We divided 49 Sprague-Dawley rats into a group of 31 high fat diet-induced obese rats and a group of 18 normal controls. The obese rats were separated into an octreotide treated group of 16 rats and an obese group of 15. The intervention group was injected with octreotide at 40 ?g/kg body weight every 12 h for 8 d. Rat body weight was measured weekly to calculate Lee's index. After euthanization, maltase and sucrase activities in the small intestine were measured by activity assays, and the fasting plasma glucose level was measured. The expression of GLUT2 in small intestinal mucosa was analyzed by immunohistochemistry, reverse transcriptase polymerase chain reaction and Western blotting assays.Body weight, Lee's index, fasting plasma glucose level, maltase activity in small intestinal mucosa, mucosa and apical GLUT2, GLUT2 mRNA and protein expression levels were all significantly higher in the obese group than in the normal control group (605.61 ± 141.00 vs 378.54 ± 111.75, 337.61 ± 10.82 vs 318.73 ± 20.10, 8.60 ± 1.38 vs 7.33 ± 0.70, 156.01 ± 58.81 vs 50.43 ± 30.49, 390 744.2 ± 62 469.21 vs 170 546.50 ± 50 646.14, 26 740.18 ± 3809.60 vs 354.98 ± 57.19, 0.26 ± 0.11 vs 0.07 ± 0.02, and 2.08 ± 0.59 vs 1.27 ± 0.38, respectively, all P < 0.01). Sucrase activity did not differ between the two groups. Octreotide intervention significantly decreased the body weight and fasting plasma glucose level of obese rats (508.27 ± 94.39 vs 605.61 ± 141.00, 7.58 ± 1.51 vs 8.60 ±1.38, respectively, all P < 0.05). The intestinal mucosa and apical GLUT2, expression of GLUT2 mRNA and protein were also significantly lower in the octreotide intervention group than in the obese group (269 975.2 ± 53 730.94 vs 390 744.2 ± 62 469.21, 3758.06 ±364.51 vs 26 740.18 ± 3809.60, 0.08 ± 0.02 vs 0.26 ±0.11, and 1.31 ± 0.27 vs 2.08 ± 0.59, respectively, all P < 0.01).High fat diet-induced obesity is associated with elevated intestinal maltase activity, GLUT2 expression, and permanent apical GLUT2 in the small intestinal mucosa of rats. Octreotide can inhibit these effects.
| S-EPMC3218159 | biostudies-literature
Project description:We investigated the mechanisms underlying damage to rat small intestine in heat- and shake-induced stress. Eighteen Sprague-Dawley rats were randomly divided into a control group and a 3-day stressed group treated 2 h daily for 3 days on a rotary platform at 35°C and 60 r/min. Hematoxylin and eosin-stained paraffin sections of the jejunum following stress revealed shedding of the villus tip epithelial cells and lamina propria exposure. Apoptosis increased at the villus tip and extended to the basement membrane. Photomicrographs revealed that the microvilli were shorter and sparser; the nuclear envelope invaginated and gaps in the karyolemma increased; and the endoplasmic reticulum (ER) swelled significantly. Gene microarray analysis assessed 93 differentially expressed genes associated with apoptosis, ER stress, and autophagy. Relevant genes were compiled from the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Forty-one genes were involved in the regulation of apoptosis, fifteen were related to autophagy, and eleven responded to ER stress. According to KEGG, the apoptosis pathways, mitogen-activated protein kinase(MAPK) signaling pathway, the mammalian target of rapamycin (mTOR) signaling pathway, and regulation of autophagy were involved. Caspase3 (Casp3), caspase12 (Casp12), and microtubule-associate proteins 1 light chain 3(LC3) increased significantly at the villus tip while mTOR decreased; phosphorylated-AKT (P-AKT) decreased. ER stress was involved and induced autophagy and apoptosis in rat intestinal damage following heat and shake stress. Bioinformatic analysis will help determine the underlying mechanisms in stress-induced damage in the small intestine.
| S-EPMC4670120 | biostudies-literature
Project description:Glucose transport and metabolism by rat small intestine was investigated by using a preparation for the combined perfusion of the lumen and the vascular bed. 2. When 5 mM-glucose was present in the lumen, only 29% was transported unchanged to the vascular side. Lactate output into the vascular and luminal fluids accounted for a further 53% and 4% respectively of the glucose taken up from the lumen. 3. Glucose was readily taken up when added at 5 mM to the vascular compartment only. Vascular lactate output accounted for approx. 33% of the glucose uptake, and luminal lactate for 6%. 4. When 2 mM-glucose was added to the lumen with 5 mM-glucose in the vascular perfusate, there was no detectable net transport of glucose to the vascular side. However, of the glucose taken up from the lumen, 31% and 2% could be accounted for by increases in vascular and luminal lactate respectively. 5. When 10 mM-glucose was added to the lumen, with 5 mM-glucose in the vascular perfusate, 33% of the glucose disappearing from the lumen was transported to the vascular side. Extra lactate output to the vascular and lumen perfusates accounted for 50% and 9% respectively of the glucose uptake from the lumen. 6. These studies indicate that at low luminal glucose concentrations no sugar is transferred to the blood unchanged, and at sugar concentrations of 5--10 mM only 25--50% of the glucose leaving the lumen reaches the serosal side intact. Furthermore, the small intestine has a greater propensity to form lactate from luminal glucose than from vascular glucose.
| S-EPMC1152027 | biostudies-other
Project description:1. beta-Galactosidase activity was studied in homogenates of the proximal and distal thirds of the small intestine from adult and infant rats. o-Nitrophenyl beta-d-galactoside served as the substrate. 2. Activity in suckling rats is highest in the distal part of the small intestine. 3. The pH optimum was 3.5 in the distal third of the small intestine in rats aged 5 and 15 days, whereas in the proximal third the maximum was not clearly defined. 4. Activity was higher in both thirds in newborn than in adult rats, expressed per wet wt. or per wt. of protein. In the proximal third activity continually decreases with age, whereas in the distal part there is a rise up to day 15 and then a sudden decrease. Total beta-galactosidase activity changes very little in the proximal third during postnatal development; the greatest changes occur in the distal third. 5. Adrenalectomy performed on day 15 postnatally slows down the decrease in beta-galactosidase activity, particularly in the distal part. 6. Feeding a lactose diet to infant rats from day 14 postnatally in the presence of the mother rat also slows down the decrease in beta-galactosidase activity and this is not found with a diet containing glucose and galactose instead of lactose.
| S-EPMC1206437 | biostudies-other