Developmental control of the expression of two ten-sugar branched chain fucolipids in rat small intestine

Two branched decaglycosylceramides, apparently identical to those identified in the small intestine of adult rats [Breimer ME, Falk K-E, Hansson GC, Karlsson K-A (1982) J Biol Chem 257:50–59], were absent during the three weeks following birth. They appeared abruptly at around 21 days. After their appearance, their tissue concentration and their base composition did not change during development. Their fatty acids were non-hydroxylated and the percentage of C₂₂–C₂₄ fatty acids, which was low at 24 days, increased and reached 48.6% by 27 days.Nomenclature Gal1-4Gal1-4GlcCer Globotriaosylceramide (GbOse₃Cer) - Il³NeuAc-LacCer M_M3-ganglioside - GalNAc1-3Gal1-4Gal1-4GlcCer globoside (globotetraosylceramide, GbOse₄Cer)     

Interaction of heparin with synthetic peptides corresponding to the C-terminal domain of intestinal mucins

Unlike most other mucins described to date, two intestinal mucins, rat MLP (rat Muc2) and human MUC2 have a C-terminal tail that is enriched in cationic amino acids. The distribution of charge in each case resembles that of several well known heparin binding proteins. Peptides designated E20-14 and F13-15, corresponding to the C-terminal 14 amino acids of the two mucins, were synthesized and shown to bind³H-labelled heparin by a process that was saturable and mediated by strong electrostatic interactions, givingK _d values of 10^–7 to 10^–8 m. Using turbidometric analyses and native gel electrophoresis, we observed that peptide-heparin mixtures formed polydisperse aggregates that dissociated with a progressive increase in the concentration of heparin. Under certain conditions heparin protected the peptide from proteolysis by trypsin. Both heparin and dextran sulfate, the latter a highly sulfated synthetic polysaccharide, were potent inhibitors of³H-heparin binding to peptide E20-14, while less sulfated glycosaminoglycans were poorly- or non-inhibitory. Mucin in tissue dispersions and homogenates, or purified from rat intestine, did not bind to heparin, and failed to interact with an antibody specific for the peptide E20-14. Both mucin samples however, reacted with antibodies that recognize regions upstream of the C-terminal 14 amino acids. Immunofluorescent localization of E20-14 was confined to the basal perinuclear regions of goblet cells, whereas localization of an antibody to a flanking sequence on the N-terminal side of the C-tail, localized to mature mucin storage granules. These findings suggest that the heparin-binding C-tail of the mucin may be removed at an early stage of biosynthesis. Heparin-mucin complexes, if they formin vivo, are thus likely to be confined to the ER and/or Golgi compartments.     

Uptake of selenite,selenomethionine and selenate by brush border membrane vesicles isolated from rat small intestine

The uptake of selenite, selenate and selenomethionine (SeMet) was performed with brush border membrane vesicles (BBMV) prepared from rats fed selenium-deficient and supplemented diets. At equilibrium (60 min), the uptake of ⁷⁵Se from [⁷⁵Se]selenite ranged from 16.5 to 18.9 nmol mg^-1 protein. There was a curvilinear relationship in the uptake of selenite over a concentration range of 10–1000 m. About 2 nmol mg^-1 protein was obtained with selenomethionine (SeMet) which occurred between 90 and 180 s. In contrast to selenite, there was a linear relationship in the initial uptake of SeMet over a concentration range of 10–1000 m. The uptake of selenate was approximately 50-fold lower than selenite, reaching 350 pmol mg^-1 protein. Dietary selenium level had no effect on the rate of ⁷⁵Se accumulation by BBMV. Dramatic differences are found in the uptake and binding of selenium by BBMV incubated with different selenocompounds.     

Suppression of mucin 2 enhances the proliferation and invasion of LS174T human colorectal cancer cells

Altered expression of MUC2 (mucin 2) is related to tumour development in colorectal cancer. Colorectal mucinous carcinomas are positive for MUC2 expression, whereas MUC2 is down-regulated in non-mucinous adenocarcinomas. In the present study, we down-regulated MUC2 expression by RNAi (RNA interference) and investigated the in vitro and in vivo effects on the proliferation and invasion/migration potential of the LS174T human colorectal cancer cells. The LS174T cell line is a goblet-cell-like colorectal cancer cell line that continuously produces high levels of MUC2. Inhibition of MUC2 expression in vitro by transfection of LS174T cells with the recombinant plasmid pcDNA6.2-GW/EmGFP-miR-MUC2 led to the production of a stably transfected MUC2-RNAi LS174T cell line. The proliferation and invasion/migration of MUC2-RNAi cells in vitro were significantly higher than those in control cells, as assessed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], colony formation and transwell assays. Subcutaneous injection of MUC2-RNAi LS174T cells into nude mice resulted in the development of subcutaneous tumours visible to the naked eye after 1 week. The growth rate of tumours derived from MUC2-RNAi LS174T cells was greater than that of tumours derived from control cells. Ki67 and matrix metalloproteinase-9 proteins were detected by immunohistochemistry in the xenografts. The expression levels of these proteins were higher in the MUC2-RNAi-derived xenografts than in xenografts derived from control cells. Although the role of MUC2 in colorectal tumorigenesis is not fully understood, these results strongly suggest a relationship between the proliferation and invasion of LS174T cells and the expression of MUC2.     

Localisation of a protein core-specific epitope from gastrointestinal mucin (MUC2). The effect of epitope immobilisation on antibody recognition

Human intestinal mucins are high molecular weight glycoproteins which protect and lubricate the epithelium of the gastrointestinal tract. In cases of malignant disease, mucins are abnormally expressed, overproduced or underglycosylated. This feature may enable the mucins to serve as tumour markers. The MUC2 mucin largely consists of a variable number of tandem repeats of a 23 amino acid sequence, ¹PTTTPITTTTTVTPTPTPTGTQT²³. In this study we have localised the minimal and the optimal epitope within this region by the previously developed protein core specific 996 monoclonal antibody using synthetic peptides. Several overlapping and truncated peptides related to the tandem repeat unit have been prepared by solid-phase methodology. Other mucin peptides were synthesised on the tips of polyethylene pins, and these remained C-terminally attached to the pins for comparative investigations. The interaction of the 996 monoclonal antibody with the synthetic peptides was studied either in solution by competition RIA or on immobilised peptides by indirect ELISA experiments. These experiments show that the minimal epitope recognised by the 996 antibody is the Ac-¹⁹TGTQ²² (IC₅₀=3100 μm in solution). For the optimal 996 antibody binding in solution the ¹⁶PTPTGTQ²² heptapeptide (IC₅₀=3 μm ) is required. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

The effect of massive small bowel resection (MSBR) and small intestinal bypass (JIB) in the rat on the enteroinsular axis

The effect of massive small bowel resection (MSBR) and jejuno-ileal bypass (JIB) on the enteroinsular axis in rats was compared. Glucose levels after an oral glucose tolerance test were determined in MSBR, JIB and control animals. The response of the beta-cell mass to glucose and gastric inhibitory polypeptide (GIP) was established in the same animals using the isolated perfused pancreas model. Immunocytochemical and morphological studies were performed to monitor the adaptive changes seen in the intestine of these animals. The glucose response to the oral glucose load was blunted in both test groups with the fasting GIP levels in the JIB group being elevated and the MSBR group being reduced. The response of the isolated perfused pancreas to GIP showed a marked (70%) reduction of insulin release in the JIB rats and a slight but non-significant reduction in the MSBR rats. In both groups the insulin response to glucose alone appeared normal. The area of the pancreatic islets and the percentage of the total area consisting of the four islet cell types (B, A, D, PP) were unchanged. In the intestine the GIP cells were markedly reduced in number in the jejunum of the functional intestine of the JIB rats and the jejunum from the MSBR rats. The GIP cells in the jejunum of the bypass loop did not differ from the control jejunum. The results indicate that the high basal GIP levels seen in the JIB rats were the result of GIP secreted from the blind loop. This study also confirmed the decreased sensitivity of the beta-cells to GIP after JIB while indicating that MSBR has little if any effect on the response of the beta-cell to GIP. These data presented further evidence that the high basal GIP levels were causally related to the decreased insulin response in the JIB rats.     

Galectin-2 at the enterocyte brush border of the small intestine

The brush border of pig small intestine is a local hotspot for β-galactoside-recognizing lectins, as evidenced by its prominent labeling with fluorescent lectin PNA. Previously, galectins 3-4, intelectin, and lectin-like anti-glycosyl antibodies have been localized to this important body boundary. Together with the membrane glycolipids these lectins form stable lipid raft microdomains that also harbour several of the major digestive microvillar enzymes. In the present work, we identified a lactose-sensitive 14-kDa protein enriched in a microvillar detergent resistant fraction as galectin-2. Its release from closed, right-side-out microvillar membrane vesicles shows that at least some of the galectin-2 resides at the lumenal surface of the brush border, indicating that it plays a role in the organization/stabilization of the lipid raft domains. Galectin-2 was released more effectively from the membrane by lactose than was galectin-4, and surprisingly, it was also released by the noncanonical disaccharides sucrose and maltose. Furthermore, unlike galectin-4, galectin-2 was preferentially coimmunoisolated with sucrase-isomaltase rather than with aminopeptidase N. Together, these results show that the galectins are not simply redundant proteins competing for the same ligands but rather act in concert to ensure an optimal cross-linking of membrane glycolipids and glycoproteins. In this way, they offer a maximal protection of the brush border against exposure to bile, pancreatic enzymes and pathogens.     

ABCG2 expression and uric acid metabolism of the intestine in hyperuricemia model rat

Abstract

To elucidate roles of the intestine in uric acid (UA) metabolism, we examined ABCG2 expression, tissue UA content and xanthine oxidoreductase (XOR) activity in different intestinal segments. Male SD rats were assigned to control group or oxonic acid-induced hyperuricemia (HUA) group. In control rats, ABCG2 was present both in villi and crypts in each segment. Tissue UA content and XOR activity were relatively high in duodenum and jejunum. However, in HUA rats, tissue UA content was significantly elevated in the ileum, whereas it remained unaltered in other segments. Moreover, ABCG2 expression in the HUA group was upregulated both in the villi and crypts of the ileum. These data indicate that the ileum may play an important role in the extra-renal UA excretion.     

Distribution,pharmacological characterization and function of the 18 kDa translocator protein in rat small intestine

Background information. The TSPO (18 kDa translocator protein) is a mitochondrial transmembrane protein involved in cholesterol transport in organs that synthesize steroids and bile salts. Different natural and synthetic high‐affinity TSPO ligands have been characterized through their ability to stimulate cholesterol transport, but also to stimulate other physiological functions including cell proliferation, apoptosis and calcium‐dependent transepithelial ion secretion. Here, we investigate the localization and functions of TSPO in the small intestine. Results. TSPO was present in enterocyte mitochondria but not in rat intestinal goblet cells. Enterocyte cytoplasm also contained the endogenous TSPO ligand, polypeptide DBI (diazepam‐binding inhibitor). Whereas intestinal TSPO had high affinity for the synthetic ligand PK 11195, the pharmacological profile of TSPO in the duodenum was distinct from the jejunum and ileum. Specifically, benzodiazepine Ro5‐4864 and protoporphyrin IX showed 5–13‐fold lower affinity for duodenal TSPO. The mRNA and protein ratios of TSPO to other mitochondrial membrane proteins VDAC (voltage‐dependent anion channel) and ANT (adenine nucleotide transporter) were significantly different. PK 11195 stimulated calcium‐dependent chloride secretion in the duodenum and calcium‐dependent chloride absorption in the ileum, but did not affect jejunum ion transport. Conclusions. The functional differences in subpopulations of TSPO in different regions of the intestine could be related to structural organization of mitochondrial protein complexes that mediate the ability of TSPO to modulate either chloride secretion or absorption in the duodenum and ileum respectively.     

Fine structural study of interstitial cells associated with the deep muscular plexus of the rat small intestine,with special reference to the intestinal pacemaker cells

Two types of interstitial cells have been demonstrated in close association in the deep muscular plexus of rat small intestine, by electron microscopy. Cells of the first type are characterized by a fibroblastic ultrastructure, i.e. a well-developed granular endoplasmic reticulum, Golgi apparatus and absence of the basal lamina. They form a few small gap junctions with the circular muscle cells and show close contact with axon terminals containing many synaptic vesicles. They may play a role in conducting electrical signals in the muscle tissue. Cells of the second type are characterized by many large gap junctions that interconnect with each other and with the circular muscle cells. Their cytoplasm is rich in cell organells, including mitochondria, granular endoplasmic reticulum and Golgi apparatus. They show some resemblance to the smooth muscle cells and have an incomplete basal lamina, caveolae and subsurface cisterns. However, they do not contain an organized contractile apparatus, although many intermediate filaments are present in their processes. They also show close contacts with axon terminals containing synaptic vesicles. These gap-junction-rich cells may be regular components of the intestinal tract and may be involved in the pacemaking activity of intestinal movement.     

Proteomic characterization of lipid rafts markers from the rat intestinal brush border

To assess intestinal lipid rafts functions through the characterization of their protein markers, we have isolated lipid rafts of rat mucosa either from the total membrane or purified brush-border membrane (BBM) by sucrose gradient fractionation after detergent treatment. In both membrane preparations, the floating fractions (4-5) were enriched in cholesterol, ganglioside GM1, and N aminopeptidase (NAP) known as intestinal lipid rafts markers. Based on MALDI-TOF/MS identification and simultaneous detection by immunoblotting, 12 proteins from BBM cleared from contaminants were selected as rafts markers. These proteins include several signaling/trafficking proteins belonging to the G protein family and the annexins as well as GPI-anchored proteins. Remarkably GP2, previously described as the pancreatic granule GPI-anchored protein, was found in intestinal lipid rafts. The proteomic strategy assayed on the intestine leads to the characterization of known (NAP, alkaline phosphatase, dipeptidyl aminopeptidase, annexin II, and galectin-4) and new (GP2, annexin IV, XIIIb, Galpha(q), Galpha(11), glutamate receptor, and GPCR 7) lipid rafts markers. Together our results indicate that some digestive enzymes, trafficking and signaling proteins may be functionally distributed in the intestine lipid rafts.     

Identification and characterization of cow's milk proteins from the rat intestinal lymph using a proteomic strategy

Food proteins were considered to be absorbed into the body after being digested to amino acids, dipeptides, and tripeptides. However, there are studies indicating that some proteins can pass through the intestinal epithelium under normal physiological conditions, perhaps not in sufficient quantities to be of nutritional importance, but in quantities that may be antigenically or biologically active. In the present study, rat intestinal lymph samples were collected using a modified lymph fistula rat model in fasting and cow's milk postprandial states. Low molecular weight proteins were enriched by ultrafiltration and differential solubilization, separated by 1D‐SDS‐PAGE, digested in‐gel based on molecular weight, and identified using nano‐LC‐MS/MS. In the postprandial rat intestinal lymph, nine bovine‐specific proteins (false discovery rate ≤1%) were identified in different molecular weight regions. Most proteins identified in lymph were highly abundant proteins in the milk, such as β‐lactoglobulin and caseins. Seven of the nine identified bovine‐specific proteins are allergens in milk. This strategy can be used to search for proteins that can enter the intestinal lymph and analyze their common features. Understanding the common features of these proteins might help to develop protein drugs taken orally, so that therapeutic proteins might embody fusion domains for cross‐barrier transport or translocation.     

Mass spectrometric analysis of combinatorial peptide libraries derived from the tandem repeat unit of MUC2 mucin.

Four 19-member synthetic peptide libraries, based on the TX1TX2T epitope motif of the mucin-2 gastrointestinal glycoprotein (MUC2) and ranging in peptide length from dipeptides to 15-mers (XT, TXT, TQTXT and KVTPTPTPTGTQTXT), were synthesized by combinatorial solid phase peptide synthesis using the portioning-mixing combinatorial approach, and analysed by electrospray ionization mass spectrometry at different (1000-10000) resolutions. Most of the components of the individual libraries could be easily identified in a single-stage molecular mass screening experiment. The resolving power of the instrument becomes an important factor above 800-1000 Da molecular mass, when predominantly multiply charged molecular ions are formed. Approaches to the identification of isobars (glutamine/lysine), isomers leucine/isoleucine) and sequence variations by tandem mass spectrometry, and/or by high-performance liquid chromatography-mass spectrometry are outlined.     

Stimulation of rat hepatocyte proliferation in vitro and in vivo by factors derived from the bovine small intestinal mucosa

Summary A factor with a molecular weight of less than 1 kDa in the mucosa of the bovine small intestine (low molecular weight factor or LMW factor) stimulated DNA synthesis in rat hepatocytes in primary culture. This factor only showed its activity when it was added with a larger factor with a molecular weight of 30 kDa that was also found in the same tissue (high molecular weight factor or HMW factor). The LMW factor probably acts to enhance the action of a hepatotrophic growth factor, since EGF and HGF can substitute for the HMW factor. The action of the LMW factor was not due to the actions of low molecular weight substances such as norepinephrine, estradiol, triiodothyronine, and putrescine, which enhance the action of EGF or HGF, since substantial amounts of these substances were not found in the extract. When intraperitoneally administered into rats, after two-thirds hepatectomy, the LMW factor enhanced hepatocyte proliferation without the administration of the HMW factor. In the regenerating liver, a hepatotrophic growth factor(s), which acts synergistically with the LMW factor, might be properly provided, but the supply of the LMW factor might be below the level that maximally stimulates hepatocyte proliferation.     

高血压大鼠心肌2型小电导-Ca2+-激活-K+通道蛋白(SK2)表达的变化*

目的:观察高血压大鼠心肌细胞2型小电导-Ca²⁺-激活-K⁺(SK2)通道蛋白表达情况。方法:12只健康成年雄性SD大鼠随机分为对照组(5只)和实验组(7只),实验组采用N’-硝基-L-精氨酸(L-NNA 15 mg/(kg·d))腹腔注射制备高血压模型,对照组以等体积生理盐水腹腔注射,每天称量大鼠体重,每周测量血压及心电图变化,4周后处死大鼠取心脏;采用Western blot的方法检测心肌SK2通道蛋白表达水平。结果:给药4周后,与对照组相比,实验组大鼠血压明显升高(P＜0.05),心电图QRS时长和R-R间期延长,实验组大鼠心房组织和心室组织SK2通道的表达均明显升高(1.12±0.18 vs 0.52±0.99,1.64±0.26 vs 0.99±0.22, P＜0.05)。结论:高血压模型大鼠心房和心室SK2通道表达增加,其可能是导致高血压模型大鼠出现心律失常的机制之一,为高血压疾病的治疗和预后提供新的思路和策略。     

Dual labeling with 5‐bromo‐2'‐deoxyuridine and 5‐ethynyl‐2'‐deoxyuridine for estimation of cell migration rate in the small intestinal epithelium

Small intestinal epithelium is a self‐renewing system in which the entire sequence of cell proliferation, differentiation, and removal is coupled to cell migration along the crypt‐villus axis. We examined whether dual labeling with different thymidine analogues, 5‐bromo‐2'‐deoxyuridine (BrdU) and 5‐ethynyl‐2'‐deoxyuridine (EdU), can be used to estimate cell migration rates on the villi of small intestines in rats. Rats received a single intraperitoneal injection of BrdU and EdU within a time interval, and signals in tissue sections were examined by immunohistochemistry and the “click” reaction, respectively. We successfully observed BrdU‐ and EdU‐positive cells on the epithelium with no cross‐reaction. In addition, we observed an almost complete overlapping of BrdU‐ and EdU‐positive cells in rats administered simultaneously with BrdU and EdU. By calculating the cell migration rate by dividing the distance between the median cell positions of the distribution of BrdU‐ and EdU‐positive cells by the time between the injection of BrdU and EdU, we estimated approximately 9 and 5 μm/h for the cell migration rates on the villi in the jejunum and ileum, respectively. We propose that dual labeling with BrdU and EdU within a time interval, followed by detecting with immunohistochemistry and the click reaction, respectively, is useful to estimate accurately the cell migration rate in the intestinal epithelium in a single animal.     

Purification and the immunological characterization of rat protein phosphatase 2A: enzyme levels in diabetic liver and heart

Summary Protein phosphatase 2A₁ was purified from rat skeletal muscle and used to produce antisera to the three subunits of the holoenzyme. Affinity purified antibodies specific for the subunits of the phosphatase enzyme were found to recognize the type 2A₁ and 2A₂ phosphatase from rat skeletal muscle, heart, liver, brain and erythrocytes and were used to investigate the effects of diabetes on the levels of this enzyme in liver and heart. Phosphorylase phosphatase assays coupled with immunoblot analysis of fractionated rat liver and heart cytosol from normal and diabetic animals show no apparent differences in the quantity or activity of these enzymes following the induction of alloxan diabetes. When considering these results and the normal physiological concentrations of known effectors of these enzymes, it is likely that protein phosphatase 2A₁ and 2A₂ are not responsible for the dephosphorylation of phosphorylase a under physiological conditions.     

Developmental expression of metabotropic P2Y(1) and P2Y(2) receptors in freshly isolated astrocytes from rat hippocampus

There are at least three subtypes of cloned metabotropic P2 receptors linked to intracellular Ca(2+) rises in rat brain cells, namely, P2Y(1), P2Y(2) and P2Y(4). In this study we explore the subtypes of the metabotropic P2 receptors seen in freshly isolated astrocytes (FIAs) from P8-P25 rats. We found by single cell RT-PCR that in process-bearing FIAs from hippocampi of P8-P12 rats, 31% of the glial fibrillary acidic protein (GFAP) mRNA (+) cells expressed P2Y(1) mRNA while only 5% of the cells tested expressed P2Y(2) mRNA. The expression of P2Y(1) receptor mRNA was not changed in FIAs from the hippocampi of P18-P25 rats, but 38% of the GFAP mRNA (+) cells in the P18-P25 age group then showed P2Y(2) mRNA. We also studied whether the mRNA was expressing functional receptor protein by measuring Ca(2+) responses to specific agonists for P2Y(1) and P2Y(2). We found that similar proportions of GFAP mRNA (+) FIAs responded to ATP or UTP as showed mRNAs for P2Y (1) and P2Y(2,) respectively. Total tissue RNA from P9 and P24 rat hippocampus showed a 2.8-fold increase in P2Y(2) mRNA levels from P9 to P24 with a decrease in P2Y(1) mRNA. Thus, this study shows a marked up-regulation of mRNA for P2Y(2) from 9 to 24 days in rat hippocampus, and some of this increase is likely due to the protoplasmic astrocytes which is being translated into functional receptor protein in these cells.