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61.
Saad Nazir Anoop Kumar Ishita Chatterjee Arivarasu N. Anbazhagan Tarunmeet Gujral Shubha Priyamvada Seema Saksena Waddah A. Alrefai Pradeep K. Dudeja Ravinder K. Gill 《PloS one》2015,10(5)
TGF-β1 is an important multifunctional cytokine with numerous protective effects on intestinal mucosa. The influence of TGF-β1 on serotonin transporter (SERT) activity, the critical mechanism regulating the extracellular availability of serotonin (5-HT), is not known. Current studies were designed to examine acute effects of TGF-β1 on SERT. Model human intestinal Caco-2 cells grown as monolayer’s or as cysts in 3D culture and ex vivo mouse model were utilized. Treatment of Caco-2 cells with TGF-β1 (10 ng/ml, 60 min) stimulated SERT activity (~2 fold, P<0.005). This stimulation of SERT function was dependent upon activation of TGF-β1 receptor (TGFRI) as SB-431542, a specific TGF-βRI inhibitor blocked the SERT stimulation. SERT activation in response to TGF-β1 was attenuated by inhibition of PI3K and occurred via enhanced recruitment of SERT-GFP to apical surface in a PI3K dependent manner. The exocytosis inhibitor brefeldin A (2.5 μM) attenuated the TGF-β1-mediated increase in SERT function. TGF-β1 increased the association of SERT with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) syntaxin 3 (STX3) and promoted exocytosis of SERT. Caco-2 cells grown as cysts in 3D culture recapitulated the effects of TGF-β1 showing increased luminal staining of SERT. Ussing chamber studies revealed increase in 3H-5-HT uptake in mouse ileum treated ex vivo with TGF-β1 (10 ng/ml, 1h). These data demonstrate a novel mechanism rapidly regulating intestinal SERT via PI3K and STX3. Since decreased SERT is implicated in various gastro-intestinal disorders e.g IBD, IBS and diarrhea, understanding mechanisms stimulating SERT function by TGF-β1 offers a novel therapeutic strategy to treat GI disorders. 相似文献
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Both the Entamoeba histolytica lectin, a virulence factor for the causative
agent of amebiasis, and the mammalian hepatic lectin bind to
N-acetylgalactosamine (GalNAc) and galactose (Gal) nonreducing termini on
oligosaccharides, with preference for GalNAc. Polyvalent GalNAc-
derivatized neoglycoproteins have >1000-fold enhanced binding affinity
for both lectins (Adler,P., Wood,S.J., Lee,Y.C., Lee,R.T., Petri,W.A.,Jr.
and Schnaar,R.L.,1995, J. Biol. Chem ., 270, 5164-5171). Substructural
specificity studies revealed that the 3-OH and 4-OH groups of GalNAc were
required for binding to both lectins, whereas only the E.histolytica lectin
required the 6-OH group. Whereas GalNAc binds with 4-fold lower affinity to
the E.histolytica lectin than to the mammalian hepatic lectin,
galactosamine and N-benzoyl galactosamine bind with higher affinity to the
E. histolytica lectin. Therefore, a synthetic scheme for converting
polyamine carriers to poly-N-acyl galactosamine derivatives (linked through
the galactosamine primary amino group) was developed to test whether such
ligands would bind the E.histolytica lectin with high specificity and high
affinity. Contrary to expectations, polyvalent derivatives including
GalN6lys5, GalN4desmosine, GalN4StarburstTMdendrimer, and
GalN8StarburstTMdendrimer demonstrated highly enhanced binding to the
mammalian hepatic lectin but little or no enhancement of binding to the
E.histolytica lectin. We propose that the mammalian hepatic lectin binds
with greatest affinity to GalNAc "miniclusters," which mimic branched
termini of N-linked oligosaccharides, whereas the E.histolytica lectin
binds most effectively to "maxiclusters," which may mimic more widely
spaced GalNAc residues on intestinal mucins.
相似文献
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Thrombosis plays a major role in acute vessel closure both after coronary balloon angioplasty and after stenting. This review will address the role of antiplatelet and anticoagulant therapy in preventing early thrombotic complications after percutaneous coronary intervention. The focus will be on agents that are routinely available and commonly used. 相似文献
65.
Annaba F Sarwar Z Kumar P Saksena S Turner JR Dudeja PK Gill RK Alrefai WA 《American journal of physiology. Gastrointestinal and liver physiology》2008,294(2):G489-G497
Apical sodium-dependent bile acid transporter (ASBT) represents a highly efficient conservation mechanism of bile acids via mediation of their active transport across the luminal membrane of terminal ileum. To gain insight into the cellular regulation of ASBT, we investigated the association of ASBT with cholesterol and sphingolipid-enriched specialized plasma membrane microdomains known as lipid rafts and examined the role of membrane cholesterol in maintaining ASBT function. Human embryonic kidney (HEK)-293 cells stably transfected with human ASBT, human ileal brush-border membrane vesicles, and human intestinal epithelial Caco-2 cells were utilized for these studies. Floatation experiments on Optiprep density gradients demonstrated the association of ASBT protein with lipid rafts. Disruption of lipid rafts by depletion of membrane cholesterol with methyl-beta-cyclodextrin (MbetaCD) significantly reduced the association of ASBT with lipid rafts, which was paralleled by a decrease in ASBT activity in Caco-2 and HEK-293 cells treated with MbetaCD. The inhibition in ASBT activity by MbetaCD was blocked in the cells treated with MbetaCD-cholesterol complexes. Kinetic analysis revealed that MbetaCD treatment decreased the V(max) of the transporter, which was not associated with alteration in the plasma membrane expression of ASBT. Our study illustrates that cholesterol content of lipid rafts is essential for the optimal activity of ASBT and support the association of ASBT with lipid rafts. These findings suggest a novel mechanism by which ASBT activity may be rapidly modulated by alterations in cholesterol content of plasma membrane and thus have important implications in processes related to maintenance of bile acid and cholesterol homeostasis. 相似文献
66.
Expression and membrane localization of MCT isoforms along the length of the human intestine 总被引:4,自引:0,他引:4
Gill RK Saksena S Alrefai WA Sarwar Z Goldstein JL Carroll RE Ramaswamy K Dudeja PK 《American journal of physiology. Cell physiology》2005,289(4):C846-C852
Recent studies from our laboratory and others have demonstrated the involvement of monocarboxylate transporter (MCT)1 in the luminal uptake of short-chain fatty acids (SCFAs) in the human intestine. Functional studies from our laboratory previously demonstrated kinetically distinct SCFA transporters on the apical and basolateral membranes of human colonocytes. Although apical SCFA uptake is mediated by the MCT1 isoform, the molecular identity of the basolateral membrane SCFA transporter(s) and whether this transporter is encoded by another MCT isoform is not known. The present studies were designed to assess the expression and membrane localization of different MCT isoforms in human small intestine and colon. Immunoblotting was performed with the purified apical and basolateral membranes from human intestinal mucosa obtained from organ donor intestine. Immunohistochemistry studies were done on paraffin-embedded sections of human colonic biopsy samples. Immunoblotting studies detected a protein band of 39 kDa for MCT1, predominantly in the apical membranes. The relative abundance of MCT1 mRNA and protein increased along the length of the human intestine. MCT4 (54 kDa) and MCT5 (54 kDa) isoforms showed basolateral localization and were highly expressed in the distal colon. Immunohistochemical studies confirmed that human MCT1 antibody labeling was confined to the apical membranes, whereas MCT5 antibody staining was restricted to the basolateral membranes of the colonocytes. We speculate that distinct MCT isoforms may be involved in SCFA transport across the apical or basolateral membranes in polarized colonic epithelial cells. monocarboxylate transporter; short-chain fatty acids; absorption; short-chain fatty acid transport; mammalian colon 相似文献
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目的构建用于呼吸道合胞病毒(respiratory syncytial virus,RSV)体外拯救的RSV基因组全长cDNA克隆,并进行鉴定。方法根据RSV Long株基因组序列设计并合成引物,利用RT-PCR技术分6段扩增RSV LZ01/09基因组序列并构建克隆载体;测序后,利用重叠PCR与酶切连接技术,根据基因组序列选择特异性酶切位点,引入Kpn I、Xma I和Sal I酶切位点,构建成4个亚克隆载体;将亚克隆载体的插入片段连接至经过改造且包含T7启动子、锤头状核酶、多克隆位点、丁肝核酶、T7终止子的p RSV1载体中,构建RSV基因组全长cDNA克隆;对克隆全长cDNA序列进行测定,与亲本RSV LZ01/09基因组进行同源性比对分析,并与RSV实验参比株进行系统进化树分析。结果测序结果显示,RSV LZ 01/09的基因组全长为15 204 bp,与GenBank公布的RSV基因组序列长度相当,将完整的序列提交GenBank,登录号为KY782635;酶切及测序结果显示,用于RSV全长cDNA克隆构建的基本载体p BSKS-MCS(简称p RSV1)与预期相符,RSV全长基因组cDNA克隆质粒(简称转录载体p RSV1-4F)酶切片段大小与预期一致;同源性比对结果显示,全长cDNA序列与亲本RSV LZ01/09基因组序列同源性高达99.83%;系统进化树分析结果显示,其与RSV-A亚型序列同属于一个分支。结论测序及酶切分析结果表明已成功构建RSVLZ01/09基因组全长cDNA克隆,为建立拯救RSV重组病毒的反向遗传学系统平台奠定了基础。 相似文献
70.
Janneke Anink Charlotte M Nusman Lisette WA van Suijlekom-Smit Rick R van Rijn Mario Maas Marion AJ van Rossum 《Arthritis research & therapy》2014,16(4)