Extract from Modified Xiao Xianxiongtang Inhibits Epithelial-mesenchymal Transition and Invasion and Migration Mediated by TGF-β1 of Human Gastric Cancer MGC-803 Cells via Wnt5a/Ca2+/NFAT Signaling Pathway / 中国实验方剂学杂志

Objective:This studu aims to investigate the effect of aqueous extract of modified Xiao Xianxiongtang on the epithelial mesenchymal transition（EMT） and the change of its invasion and migration ability of human gastric cancer MGC-803 cells mediated by transforming growth factor-<italic>β</italic>₁（TGF-<italic>β</italic>₁），and to explore the mechanism of regulating Wnt5a/Ca²⁺/ activated T-cell nuclear factor（NFAT） signaling pathway to inhibit EMT and invasion and metastasis of MGC-803 cells. Method:TGF-<italic>β</italic>₁（10 μg·L^-1）was used to induce EMT and the invasion and metastasis model of human gastric cancer MGC-803 cells. Transwell chamber experiment， scratchhealing experiment， Western blot and immunofluorescence assay were used to detect cell invasion and migration ability， expression of EMT marker protein and key protein expression of Wnt5a/Ca²⁺/NFAT signaling pathway， and intracellular Ca²⁺ concentration. Result:Compared with the blank group， TGF-<italic>β</italic>₁ could significantly enhance the invasion and migration ability of MGC-803 cells（<italic>P</italic><0.01）， down-regulate the level of E-cadherin（<italic>P</italic><0.01）， up-regulate protein expressions of N-cadherin， Snail and Vimentin（<italic>P</italic><0.01）， and induce cell Wnt5a， calcineurin （CaN）， total protein of activated T-cell nuclear factor 1（NFAT1）， up-regulation of phosphorylated proteins p-NFAT1 and NFAT1 nucleoprotein and intracellular accumulation of Ca²⁺（<italic>P</italic><0.01）. Compared with the TGF-<italic>β</italic>₁ group， modified Xiao Xianxiongtang （10， 20， 40 mg·L^-1） could significantly inhibit this phenomenon，and 40 mg·L^-1 had the best effect（<italic>P</italic><0.05，<italic>P</italic><0.01）.The specific inhibitors of Wnt5a/Ca²⁺/NFAT signaling pathway （<italic>R</italic>）-（+）-Bay-K-8644 and modified Xiao Xianxiongtang （40 mg·L^-1） could significantly inhibit theinvasion and migration of MGC-803 cells mediated by TGF-<italic>β</italic>₁， up-regulate the level of E-cadherin， and down-regulate expressions of N-cadherin， Snail， Vimentin， Wnt5a， CaN and NFAT1 proteins and reduce the intracellular accumulation of Ca²⁺（<italic>P</italic><0.05，<italic>P</italic><0.01）.Moreover， （<italic>R</italic>）-（+）-Bay-K-8644 combined with modified Xiao Xianxiongtang （40 mg·L^-1） had stronger inhibitory effect（<italic>P</italic><0.05，<italic>P</italic><0.01）. Conclusion:These results suggest that modified Xiao Xianxiongtang can inhibit the EMT mediated by TGF-<italic>β</italic>₁ via Wnt5a/Ca²⁺/NFAT signaling pathway，thereby reducing the invasion and migration ability of MGC-803 cells.

Effect of Paridis Rhizoma total saponins on apoptosis of human gastric cancer cell MKN-45 and Fas/FasL signaling pathway / 中国中药杂志

<p><b>OBJECTIVE</b>The study aimed to test if Paridis Rhizoma total saponins (PRTS) could induce apoptosis of human gastric cancer cell MKN-45.</p><p><b>METHOD</b>Based on the previous researches, PRTS was set by different concentrations to treat human gastric cancer cell for 12 h (5, 10, 20 mg x L(-1)). Fluorescent staining methods were adopted to observe apoptotic morphological changes of MKN-45. The apoptosis rates were analyzed by flow cytometry with Annexin V-FITC/PI staining. The enzymatic activities of caspase-3 and caspase-8 were measured by ELISA. The protein levels of Fas and FasL were detected by Western blotting.</p><p><b>RESULT</b>Under a fluorescence microscope, MKN-45 treated by PRTS was seen typical apoptotic morphological features. PRTS significantly increased the rate of apoptosis. Compared with the control group, there exsited significant differences in apoptosis rate of PRTS concentration of 20 mg x L(-1) (P < 0.01); besides, the enzymatic activities of caspase-3 and caspase-8 were promoted obviously after the effect of PRTS on MKN-45 cells for 12 h (P < 0.01). The protein levels of Fas and FasL in the MKN-45 were upgraded significantly.</p><p><b>CONCLUSION</b>PRTS can induce apoptosis of human gastric cancer cell MKN-45 , which is concerned with caspase-3 and caspase-8 and upgraded Fas and FasL.</p>

Cloning and localization of A3IP -a novel protein that interacts with ataxin-3 / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To clone an A3IP gene and investigate its cellular and histological localization based on previous research which has identified part of A3IP sequence interacting with carboxyl-terminal of ataxin-3.</p><p><b>METHODS</b>Bioinformatic and Northern blotting were applied to clone the A3IP gene and detect the expression of its transcripts in various human tissues and brain regions. Western blotting and immunofluorescence staining were applied to detect expression of A3IP protein in cultured cells. Immunohistochemistry staining was applied to study the expression of A3IP protein in various human tissues and brain regions.</p><p><b>RESULTS</b>cDNA cloning of A3IP gene's reading frame and its sequence assembly were completed. Three transcripts (1 kb, 1.35 kb and 6 kb, respectively) of A3IP were found to express in various human tissues and brain regions. A3IP pEGFP expresses in cytoplasm of cultured COS-7 cells and various human tissues and brain regions including cerebral cortex, cerebellum, muscle, peripheral nerve, liver and kidney.</p><p><b>CONCLUSION</b>The cloned A3IP gene encodes A3IP, a novel ataxin-3 interacting protein. Three transcripts of A3IP are expressed in various human tissues and brain regions. A3IP is a cytosolic protein.</p>

Screening for proteins interacting with ataxin-3, the gene product of SCA3/MJD / 中南大学学报(医学版)

OBJECTIVE@#To screen for proteins interacting with ataxin-3 by yeast two-hybrid system 3, and to discuss the function of ataxin-3 and pathogenesis of spinocerebellar ataxia type 3 and Machado-Joseph disease (SCA3/MJD).@*METHODS@#First we sub-cloned the full reading frame of both wild-type and mutant ataxin-3 into carrier pGBKT7 (ataxin-3-bait), and then screened human brain cDNA library with ataxin-3-bait.@*RESULTS@#We found five positive clones in 6.5 x 10(6) transformers. After sequencing, we knew all of them were novel ataxin-3 interacting proteins. Three were corresponded to the known sequences coding the known proteins, which were human Rho GDP dissociation inhibitor alpha, small ubiquitin-like modifier 1, and human neuronal amiloride-sensitive cation channel 2. Another two of the five were unknown.@*CONCLUSION@#Small ubiquitin-like modifier 1 probably interacted with ataxin-3, suggesting that the sumoylation probably participated in post-translation modifying of ataxin-3 and pathogenesis of SCA3/MJD.

Construction of middle fragment deletion mutant with improved gene splicing by overlap extension / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To construct a phosphatidylinositol 4-kinase beta (PI4K-beta) mutant with the 325th to 373rd amino acid codons deleted, and try to develop a simple method for constructing middle fragment deletion mutant.</p><p><b>METHODS</b>In line with the mechanism of gene splicing by overlap extension(SOE), an additional PCR was used to get the PI4K-beta mutant in which the 325th to 373rd amino acid codons were deleted. Then the mutated gene was cloned into pCMV-Tag4A mammalian expression vector.</p><p><b>RESULTS</b>A mutant with the 325th to 373rd amino acid codons deleted was constructed successfully.</p><p><b>CONCLUSION</b>The improved SOE is a very effective and reliable method to construct middle fragment deletion mutant. It is worthy to be popularized.</p>