Study on the regulatory effect of liver X receptor in HEK293 cells by six main diterpene esters in Semen Euphorbiae

Background:To study the effects of the main diterpene esters in Euphorbia factor L_(1),L_(2),L_(3),L_(7a),L_(7b)and L_(8)on the transcriptional activity and protein expression of liver X receptor(LXR).Methods:The effect of the main diterpene ester components in Semen Euphorbiae on the viability of HEK293 cells were studied by MTT assay.The LXR-Luc plasmid vector was transfected into HEK293 cells and treated with Euphorbia factor L_(1),L_(2),L_(3),L_(7a),L_(7b)and L_(8)for 24 h.The effect of the main diterpene ester components of Semen Euphorbiae on LXR-Luc luciferase activity was investigated by dual luciferase reporter gene system,and the expression of LXRαprotein was detected by Western Blot.Results:Euphorbia factor L_(1),L_(2),L_(3),L_(7a),L_(7b)and L_(8)could significantly reduce the relative luciferase activity(RLU)of LXRα,and the expression level of LXRαprotein was significantly down-regulated.Conclusion:Euphorbia factor L_(1),L_(2),L_(3),L_(7a),L_(7b)and L_(8)can inhibit the expression of LXR protein level,which may be achieved by inhibiting the transcriptional activity of LXR.

Phylogenetic Relationship of the Firefly,Diaphanes pectinealis(Insecta,Coleoptera,Lampyridae) Based on DNA Sequence and Gene Structure of Luciferase

Diaphanes is the fourth largest genus in Lampyridae, but no luciferase gene from this genus has been reported. In this paper, by PCR amplification of the genomic DNA, the luciferase gene of Diaphanes pectinealis, which is the first case from Diaphanes, was identified and sequenced. The luciferase gene from D. pectinealis spans 1958 base pairs （bp） from the start to the stop codon, including seven exons separated by six introns, and encoding a 547-residuelong polypeptide. Its deduced amino acid sequence showed high protein similarity to those of the Lampyrini tribe （93 - 94% ） and the Cratomorphini tribe （92%）, while low similarity was found with the North American firefly Photinus pyralis （83%） of the Photinini tribe within the same subfamily Lampyrinae. The phylogenetic analysis performed with the deduced amino acid sequences of the luciferase gene further confirms that D. pectinealis, Pyrocoelia, Lampyris, Cratomorphus, and Photinus belong to the same subfamily Lampyrinae, and Diaphanes is closely related to Pyrocoelia, Lampyris, and Cratomorphus. Furthemore, the phylogenetic analysis based on the nucleotide sequences of the luciferase gene indicates Diaphanes is a sister to Lampyris. The phylogenetic analyses are partly consistent with morphological （Branham ＆ Wenzel, 2003） and mitochondrial DNA analyses （Li et al, 2006）.

Cloning,Expression and Sequence Analysis of A Luciferase Gene from the Chinese Firefly Pyrocoelia pygidialis

The cDNA encoding the luciferase from lantern mRNA of one diurnal firefly Pyrocoelia pygidialis Pic, 1926 has been cloned, sequenced and functionally expressed. The cDNA sequence of P pygidialis luciferase is 1647 base pairs in length, coding a protein of 548 amino acid residues. Sequence analysis of the deduced amino acid sequence showed that this luciferase had 97.8% resemblance to luciferases from the fireflies Lampyris noctiluca, Lampyris turkestanicus and Nyctophila cf. caucasica. Phylogenetic analysis using deduced amino acid sequence showed that P pygidialis located at the base of Lampyris＋Nyctophila clade with robust support （BP=97%）; but did not show a monophyletic relationship with its congeneric species P pectoralis, P tufa and P miyako, all three are strong luminous and nocturnal species. The expression worked in recombinant Escherichia coli. Expression product had a 70kDa band and emitted yellow-green luminescence in the presence of luciferin. Five loops in the P pygidialis luciferase, L1 （NI98-G208）, L2 （T240-G247）, L3 （G317-K322）, L4 （L343-I350） and L5 （G522-D532）, were found from the structure modeling analysis in the cleft, where it was considered the active site for the substrate compound entering and binding. Different amino acid residues between the luciferases of P. pygidialis and the three other known strong luminous species can not explain the situation of weak or strong luminescence. Future study of these loops, residues or crystal structure analysis may be helpful in understanding the real differences between the luciferases between diurnal and nocturnal species.

Roles of phosphatidylinositol-3-kinases signaling pathway in inflammation-related cancer:Impact of rs10889677 variant and buparlisib in colitis-associated cancer

BACKGROUND Phosphatidylinositol-3-kinases(PI3K)is a well-known route in inflammationrelated cancer.Recent discovery on PI3K-related genes revealed a potential variant that links ulcerative colitis(UC)and colorectal cancer(CRC)with colitisassociated cancer(CAC).PI3K/AKT pathway has been recommended as a potential additional therapeutic option for CRC due to its substantial role in modifying cellular processes.Buparlisib is a pan-class I PI3K inhibitor previously shown to reduce tumor growth.AIM To investigate the regulation of rs10889677 and the role of buparlisib in the PI3K signaling pathway in CAC pathogenesis.METHODS Genomic DNA from 32 colonic samples,including CAC(n=7),UC(n=10)and CRC(n=15),was sequenced for the rs10889677 mutation.The mutant and wildtype fragments were amplified and cloned in the pmirGLO vector.The luciferase activity of cloned vectors was assessed after transfection into the HT29 cell line.CAC mice were induced by a mixture of a single azoxymethane injection and three cycles of dextran sulphate sodium,then buparlisib was administered after 14 d.The excised colon was subjected to immunohistochemistry for Ki67 and Cleaved-caspase-3 markers and quantitative real-time polymerase chain reaction analysis for Pdk1 and Sgk2.RESULTS Luciferase activity decreased by 2.07-fold in the rs10889677 mutant,confirming the hypothesis that the variant disrupted miRNA binding sites,which led to an increase in IL23R expression and the activation of the PI3K signaling pathway.Furthermore,CAC-induced mice had a significantly higher disease activity index(P<0.05).Buparlisib treatment significantly decreased mean weight loss in CAC-induced mice(P<0.05),reduced the percentage of proliferating cells by 5%,and increased the number of apoptotic cells.The treatment also caused a downward trend of Pdk1 expression and significantly decreased Sgk2 expression.CONCLUSION Our findings suggested that the rs10889677 variant as a critical initiator of the PI3K signaling pathway,and buparlisib had the ability to prevent PI3K-non-AKT activation in the pathophysiology of CAC.

一种可药物筛选及体内检测的新型慢病毒基因治疗载体的构建

为实现基因治疗过程中的有效药物筛选及体内检测,首次利用核糖体内部进入位点(IRES)构建了同时携带O6-烷基鸟嘌呤-DNA烷基转移酶(MGMT)的突变型P140K基因和荧光素酶(Luciferase)基因的慢病毒载体pBobi-MIL。RT-PCR、免疫荧光、药物筛选克隆形成及化学发光检测等实验结果表明感染重组慢病毒L-MIL的细胞能同时表达MGMT及Luciferase。构建成功的新型慢病毒载体为今后的基因治疗奠定了基础,也为慢病毒滴度的确定提供了一种新的可能。

RCAS(J)-Luciferase重组病毒拯救及其相关生物学功能鉴定

RCAS载体系列是基于A型罗斯肉瘤病毒(RSV-A)所改造而成的反转录病毒感染性克隆,转染易感细胞后,能够产生高滴度的RSV-A病毒。同时,RCAS载体可携带外源基因进入靶细胞,从而进行蛋白的高效表达。为了获取滴度高且易于检测的RSV,进行ALV-J受体方面的研究,本研究将RCAS(A)-GFP载体中RSV-A的囊膜蛋白基因替换为ALV-J的囊膜蛋白基因,并将绿色荧光蛋白报告基因(gfp)替换为海参荧光素酶(luciferase)报告基因,构建带有luciferase报告基因的囊膜蛋白为ALV-J囊膜蛋白的RSV重组病毒。经验证,本研究所构建的重组病毒,与传统的ALV-J株相比,具有复制能力强,病毒滴度高等优点,而且能够通过ALV-J特异性受体ch NHE1感染ALV-J非允许细胞系,有利于病毒感染初期的检测和定量,为病毒与其受体之间相互作用的研究奠定了基础。

利用生物发光成像示踪大鼠颅骨缺损中移植的骨髓间充质干细胞

背景:生物发光成像是一种新兴的活体成像技术,目前尚未有运用该技术对移植干细胞在颅骨缺损活体示踪的研究。目的:探究Luciferase慢病毒转染大鼠骨髓间充质干细胞的最佳转染复数,进一步研究表达荧光素酶骨髓间充质干细胞在体外的有效性和稳定性以及在大鼠颅骨缺损内的示踪作用。方法:以不同感染复数转染对数生长期的大鼠骨髓间充质干细胞,并在活体成像系统中定量分析荧光值以确定最佳感染复数;在最佳感染复数下构建表达荧光素酶的骨髓间充质干细胞,分析不同数量细胞和不同代数细胞对应的荧光值变化,以评估转染干细胞的有效性和稳定性;最后在颅骨缺损中示踪表达荧光素酶的骨髓间充质干细胞。结果与结论:①感染复数在10-50区间递增时,荧光定量随感染复数同比例增加,而感染复数增加至100时,荧光定量增幅不明显,证明Luciferase慢病毒转染大鼠骨髓间充质干细胞的最佳感染复数为50;②表达荧光素酶的骨髓间充质干细胞在体外具有良好的细胞-生物荧光量效关系和增殖稳定性,证明荧光定量能指示细胞的相对数量;③表达荧光素酶的骨髓间充质干细胞在颅骨缺损区局限分布,其生物荧光强度逐渐减弱,最终在术后35 d全部消失;④结果说明,生物发光成像是一种高效灵敏的活体示踪技术,有利于表征移植干细胞在体内的分布和数量变化,并为解释干细胞功能和机制研究提供有力证据。

Gilbert's syndrome: High frequency of the (TA)_7 TAA allele in India and its interaction with a novel CAT insertion in promoter of the gene for bilirubin UDP-glucuronosyltransferase 1 gene

AIM： To identify the variants in U rase 1 （UGT1A1） gene in Gilbert＇s syndrome （GS） and to estimate the association between homozygosity for TA insertion and GS in India, as well as the frequency of TA insertion and its impact among normal controls in India. METHODS： Ninety-five GS cases and 95 normal controls were selected. Liver function and other tests were done. The promoter and all 5 exons of UGT1A1 gene were resequenced. Functional assessment of a novel trinucleotide insertion was done by in silico analysis and by estimating UGT1A1 promoter activity carried out by ludferase reporter assay of appropriate constructs in Hep G2 cell line. RESULTS： Among the GS patients, 80% were homozygous for the TA insertion, which was several-fold higher than reports from other ethnic groups. The mean UCB level was elevated among individuals with only one copy of this insertion, which was not significantly different from those with two copies. Many new DNA variants in UGT1A1 gene were discovered, including a trinucleotide （CAT） insertion in the promoter found in a subset （10%） of GS patients, but not among normal controls. In-silico analysis showed marked changes in the DNA-folding of the promoter and functional analysis showed a 20-fold reduction in transcription efficiency of UGT1A1 gene resulting from this insertion, thereby significantly elevating the UCB level. CONCLUSION： The genetic epidemiology of GS is variable across ethnic interactions among UGT1A1 groups and the epistatic promoter variants modulate bilirubin glucuronidation.

人Tim-3启动子的克隆及其真核报告质粒的构建

目的克隆人Tim-3基因5′端非编码区处具有启动子活性的片段,构建其真核报告质粒,为进一步研究Tim-3表达调控奠定基础。方法以人基因组DNA为模板,针对人Tim-3启动子5′端非编码区处包括翻译起始点ATG和转录起始点TSS在内的-898^+242片段,设计特异性引物,PCR扩增该片段,命名为Tim-3P2,经双酶切后克隆入pGL3-Basic载体SacⅠ、XhoⅠ位点,构建pGL3-Tim-3P2荧光素酶报告质粒,与内参照pRL-TK用脂质体法瞬时共转染COS-7、RAW264.7和B16细胞系,通过双荧光素酶活性检测确定其启动子活性。结果pGL3-Tim-3P2经PCR、双酶切及DNA测序分析鉴定正确无误;pGL3-Tim-3P2转染RAW264.7和B16细胞(两种细胞均可表达内源性Tim-3)24 h和48h,双荧光素酶活性检测显示其启动子相对活性约为pGL3-Basic空载体的2倍;而pGL3-Tim-3P2转染不表达内源性Tim-3的COS-7细胞后检测不到荧光素酶活性。结论成功克隆并构建含有人Tim-3启动子的真核报告质粒,该载体具有特异性Tim-3启动子活性,为进一步研究Tim-3表达调控奠定了基础。

Tet-on控制的Luciferase和SupF突变报告基因质粒的构建及其在顺铂致突变作用中的应用研究

目的研究TCR在顺铂导致的细胞内DNA损伤和突变过程中的分子机制,并为进一步研究转录偶联修复与突变发生的分子机制提供重要分子生物学依据。方法首先将SupF突变报告基因克隆到带有双向真核启动子的含有Tet调控元件TRE的表达载体pBI-L的多克隆位点(MCS)上,获得pTCR-1,再将SV40ori元件插入pTCR-1载体,最终获得pTCR-2质粒。酶切和DNA测序证实后,用顺铂将pTCR-2进行体外DNA损伤后瞬时转染至Tet-on293细胞,在Dox诱导下培养48h,从细胞内提取质粒,纯化后转化SY204菌株,蓝白斑筛选挑取白色突变斑,计算突变频率并进行DNA测序检测突变频谱。结果经酶切鉴定和测序分析,插入片段长度和序列正确。在Dox诱导下取得了SupF报告基因在转录偶联修复途径中的突变频率与频谱。结论重组的pTCR-2质粒具有在真核细胞中Tet启动的转录活性,应用于顺铂致突变研究中,使转录条件下的突变情况能够得以反映。

表达Renilla Luciferase重组流感病毒的构建及生物学特性研究

本研究构建了表达Renilla Luciferase的重组流感病毒,并运用Luciferase报告基因检测系统对该病毒的生物学特性进行了研究。研究发现该病毒复制时能稳定表达Renilla Luciferase,添加磷酸奥斯他韦抑制病毒复制后Luciferase表达量明显低,而且表达量与磷酸奥司他韦浓度成反比。结果表明检测Luciferase表达量可以直接反应病毒的复制,将该重组病毒与Luciferase报告基因系统运用到抗流感药物的大规模筛选中,可为抗流感药物的筛选提供一种快速且灵敏的检测方法。

酿造罐体清洗优化及监控

本文主要阐述了通过ATP快速检测．快速、详细地了解了罐体清洗中的问题，针对问题逐一解决后，实现了罐体清洗工艺的优化。

Protective mechanisms of micro RNA-27a against oxygen-glucose deprivation-induced injuries in hippocampal neurons

Hypoxic injuries during fetal distress have been shown to cause reduced expression of micro RNA-27a（mi R-27a）,which regulates sensitivity of cortical neurons to apoptosis.We hypothesized that miR-27 a overexpression attenuates hypoxia- and ischemia-induced neuronal apoptosis by regulating FOXO1,an important transcription factor for regulating the oxidative stress response.miR-27 a mimic was transfected into hippocampal neurons to overexpress miR-27 a.Results showed increased hippocampal neuronal viability and decreased caspase-3 expression.The luciferase reporter gene system demonstrated that mi R-27 a directly binded to FOXO1 3′UTR in hippocampal neurons and inhibited FOXO1 expression,suggesting that FOXO1 was the target gene for mi R-27 a.These findings confirm that mi R-27 a protects hippocampal neurons against oxygen-glucose deprivation-induced injuries.The mechanism might be mediated by modulation of FOXO1 and apoptosis-related gene caspase-3 expression.

Improvement of Chemically-activated Luciferase Gene Expression Bioassay for Detection of Dioxin-Iike Chemicals

Objective To improve the chemically-activated luciferase expression (CALUX)bioassay for detection of dioxin-like chemicals (DLCs) based on the toxicity mechanisms ofDLCs. Method A recombinant vector was constructed and used to transfect humanhepatoma (HepG2). The expression of this vector was 10-100 folds higher than that of pGL2used in previous experiments. The transfected cells showed aromatic hydrocarbon receptor(AhR)-meditated luciferase gene expression. The reliability of luciferase induction in thiscell line as a reporter of AhR-mediated toxicity was evaluated, the optimal detection timewas examined and a comparison was made by using the commonly used ethoxyresoufin-O-deethylase (EROD) activity induction assay. Result The results suggested that theluciferase activity in recombinant cells was peaked at about 4 h and then decreased to astable activity by 14 h after TCDD treatment. The detection limit of this cell line was0.11pmol/L, or 10-fold lower than in previous studies, with a linear range from 1 to 100pmol/L, related coefficient of 0.997, and the coefficient of variability (CV) of 15-30%.Conclusion The luciferase induction is 30-fold more sensitive than EROD induction, thedetection time is 68 h shorter and the detection procedure is also simpler.

An antioxidant resveratrol significantly enhanced replication of hepatitis C virus

AIM:To elucidate the effect of antioxidants,resveratrol (RVT)and astaxanthin(AXN),on hepatitis C virus(HCV) replication. METHODS:We investigated the effect of recent popular antioxidant supplements on replication of the HCV replicon system OR6.RVT is a strong antioxidant and a kind of polyphenol that inhibits replication of various viruses.AXN is also a strong antioxidant.The replication of HCV RNA was assessed by the luciferase reporter assay.An additive effect of antioxidants on antiviral effects of interferon(IFN)and ribavirin(RBV) was investigated.RESULTS:This is the first report to investigate the effect of RVT and AXN on HCV replication.In contrast to other reported viruses,RVT significantly enhanced HCV RNA replication.Vitamin E also enhanced HCV RNA replication as reported previously,although AXN didnot affect replication.IFN and RBV significantly reduced HCV RNA replication,but these effects were dose-dependently hampered and attenuated by the addition of RVT.AXN didnot affect antiviral effects of IFN or RBV. CONCLUSION:These results suggested that RVT is not suitable as an antioxidant therapy for chronic hepatitis C.

Protective effects of anti-ricin A-chain RNA aptamer against ricin toxicity

AIM： To investigate the therapeutic potential of an RNA ligand （aptamer） specific for the catalytic ricin A-chain （RTA）, the protective effects of a 31-nucleo- tide RNA aptamer （31RA）, which formed a high affinity complex with RTA, against ricin-induced toxicity in cell- based luciferase translation and cell cytotoxicity assays were evaluated. METHODS： To test the therapeutic potential of anti- RTA aptamers in Chinese hamster ovary （CliO） AA8 cells stably transfected with a tetracycline regulatable promoter, ricin ribotoxicity was measured us- ing luciferase and ricin-induced cytotoxicity was ascertained by MTS cell proliferation assay with tet- razolium compound [3-（4,5-dimethylthiazol-2-yl）- 5-（3-carboxymethoxyphenyl）-2-（4-sulfophenyl）-2H- tetrazolium]. RESULTS： Inhibition of protein synthesis by ricin in CliO AA8 cells resulted in diminished luciferase activity and treatment with polyclonal antibody against degly- cosylated RTA （dgA） neutralized the inhibitory effects of ricin on luciferase activity and protected against ricin-induced cytotoxicity as measured by MTS assay. The 31RA anti-RTA aptamer inhibited the translation of luciferase mRNA in cell-free reticulocyte translation assay. 31RA aptamer also partially neutralized the inhibitory effects of ricin on luciferase activity and partially protected against ricin-induced cytotoxicity in CliO AA8 cells. CONCLUSION： We have shown that anti-RTA RNA aptamer can protect against ricin ribotoxicity in cell- based luciferase and cell cytotoxicity assays. Hence, RNA aptamer that inhibits RTA enzymatic activity represents a novel class of nucleic acid inhibitor that has the potential to be developed as a therapeutic agent for the treatment of ricin intoxication.

Functional analysis of the promoter of the dmrt1 gene in Chinese tongue sole,Cynoglossus semilaevis

The dmrt1 (doublesex and mab-3 related transcription factor 1) gene is considered to play an essential role in testis differentiation and development among metazoan species. As an economically important marine fish in China, Chinese tongue sole (Cynoglossus semilaevis) possess a ZW/ZZ sex-determining system, and the females usually grow two to four times bigger than males. Previous studies have confirmed that Z-linked dmrt1 gene is the male determining gene in C. semilaevis and TALENs-mediated knocking-out individuals showed increased growth phenotype. In order to investigate the function of dmrt1 regulatory regions and its potential application, we have cloned the promoter of C. semilaevis dmrt1. The acquired sequence consisted of a 5'-upstream sequence and a partial exon1;functional analysis showed that there were two positive regulatory regions in the promoter, as well as one negative region and one neutral region. These positive regions were then combined to obtain several newly constructed promoters. Both in vitro and in vivo analysis confirmed one of them (dmrt1-Δ3+Δ4) showed elevated promoting activity compared to the full-length promoter and could drive exogenous gene expression in tongue sole gonads. Our results will provide useful information for understanding the regulatory mechanism of dmrt1 gene during C. semilaevis sex determination, and the endogenous promoter will facilitate the transgenic research for analyzing sex- and growth-related genes.

miR-15b-5p targeting amyloid precursor protein is involved in the anti-amyloid eflect of curcumin in swAPP695-HEK293 cells

Curcumin exerts a neuroprotective effect on Alzheimer’s disease;however,it is not known whether microRNAs are involved in this protective effect.This study was conducted using swAPP695-HEK293 cells as an Alzheimer’s disease cell model.swAPP695-HEK293 cells were treated with 0,0.5,1,2,5,and 10μM curcumin for 24 hours.The changes in miR-15b-5p,miR-19a-3p,miR-195-5p,miR-101-3p,miR-216b-5p,miR-16-5p and miR-185-5p expression were assessed by real-time quantitative polymerase chain reaction.The mRNA and protein levels of amyloid precursor protein,amyloid-β40 and amyloid-β42 were evaluated by quantitative real-time polymerase chain reaction,western blot assays and enzyme-linked immunosorbent assays.swAPP695-HEK293 cells were transfected with miR-15b-5p mimic,or treated with 1μM curcumin 24 hours before miR-15b-5p inhibitor transfection.The effects of curcumin on amyloid precursor protein,amyloid-β40 and amyloid-β42 levels were evaluated by western blot assays and enzyme-linked immunosorbent assay.Luciferase assays were used to analyze the interaction between miR-15b-5p and the 3′-untranslated region of amyloid precursor protein.The results show that amyloid precursor protein and amyloid-βexpression were enhanced in swAPP695-HEK293 cells compared with HEK293 parental cells.Curcumin suppressed the expression of amyloid precursor protein and amyloid-βand up-regulated the expression of miR-15b-5p in swAPP695-HEK293 cells.In addition,we found a negative association of miR-15b-5p expression with amyloid precursor protein and amyloid-βlevels in the curcumin-treated cells.Luciferase assays revealed that miR-15b-5p impaired the luciferase activity of the plasmid harboring the 3′-untranslated region of amyloid precursor protein.These findings indicate that curcumin down-regulates the expression of amyloid precursor protein and amyloid-βin swAPP695-HEK293 cells,which was partially mediated by miR-15b-5p via targeting of the 3′-untranslated region of amyloid precursor protein.

Novel CDH1 germline mutations identified in Chinese gastric cancer patients

AIM:To give a comprehensive report of E-cadheringene (CDH1) variations in a population at a high risk for gastric cancer (GC).METHODS:The samples consisted of 178 men and 58 women with a mean age of 62.3 ± 9.4 years and an age range of 30-84 years.A total of 240 cancerfree controls were recruited (mean age of 61.8 ± 10.1 years,age range of 26-82 years).Samples were screened for CDH1 germline mutations by high-resolution melting analysis or directly sequencing.Luciferase reporter assay,RNA splicing assay and bioinformatic analysis were used to evaluate the effect of mutations.RESULTS:Four novel CDH1 sequence alterations were identified in GC patients including a G>T transition 49 bp before the start codon;a three-nucleotide deletion,c.44_46del TGC;one missense mutation,c.604G>A (V202I);and one variation in the intron,c.1320+7A>G.In addition,polymorphism frequencies were observed for CDH1-164delT,-161C>A,-73A>C,c.48+6C>T,c.48+62_48+63delinsCGTGCCCCAGCCC,c.894C>T (A298A),c.1224G>A (A408A),c.1888C>G (L630V),c.2076T>C (A692A),and c.2253C>T (N751N) which is similar to the data reported in http://www.ncbi.nlm.nih.gov/projects/SNP/.RNA splicing analysis suggested that the c.1320+7A>G and c.1224G>A variations did not affect exon splicing ability.Luciferase reporter assay demonstrated that the c.-49T variation might be helpful for E-cadherin transcription,though the increase in transcription activity is limited (only 33%).SIFT score and PolyPhen analysis both demonstrated that the L630V missense mutation probably damages protein function,while the V202I variant does not.CONCLUSION:This study reveals novel mutations in sporadic GC patients which had been poorly investigated for susceptibility genes.

Cycloartane Glycosides from the Roots of Cimicifuga foetida with Wnt Signaling Pathway Inhibitory Activity

Four new 9,19-cycloartane triterpenoids,cimilactone E(1),cimilactone F(2),20-O-(E)-butenoyl-23-epi-26-deoxyactein(3),and 20,12b-O-diacetylcimiracemonol-3-O-b-D-xylopyranoside(4),together with four known constituents(5–8)were isolated from the roots of Cimicifuga foetida.The new structures were elucidated by extensive spectroscopic analysis.In addition,compounds 7 and 8 showed significant Wnt signaling pathway inhibitory activity,with IC50 values of 3.33 and 13.34 lM,respectively,using the luciferase reporter gene assay.