椿叶花椒叶挥发油化学成分的研究

采用气相色谱-质谱联用仪分析,研究了用水蒸气蒸馏法提取的椿叶花椒叶挥发油的化学成分。结果表明:鉴定出33种成分,占挥发油总离子流色谱峰面积的99.99%。椿叶花椒叶挥发油的主要成分为2-壬酮,芳樟醇,β-水芹烯。     

Assembly mechanism of [Fe2S2] cluster in ferredoxin from Acidithiobacillus ferrooxidans

Ferredoxin is a typical iron-sulfur protein that is ubiquitous in biological redox systems. This study investigates the in vitro assembly of a [Fe2S2] cluster in the ferredoxin from Acidithiobacillus ferrooxidans in the presence of three scaffold proteins: IscA, IscS, and IscU. The spectra and MALDI-TOF MS results for the reconstituted ferredoxin confirm that the iron-sulfur cluster was correctly assembled in the protein. The inactivation of cysteine desulfurase by L-allylglycine completely blocked any [Fe2S2] cluster assembly in the ferredoxin in E. coli, confirming that cysteine desulfurase is an essential component for iron-sulfur cluster assembly. The present results also provide strong evidence that [Fe2S2] cluster assembly in ferredoxin follows the AUS pathway.     

CDH1 -160C>A gene polymorphism is an ethnicity-dependent risk factor for gastric cancer

The associations between E-cadherin (CDH1) gene polymorphisms and gastric cancer (GC) susceptibility are still controversial. Given this uncertainty, we carried out a meta-analysis of published case-control studies to derive more precise estimations of these relationships. Relevant studies were identified from PubMed and EMBASE up to March 2011. Seventeen studies with 3511 GC cases and 4826 controls were selected. Crude odds ratios (OR) and 95% confidence intervals (CI) were used to investigate the strength of the associations. No associations between CDH1 (+54T>C, -160C>A, -347G>GA, -616G>C, -2076C>T and -3159T>C) gene polymorphisms and GC risk for all genetic models were found. As for CDH1 -160C>A polymorphism, subgroup analyses by country, gender, study design, smoking status, Helicobacter pylori infection, and the Lauren classification of GC did not change the results. When stratified by ethnicity, we found the A allele carriers had a significantly increased risk of GC among Caucasians (AA vs. CA+CC: OR=1.50, 95% CI=1.03-2.19, P=0.03), but not among Asians (AA vs. CA+CC: OR=0.87, 95% CI=0.56-1.37, P=0.56). No publication bias was found in the present study. This meta-analysis suggests that CDH1 -160C>A gene polymorphism may contribute to increased risk of GC among Caucasians.     

miR-223 regulates migration and invasion by targeting Artemin in human esophageal carcinoma

Background

Artemin (ARTN) is a neurotrophic factor belonging to the glial cell-derived neurotrophic factor family of ligands. To develop potential therapy targeting ARTN, we studied the roles of miR-223 in the migration and invasion of human esophageal carcinoma.

Methods

ARTN expression levels were detected in esophageal carcinoma cell lines KYSE-150, KYSE-510, EC-9706, TE13, esophageal cancer tissues and paired non-cancerous tissues by Western blot. Artemin siRNA expression vectors were constructed to knockdown of artemin expression mitigated migration and invasiveness in KYSE150 cells. Monolayer wound healing assay and Transwell invasion assay were applied to observe cancer cell migration and invasion. The relative levels of expression were quantified by real-time quantitative PCR.

Results

ARTN expression levels were higher in esophageal carcinoma tissue than in the adjacent tissue and was differentially expressed in various esophageal carcinoma cell lines. ARTN mRNA contains a binding site for miR-223 in the 3'UTR. Co-transfection of a mir-223 expression vector with pMIR-ARTN led to the reduced activity of luciferase in a dual-luciferase reporter gene assay, suggesting that ARTN is a target gene of miR-223. Overexpression of miR-223 decreased expression of ARTN in KYSE150 cells while silencing miR-223 increased expression of ARTN in EC9706 cells. Furthermore, overexpression of miR-223 in KYSE150 cells decreased cell migration and invasion. Silencing of miR-223 in EC9706 cells increased cell migration and invasiveness.

Conclusions

These results reveal that ARTN, a known tumor metastasis-related gene, is a direct target of miR-223 and that miR-223 may have a tumor suppressor function in esophageal carcinoma and could be used in anticancer therapies.     

Galactosylated N-2-hydroxypropyl methacrylamide-b-N-3-guanidinopropyl methacrylamide block copolymers as hepatocyte-targeting gene carriers

As alternatives of viral and cationic lipid gene carriers, cationic polymer-based vectors may provide flexible chemistry for the attachment of targeting moieties. In this report, galactosylated N-2-hydroxypropyl methacrylamide-b-N-3-guanidinopropyl methacrylamide block copolymers (galactosylated HPMA-b-GPMA block copolymers, or abbreviated as GHG) were prepared in order to develop hepatocyte targeting gene transfection carriers. The block copolymers were synthesized by aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization of N-2-hydroxypropyl methacrylamide (HPMA) and N-3-aminopropyl methacrylamide (APMA), followed by galactosylation and guanidinylation. The molecular weight of GHG copolymers determined by static light scattering method was in the range from 48?600 to 76?240 g/mol. In addition, the galactose content in the GPMA block in the copolymers was determined to be 6.5-8.0 mol % according to the sulfuric acid method. The GHG copolymers complexed completely with plasmid DNA (pDNA) to show positive zeta-potential values with diameter 100-250 nm from charge ratio of 4, which demonstrated the excellent DNA condensing ability of guanidino groups. Furthermore, the MTT assay data of GHG/pDNA complexes on HepG2 cells and HeLa cells indicated that GHG copolymers had significantly lower cytotoxicity than PEI. In addition, the copolymers with GPMA component from 30.23% showed higher transfection efficiency than PEI at charge ratio of 12 in HepG2 cells. The result revealed that the conjugation of galactose groups in the copolymers brought asialoglycoprotein-receptor (ASGP-R) mediated transfection. The employing of HPMA component decreased the aggregation of protein in transfection presence of serum. The GHG copolymers combined the advantages of galactose moieties, guanidino groups, and HPMA component might show potential in safe hepatocyte targeting gene therapy.     

流感疫苗单向免疫扩散试验中抗血清制备新方法的研究

流感疫苗血凝素含量检测方法为单向免疫扩散试验,其抗血清通常由WHO参比实验室提供,通过纯化病毒、蛋白酶切获得血凝素主要抗原片段,然后再免疫动物获得抗血清。该方法制备时间较长,是制约流感疫苗研发及检测的主要因素。以RT-PCR方法获取甲型H1N1流感病毒血凝素中主要抗原片段基因,构建重组质粒并在大肠杆菌中表达,表达产物经纯化、复性后,以蛋白电泳及免疫印迹方法进行了鉴定。以纯化蛋白免疫家兔,制备了相应的抗体,初步证明可用于常规的单向免疫扩散试验。以该法可以快速获得抗血清,尤其是在大流行流感疫苗的研发中,加快疫苗研发进程。     

广西巴马小型猪血清淀粉样P物质基因真核载体构建及细胞转染

血清淀粉样P物质(Serum Amyloid Pcomponent,SAP)是一种在进化上高度保守的血清糖蛋白,它可与各种类型的原纤维结合,在免疫应答和炎症反应等多种免疫疾病中发挥作用.以广西巴马小型猪肝组织总RNA为模板,利用RT-PCR技术扩增出相应cDNA片段,连接到克隆载体pMD18-T上进行检测,测序结果为675 bp,与GenBank所提供相关序列同源性为100％,并成功构建pEGFP-N1 -SAP重组真核表达载体,利用脂质体(Lipofectamine 2000)介导法将重组质粒导入到NIH-3T3细胞中培养,经转染24 h后,置于倒置荧光显微镜下观察,发现含有重组质粒的NIH-3T3细胞中表达出绿色荧光,为进一步研究SAP基因的功能特点及在试验动物相关疾病模型的应用提供务件.     

原油微生物群落构成多样性及降解菌DYL-1降解原油的研究

为分离得到高效原油降解菌,直接向原油中加入营养物刺激,培养一段时间后原油乳化降解,傅里叶红外光谱显示,2957cm-1、723cm-1处吸收峰消失;2855cm-1、1377cm-1处吸收峰减弱;880cm-1以及800cm-1吸收峰几乎消失,表明原油发生降解,效果明显。同时分离得到一株降解菌,经分子鉴定为芽孢杆菌属(Bacillus sp.),红外光谱法和紫外吸收法分析表明其具有较强的降解原油烃的能力。根据传统分子生物学的方法,构建原油菌16S rDNA克隆文库,限制性酶切片段长度多态性(Restriction Fragment Length Polymorphism,RFLP)分析了原油中的细菌多样性。结果表明此方法有效地评估了原油中的细菌群落和多样性。     

扩增和纯化携带肌浆网钙ATP酶重组腺病毒的实验方法

目的:通过扩增和纯化rAd.SERCA2a,为转SERCA2a基因研究提供实验基础,并为建立基因库提供稳定可靠的实验方法.方法:用100μL 1.9×10<'12>pfu/ml rAd.SERCA2a感染HEK293细胞,出现细胞病变效应时收获细胞,经物理反复冻融方法及两步氯化铯超速离心方法获得纯化的rAd.SERCA2a,紫外分光光度计比色法测定病毒DNA质粒数.结果:rAd.SERCA2a成功在HEK293细胞表达呈现绿色荧光,纯化的rAd-SERCA2a-GFP DNA质粒数为1.3±0.58×10<'12>pfu/mL,OD<,260>/OD<,280>比值为1.57±0.49(n=50).结论:建立了稳定可靠的借助HEK293细胞培养扩增rAd.SERCA2a的实验方法,纯化后的高效价的SERCA2a基因的重组腺病毒可直接用于心力衰竭的实验研究,对重组腺病毒携带其他基因的扩增与提纯方法也具有一定的参考价值.     

Identification and characterization of wheat long non-protein coding RNAs responsive to powdery mildew infection and heat stress by using microarray analysis and SBS sequencing

Background  

Biotic and abiotic stresses, such as powdery mildew infection and high temperature, are important limiting factors for yield and grain quality in wheat production. Emerging evidences suggest that long non-protein coding RNAs (npcRNAs) are developmentally regulated and play roles in development and stress responses of plants. However, identification of long npcRNAs is limited to a few plant species, such as Arabidopsis, rice and maize, no systematic identification of long npcRNAs and their responses to abiotic and biotic stresses is reported in wheat.