大肠杆菌DNA和RNA中核苷的高效液相色谱分析

建立了用反相高效液相色谱法（RP-HPLC）测定大肠杆菌（E.coli）脱氧核糖核酸（DNA）和核糖核酸（RNA）水解产物中核苷的方法，色谱柱为Supelco Inc.c18反相柱，254nm和280nm紫外吸收波长同时检测，分析条件经选择在流速为1mL/min，流动相A（2.5%甲醇 0.01mol/L KH2PO4,PH4.6)28min，流动相B（8.0%甲醇 0.01mol/L KH2PO4，PH4.6)37min，柱温为20℃条件下65min可实现核苷的完全分离，回收率为80.19%-99.31%，相对标准偏差RSD为0.86%-2.62%，该方法具有高灵敏度、高选择性的特点，得到的结果与十分满意。     

基于分子信标实时监测大肠杆菌DNA连接酶催化的DNA连接过程

报道了一种对DNA连接过程进行实时监测的方法,利用分子信标核酸探针作为DNA连接反应的模板和检测探针,实时监测了 E.coli DNA连接酶催化的DNA连接反应,克服了传统的凝胶电泳技术操作复杂、周期长及无法实时监测DNA连接过程的缺点,为核酸连接过程的实时监测和连接酶催化机理的研究提供了更为丰富的信息.在此基础上,发展了一种快速、准确测定 E.coli DNA连接酶的方法,线性响应范围为4.0×10^-6～2.0×10^-4U/μL,检测下限为4.0×10^-6U/μL.     

E.coli O86 O-Antigen全保护五糖重复单元的化学简易合成

以5个单糖组分为原料, 经过7步, 以21%的总产率得到E.coli O86抗原全保护的五糖重复单元.在合成路线中, 充分利用糖基化反应的立体选择性原则, 结合HClO4-SiO2固体催化剂和“IP”策略, 大大提高了合成的效率. 整个合成路线设计操作简单, 选择性高, 消耗低, 产率高, 可以用于快速高效地合成其它一些具有生物活性的寡糖分子.     

Expression of Shiga Toxin B Subunit at Cell Surface in E. coli K-12

The three parts(Stx17B, Stx27B and StxB) of Shiga toxin B subunit have been fused into a cell surface exposed loop of the LamB protein at a BamH I site between residues 153 and 154. Western blotting revealed that the three parts of Shiga toxin B subunit could be expressed as the Lamb fusion proteins in E. coli. Indirect immunofluorescence and immunoelectron microscopy analyses showed fusion proteins LamB/Stx17B and LamB/Stx27B could be expressed at cell surface in E. coli, but fusion protein LamB/StxB could not be expressed at cell surface; it was aggregated in cytoplasm and was toxic to host. This expression system provided a new way to construct an oral live vaccine against Shigella dysenteriae 1.     

Induction of phr gene expression in E. coli strain KY706/pPL-1 by He-Ne laser (632.8 nm) irradiation

We have observed that He–Ne laser irradiation of E. coli strain KY706/pPL-1 leads to induction of photolyase gene, phr. The magnitude of induction was found to depend on the He–Ne laser fluence, fluence rate and post-irradiation incubation period in the nutrient medium. The optimum values for fluence and fluence rate were 7×10³ J/m² and 100 W/m², respectively, and the induction of phr gene was observed to saturate beyond an incubation period of 2 h. Experiments carried out with singlet oxygen quenchers and with D₂O suggest that the effect is mediated via singlet oxygen. Photoreactivation studies carried out after UVC exposure of both the He–Ne laser-exposed as well as unexposed cells showed a larger surviving fraction in the He–Ne laser pre-irradiated cells. This can be attributed to He–Ne laser irradiation-induced induction of phr expression. However, since even without photoreactivating light He–Ne laser pre-irradiated cells show higher survival against UVC radiation it appears that He–Ne laser irradiation induces both light-dependent as well as dark DNA repair processes.     

Binding Characteristics Study of DNA based Aptamers for E. coli O157:H7

E. coli O157:H7 is a pathogenic bacterium producing verotoxins that could lead to serious complications such as hemolytic uremia syndrome. Fast detection of such pathogens is important. For rapid detection, aptamers are quickly gaining traction as alternative biorecognition molecules besides conventional antibodies. Several DNA aptamers have been selected for E. coli O157:H7. Nonetheless, there has not been a comparative study of the binding characteristics of these aptamers. In this work, we present a comprehensive analysis of binding characteristics including binding affinity (K_d) and binding capacity (B_max) of DNA-based aptamers for E. coli O157:H7 using qPCR. Our results show that aptamer E18R has the highest binding capacity to E. coli 157:H7 and the highest specificity over non-pathogenic E. coli strains K12 and DH5α. Our study also finds that the common biotin-tag modification at 5′ end typically changes the binding capacity significantly. For most of the selected aptamers, the binding capacity after a biotin-tag modification decreases. There exists a discrepancy in the binding capability between the selected aptamer and the aptamer used for detection. Our study also shows that a lower concentration of Mg²⁺ ions in the binding buffer leads to a decrease in the binding capacity of E17F and E18R, while it does not affect the binding capacity of S1 and EcoR1.     

纳米Ag2O2-PbO2化学修饰电极对大肠杆菌细胞膜壁和DNA损伤的研究

制备了一种新型纳米AgO2-PbO2修饰电极,在选定的正电位下,电极表面产生羟基自由基(.OH).通过测定产生的脂质过氧化物和漏出蛋白质的量来研究羟基自由基对大肠杆菌细胞膜壁损伤的情况,并运用电泳和DNA测序的方法对大肠杆菌质粒DNA的损伤及其对序列变化进行了研究.     

Electrochemical Immunoassay of E. coli in Urban Sludge Using Electron Mediator-mediated Enzymatic Catalysis and Gold Nanoparticles for Signal Amplification

An electrochemical immunosensor was developed for sensitive assay of E. coli in urban sludge, in which electron mediator-mediated enzymatic catalysis and gold nanoparticles(AuNPs) were utilized for signal amplification. The immnuosensing platform chitosan-thionine(chit-thio)/poly(amidoamine) dendrimer-encapsulated AuNPs [PAMAM(Au)] composites were first prepared from chit-thio and PAMAM(Au) using the layer-by-layer method to provide a matrix for high-stability and high-bioactivity bindings of the capture antibody(_cAb). Moreover, the {_dAb-AuNPs-HRP} nanoprobes were designed to exploit the amplification effect of the carrier AuNPs due to the loading amounts of horseradish peroxidase(HRP) and the detection antibody(_dAb). The sandwich-type immunoassay was then successfully used to assay E. coli based on the oxidation of thionine as a result of H₂O₂-induced enzymatic catalytic reaction by HRP. This study presents a powerful tool in electrochemical immunoassay for E. coli detection with rapid response, high-sensitivity and high-specificity, providing a potential new tool for feasibility assessment of sludge recycle.     

An Efficient Method for the Synthesis of 3-Cyano-6-hydroxy-2(1H)-pyridinones under Microwave Irradiation and Solvent-free Conditions

Summary. Three component condensation of alkylacetoacetates, primary amines, and alkyl cyanoacetates catalyzed by solid supports under microwave irradiation gave N-alkyl 3-cyano-6-hydroxy-2(1H)-pyridinones with high yields. Upon carrying out the reaction under the same condition on acidic alumina, zeolite HY, silica gel, and montmorillonite K-10, the best yields were achieved by silica gel. Corresponding author. E-mail: balalaie@yahoo.com Received August 28, 2002; accepted September 3, 2002     

一种新型脱氧核糖核酸序列表征子及其应用于E.coli启动子对RNA转录启动强度的QSAM研究

从天然碱基的36种性质参数出发,通过主成分分析(PCA)技术处理得到了1个显著的主成分得分,并将该得分作为单个碱基的信息描述子———VBPV。进而使用VBPV对38个大肠杆菌(E.coli)启动子序列一级结构进行表征,并结合多元统计方法将表征参数与转录启动强度(PS)成功地建立了定量序列活性模型(QSAM),该模型拟合复相关系数Rcum与交叉检验复相关系数Qcum分别为0.97和0.95。     

The Reaction of N-Isocyaniminotriphenylphosphorane With Biacetyl in the Presence of (E)-Cinnamic Acids: Synthesis of Fully Substituted 1,3,4-Oxadiazole Derivatives via Intramolecular AZA-Wittig Reactions of in Situ Generated Iminophosphoranes

Abstract

Reaction of N-isocyaniminotriphenylphosphorane with biacetyl in the presence of (E)-cinnamic acids proceeds smoothly at room temperature and under neutral conditions to afford sterically congested 1,3,4-oxadiazole derivatives in high yields. The reaction proceeds smoothly and straightforward under mild conditions and no side reactions are observed.     

Traceless Sulfone Linker for the Solid-Phase Organic Synthesis of 1-(E)-Styryl-4-substituted-1,2,3-triazoles

A novel, facile, solid-phase, organic synthesis of 1-(E)-styryl-4-substituted-1,2,3-triazoles in good yields and purities via traceless sulfone linker has been developed. Key steps involved in this synthetic procedure include (i) sulfone alkylation of sulfinate resin with (2-azido-1-iodoethyl)benzene, (ii) [3 + 2] cycloaddition with terminal alkynes in the presence of CuI, and (iii) traceless product release by base-mediated elimination process.     

Boron Clusters as a Platform for New Materials: Synthesis of Functionalized o‐Carborane (C2B10H12) Derivatives Incorporating DNA Fragments

A synthetic strategy for functionalization of the three vertices of o‐carborane and the attachment of the obtained triped to the solid support was developed. Further functionalization of the triped with short DNA sequences by automated DNA synthesis was achieved. The proposed methodology is a first example of boron cluster chemistry on a solid support opening new perspectives in boron cluster functionalization.     

Reactions of Polymer‐Supported α‐Selenoaldehydes with Grignard Regents. A Facile Solid‐Phase Stereoselective Synthesis of (E)‐1,2‐Disubstituted Ethenes

Polymer‐supported α‐selenoaldehydes easily obtained by reaction of polymer‐supported 4‐(phenylseleno)morpholine with aldehydes react with Grignard reagents to form polymer‐supported β‐hydroxyalkyl selenides, which were treated with thionyl chloride/triethylamine leading to (E)‐1,2‐disubstituted ethenes in good yields.