GR CALUX assay detects synthetic glucocorticoids in calf urine: a validation study

Member States of the EU are required to monitor the use of pharmacologically active substances in food-producing animals. There is evidence, however, that the target-based approach currently applied in official monitoring plans might under-estimate the real incidence of growth promoter abuse in livestock. As demonstrated for sex hormones, the association of effect-oriented biological screening with chemical confirmatory techniques could be the best strategy in revealing the abuse of veterinary drugs. Here we demonstrate the reliability of a cell-based assay to screen calf urine samples for synthetic glucocorticoids. The validation included the most widely used synthetic drugs (flumethasone, dexamethasone, betamethasone, methylprednisolone and prednisolone) and was developed according to the Commission Decision 2002/657/EC, thus including the verification of cut-off level, the β error, the specificity, ruggedness and stability. The study was carried out using prednisolone as representative substance at 5 ng mL^?1 concentration. All blank and spiked urine fulfilled the EU criteria, moreover the method resulted in being specific and sound, and the analytes in urine were stable for at least 30 days. The assay results indicated its suitability for a qualitative analysis of calf urine samples. This method enabled the detection of low doses of synthetic glucocorticoids (GCs) in matrix (<2 ng mL^?1 for flumethasone, dexamethasone, betamethasone; < 4 ng mL^?1 for methylprednisolone; 5 ng mL^?1 for prednisolone), with the possibility of detecting new or unknown molecules and cumulative effects of low-level mixtures with glucocorticoid bioactivity.     

A Saccharomyces cerevisiae cell-based quantitative beta-galactosidase assay compatible with robotic handling and high-throughput screening

Reporter-gene assays that employ the Escherichia coli lacZ gene are ubiquitously employed in biological research. However, we were not able to readily identify a quantitative method that worked reliably with yeast (Saccharomyces cerevisiae) cells and that was compatible with high-throughput screening and robotic liquid handling tools. We have therefore adapted a commercially available assay employing a 6-O-beta-galactopyranosyl-luciferin substrate to provide the required sensitivity with minimal sample handling times. Our assay uses only one-tenth of the reagents suggested by the reagent manufacturer (Promega) for equivalent assays with mammalian cell cultures and produces rapid, sensitive and reproducible analysis with as little as 1 microl yeast cell culture and with < 100 cells. We demonstrate that the assay is compatible with yeast strains generated by the systematic yeast deletion project and functions equally well with genomically integrated or plasmid-encoded lacZ reporters and with cells grown in complex or defined media. The high-sensitivity, miniaturized format reduced sample handling required will make this assay useful for a wide range of applications.     

Sensitive detection of chemical-induced genotoxicity by the Cypridina secretory luciferase reporter assay, using DNA repair-deficient strains of Saccharomyces cerevisiae

Yeast-based reporter assays are useful for detecting various genotoxic chemicals. We established a genotoxicity assay using recombinant strains of Saccharomyces cerevisiae, each containing a reporter plasmid with the secretory luciferase gene from Cypridina noctiluca, driven by a DNA damage-responsive promoter of the yeast RNR3 gene. This system detected the genotoxicity of methyl methanesulphonate (MMS) as sensitively as conventional yeast-based reporter assays, using the β-galactosidase gene in a concentration-dependent manner; it also detects four other genotoxic chemicals, allowing us to monitor DNA damage easily by skipping the cell extraction process for the assay. We examined Cypridina luciferase levels induced by MMS and three antitumour agents using a set of BY4741-derived deletion mutants, each defective in a DNA repair pathway or DNA damage checkpoint. Luciferase activities were particularly enhanced in mutant strains with mms2 Δ and mag1 Δ by exposure to MMS, rad59 Δ and mlh1 Δ to camptothecin and mms2 Δ and mlh1 Δ to mitomycin C, respectively, compared with their parent strains. Enhanced reporter activities were also found in some DNA repair mutants with cisplatin. These observations suggest that this Cypridina secretory luciferase reporter assay using yeast DNA repair mutants offers convenient and sensitive detection of the potential genotoxicity of numerous compounds, including antitumour drugs and studying the mechanisms of DNA damage response in yeast.     

降血脂药非诺贝特的绿色合成

以一水合硫酸氢钠为催化剂,二甲苯为带水剂,对非诺贝特酸与异丙醇的酯化反应进行了研究,考察了醇酸摩尔比、催化剂用量、带水剂用量等因素对非诺贝特产率的影响。结果表明:硫酸氢钠有着良好的催化活性,在非诺贝特酸用量0.1 mol,n(异丙醇)∶n(非诺贝特酸)=3.5∶1,催化剂一水合硫酸氢钠用量3.5 g,二甲苯40 m L,反应时间3 h的条件下,非诺贝特产率可达88.6%,催化剂重复使用5次仍保持较高的活性,其结构经IR确证。     

First Domestic Third-Party Tire Testing Ground to Be Completed in October

The reporter learned from the China Rubber Annual Conference held in Qingdao at the end of March that CATRC Yancheng Automobile Testing Ground Co., Ltd., the largest and most comprehen- sive domestic public automobile testing ground invested with RMB 2 billion yuan by CATRC would be completed and put into use at the end of this year. Thereinto, the Automobile Tire Test Center with an investment of RMB 40 million yuan would be completed in October this year, in which the tires interior and exterior projects test could be carried out, and the testing projects cover EU Tire Labeling Act test, domestic 3C authentication test,     

基于表面增强拉曼光谱标记技术的生物传感器用于农药残留检测的研究进展

近年来,表面增强拉曼光谱（surface enhancement Raman spectroscopy,SERS）标记技术因具有高灵敏度、可多路复用、强抗光漂白性和较好的分子指纹保真度等特性而备受瞩目,并已成功应用于传感分析及生物成像领域。本综述主要总结了基于SERS标记技术的生物传感器的原理及其在农药残留快速检测领域中的最新研究进展,重点介绍了SERS标签的设计与制备及基于不同识别元件传感系统的研究现状。同时,对其面临的挑战和解决方案进行了探讨,以期促进SERS标记技术在农药残留检测及食品安全检测方面的应用。     

Structural Elucidation and Biological Activity of Acyl-homoserine Lactones from the Phytopathogen Pantoea ananatis Serrano 1928

In Gram-negative bacteria, the acyl-homoserine lactones (acyl-HSLs) are the main signaling substances employed in cell-to-cell communication systems. This paper describes the chemical characterization of acyl-HSLs produced by the worldwide-spread phytopathogen Pantoea ananatis (Serrano 1928) by using gas chromatography-electron impact mass spectrometry. The absolute configuration of the major identified substance, (S)-(−)-N-hexanoyl-HSL, was determined with gas chromatography-flame ionization detection with a chiral capillary column. Biological activities of extracts, fractions, and synthetic products were evaluated with the specific reporter Agrobacterium tumefaciensNTL4(pZLR4) in β-galactosidase expression assays.     

菜热休克蛋白基因HSP81.1启动子的克隆与功能验证

本研究从甘蓝型油菜（Brassica napus）基因组中扩增获得热休克蛋白基因启动子,并将其克隆至双元载体pBI121的GUS报告基因上游,构成双元表达载体pBI121 HSP GUS。通过农杆菌（Agrobacterium tumefaciens）介导法转化烟草后获得转基因植株。对T1转基因植株研究表明,外源基因在42℃处理1h后开始表达,且即使用同样条件诱导,GUS基因的表达率和表达量在不同生长时期也有差异。在第50d左右的苗期,GUS基因的表达率和表达量高,但随植株老化而逐渐下降。研究显示,利用油菜的热休克蛋白启动子可以达到通过温度控制烟草基因表达的目的。     

基因枪法建立紫菜丝状体遗传转化模式

以坛紫菜和条斑紫菜的自由丝状体为材料,利用基因枪法分别转化CaMV35S、SV40、FCP、Amt、Ubi 5种启动子与报告基因组合,转化后48h进行原位组织化学染色检测,首次针对重要经济红藻紫菜的自由丝状体建立并优化遗传转化技术,为紫菜研究提供了新工具。结果显示,SV40启动子可以驱动laeZ报告基因(编码β-半乳糖苷酶)在紫菜丝状体中的瞬间表达,空白与阴性对照未检测到本底;其它4种启动子未检测到基因表达。进一步优化实验发现,在可裂膜650psi、轰击距离6cm下获得最高转化效率为8.0×10~(-5),经定量检测与双因子方差分析,是最佳转化参数,提示基因枪参数对外源基因转化效率具显著影响。