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。     

A Thioacetal Photocage Designed for Dual Release: Application in the Quantitation of Therapeutic Release by Synchronous Reporter Decaging

Despite the immense potential of existing photocaging technology, its application is limited by the paucity of advanced caging tools. Here, we report on the design of a novel thioacetal ortho‐nitrobenzaldehyde (TNB) dual arm photocage that enabled control of the simultaneous release of two payloads linked to a single TNB unit. By using this cage, which was prepared in a single step from commercial 6‐nitroverataldehyde, three drug–fluorophore conjugates were synthesized: Taxol‐TNB‐fluorescein, Taxol‐TNB‐coumarin, and doxorubicin‐TNB‐coumarin, and long‐wavelength UVA light‐triggered release experiments demonstrated that dual payload release occurred with rapid decay kinetics for each conjugate. In cell‐based assays performed in vitro, dual release could also be controlled by UV exposure, resulting in increased cellular fluorescence and cytotoxicity with potency equal to that of unmodified drug towards the KB carcinoma cell line. The extent of such dual release was quantifiable by reporter fluorescence measured in situ and was found to correlate with the extent of cytotoxicity. Thus, this novel dual arm cage strategy provides a valuable tool that enables both active control and real‐time monitoring of drug activation at the delivery site.     

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.     

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.     

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.     

中慢生型天山根瘤菌MsiR组成型蛋白突变株的筛选

MsiR蛋白来源于中慢生型天山根瘤菌（Mesorhizobium tianshanense）,是LysR转录调控蛋白家族中的一员,它可以响应宿主植物释放的抗代谢物-刀豆氨酸,激活外运蛋白msiA编码基因的转录表达。通过构建MsiR突变文库,筛选得到了7个MsiR组成型突变蛋白,L166P、A147V、P83L、A278T组成型突变蛋白丧失了对刀豆氨酸的响应,在没有刀豆氨酸时的荧光值为800左右;A147T、E59G组成型突变蛋白仍然可以响应刀豆氨酸的诱导信号,添加刀豆氨酸时荧光值是未添加时的1.7倍。通过蛋白同源建模分析了组成型突变的氨基酸残基在MsiR蛋白上的空间分布及其对MsiR蛋白调控功能可能的影响。组成型突变蛋白的研究对进一步揭示LysR家族蛋白转录调控的机理有重要意义。     

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。