凡纳滨对虾优势腐败菌鉴定及其群体感应现象

为了鉴定凡纳滨对虾的优势腐败菌并研究其是否存在以N-酰基高丝氨酸内酯类化合物(AHLs)介导的群体感应系统,采用16S rRNA序列鉴定凡纳滨对虾的优势腐败菌,并采用紫色杆菌CV026对优势腐败菌的AHLs活性进行检测.结果发现凡纳滨对虾优势腐败菌菌株1(Aci-1)和菌株2 (Aci-2)均为不动杆菌属,均存在以AHLs为信号分子的群体感应系统.添加外源信号分子AHLs能促进Aci-1菌株生物膜的形成,且呈浓度依赖性.在一定的贮藏范围内,凡纳滨对虾腐败菌信号分子AHLs浓度与细菌总数、挥发性盐基氮含量存在正相关性,其相关系数r分别为0.846 6和0.986 7,分别在P＜0.05与P＜0.01水平上显著,结论是凡纳滨对虾优势腐败菌不动杆菌菌株存在以AHLs介导的群体感应系统,且与凡纳滨对虾的腐败密切相关.     

食源性致病菌群体感应信号分子的检测

群体感应(Quorum sensing,QS)在食物中毒导致的食源性疾病暴发机制和食物腐败变质中起主要作用,QS影响致病菌的细胞被膜形成和致病性。文中通过深入了解食源性致病菌的QS信号分子,综述了革兰氏阴性和革兰氏阳性菌产生的信号分子类型,同时介绍了检测QS信号分子的不同技术,并根据QS机制在食品中的影响提出了思考和建议,为监控食源性致病菌提供依据。     

细菌群体感应信号分子的光电检测技术

群体感应（quorum sensing，QS）是一种依赖菌群密度的细菌交流系统。在探究细菌群体感应系统的调控机制中，对QS信号分子的鉴别和检测是不可或缺的环节，其对生命科学、药学等领域涉及细菌等微生物的相互作用、高效检测和作用机制解析等具有重要的参考意义。本文在总结不同类型细菌QS信号分子来源和结构的基础上，对QS信号分子的光电检测方法和技术进行了综述，重点对光电传感检测的敏感介质、传感界面、传感机制及测试效果进行探讨，同时关注了将微流控芯片分析技术应用于细菌QS信号分子原位监测的相关研究进展。     

群体感应信号分子AI-2研究进展

群体感应(QS)是细菌根据种群密度的变化调控基因表达,协调群体行为的机制。除具有种特异性的信号分子AI-1外,近年来发现一类新的信号分子AI-2在调控细菌基因表达中起重要作用。AI-2的结构和生物合成途径已被确定,其产生依赖于一种称为LuxS的蛋白。目前认为AI-2在细菌种间交流中起通用信号分子(universalsignal)的作用。了解细菌的QS调控过程以及种间细胞交流的新机制,有助于对细菌病害进行防治。     

细菌群体感应信号分子N-酰基高丝氨酸内酯的检测

群体感应是细菌生长到一定密度时相互感应,并进行基因表达及调控产生的独特、多样的群体行为现象。N-酰基高丝氨酸内酯(AHL)类化合物是革兰阴性菌群体感应中最重要的一类信号分子,调控许多生理特性基因的表达。快速、简便、有效地检测细菌能否产生AHL或产生何种信号分子,成为深入研究和了解细菌群体感应的重要手段。我们就细菌群体感应信号分子AHL检测的基本原理和方法及国内外研究进展进行了总结。     

用气相色谱-质谱联用比较牛耳草代谢物的提取方法

通过比较不同的提取方法对牛耳草新鲜和脱水叶片中代谢物的提取效率,旨在建立一种可以有效鉴定并分析牛耳草脱水过程中关键小分子代谢物的种类和含量变化的方法,为研究植物耐脱水分子机制提供技术方法。本研究以气相色谱-质谱联用(GC-MS)为分析方法,对复苏植物牛耳草代谢物提取方法进行比较。从提取总色谱峰数目、提取效率、代谢物保留时间和提取效率稳定性等方面比较甲醇溶液(A法)和甲醇-氯仿-水溶液(B法)两种提取方法的提取效果。对牛耳草新鲜样品提取结果表明,B法提取的总色谱峰数目多于A法;对9种共有代谢物的提取效率比较结果表明,B法的提取效率高于A法;对10种色谱峰的保留时间和提取效率的方法学考察结果表明,两者保留时间RSD(相对标准偏差)值均小于1%,A法提取效率的RSD值≤10%的比例为50%,B法的为100%。A法对干样的提取色谱峰数目远少于鲜样,而B法对干样的提取色谱峰数目和鲜样没有显著差异,保留时间RSD值均小于1%,提取效率的RSD值与鲜样没有差异,稳定性良好。     

越鞠丸石油醚提取物的气相色谱-质谱分析

采用气相色谱-质谱联用技术对中药复方越鞠丸石油醚提取物中的成分进行鉴定,共鉴定出60种成分,并采用峰面积归一化法确定了各成分的相对含量。     

细菌群体感应调控多样性及群体感应淬灭

群体感应(Quorum sensing, QS)是细菌通过信号分子分泌、识别,从而调控基因水平转移、毒力因子分泌、芽孢产生及生物膜形成等群体行为的细胞交流机制。干扰信号分子的分泌、识别,可以阻断群体感应,实现群体淬灭。群体淬灭(Quorum quenching, QQ)是目前致病性控制、致腐性预防以及生物膜污染削减的重要策略之一。本文以群体感应信号分泌-识别-响应为主线,将群体感应分为等级、平行及竞争型三类调控方式,并对其特征进行了详细阐述;同时,探讨了信号分子类似物、信号分子降解酶剂、信号受体激活剂/抑制剂等策略在不同调控方式淬灭中的适用性;最后,对群体感应调控及淬灭进行了展望,以期为丰富细菌群体感应认知、促进群体淬灭应用提供参考。     

细菌群体感应信号分子与抑制剂研究进展

具有群体感应系统的细菌通过相互交换一种自动诱导（autoinducer）信号分子来实现彼此问的信息交流。当信号分子积累到一定浓度时会改变细菌特定基因的表达,如生物膜的形成、生物发光行为、毒性基因的表达、孢子的形成等。近年来,人们发现了多种天然或者人工合成的群体感应抑制剂,可以干扰群感系统的信息回路。本文系统地阐述了细菌群体感应信息系统的划分、自体诱导分子及其抑制剂的研究进展。     

群体感应信号分子及其抑制剂快速检测方法的建立

细菌能自发产生、释放一些特定的信号分子,并能感知其浓度变化,调节微生物的群体行为,这一调控系统称为群体感应。细菌群体感应参与包括人类、动植物病原菌致病力在内的多种生物学功能的调节,群体感应抑制剂成为抗感染药物开发的靶点。利用紫色色杆菌(Chromobacterium violaceum)和根癌农杆菌(Agrobacterium tumefaciens)作为指示菌,建立检测高丝氨酸内酯(AHLs)及其抑制剂的简便方法。结果表明,通过平板交叉划线接种,使用指示菌能够有效地检测AHLs,并且通过薄层层析(TLC)与细菌生物感应器相结合的方法可以快速、方便地鉴定AHLs的种类;通过双层平板法观察指示菌色素产生情况,能够有效地检测群体感应信号分子AHLs抑制剂,且该方法简单易行。     

细菌群体感应及其在食品变质中的作用

食品相关细菌引起的生物被膜形成和食品变质是食品工业中的重大问题。研究表明细菌群体感应(Quorum sensing,QS)与被膜形成、食品腐败变质密切相关。重点对细菌产生的各种QS信号分子及其在被膜形成的作用和被膜在食品工业中的重要性做了介绍。QS信号分子与食品变质密切相关,故对QS抑制剂作为新型食品防腐剂以延长储存期限及加强食品安全的前景进行了概述。     

Quantification of isoflavones and lignans in urine using gas chromatography/mass spectrometry

Phytoestrogens (isoflavones and lignans) are of increasing interest due to their potential to prevent certain types of complex diseases. However, epidemiological evidence is needed on the levels of phytoestrogens and their metabolites in foods and biological fluids in relation to risk of these diseases. We report an assay for phytoestrogens which is sensitive, accurate, and uses low volumes of sample. Suitable for epidemiological studies, the assay consists of a simple sample preparation procedure and has been developed for the analysis of five isoflavones (daidzein, O-desmethylangolensin, equol, genistein, and glycitein) and two lignans (enterodiol and enterolactone), which requires only 200 microl of urine and utilizes one solid-phase extraction stage for sample preparation prior to derivatization for GC/MS analysis. Limits of detection were in the region 1.2 ng/ml (enterodiol) to 5.3ng/ml (enterolactone) and the method performed well in the UK Government's Food Standards Agency-sponsored quality assurance scheme for phytoestrogens. For the first time, average levels of all the above phytoestrogens were measured in samples of urine collected from a free living population sample of women. Results show a large range in both the amount and the type of phytoestrogens excreted.     

Quantitative gas chromatography/mass spectrometry determination of C-mannosylation of tryptophan residues in glycoproteins

C-mannosylation of Trp residue is one of the most recently discovered types of glycosylation, but the identification of these mannosylated residues in proteins is rather tedious. In a previous paper, it was reported that the complete analysis of all constituents of glycoproteins (sialic acids, monosaccharides, and amino acids) could be determined on the same sample in three different steps of gas chromatography/mass spectrometry of heptafluorobutyrate derivatives. It was observed that during the acid-catalyzed methanolysis step used for liberation of monosaccharide from classical O- and N-glycans, Trp and His were quantitatively transformed by the addition of a methanol molecule on their indole and imidazole groups, respectively. These derivatives were stable to acid hydrolysis used for the liberation of amino acids. Since monosaccharide derivatives were also stabilized as heptafluorobutyrate derivatives of O-methyl-glycosides, it was suggested that C-mannosides of Trp residues could quantitatively be recovered. Based on the analyses of standard compounds, peptides and RNase 2 from human urine, we report that C((2))-mannosylated Trp could be quantitatively recovered and identified during the step of amino acid analysis. Analyses of different samples indicated that this type of glycosylation is absent in bacteria and yeasts.     

Global analysis of metabolites in rat and human urine based on gas chromatography/time-of-flight mass spectrometry

Sediment in urine may contain low-molecular-weight compounds that should be included in the analysis. To date, no systematic investigation has addressed this issue. We investigated three primary factors that influence the extraction efficiency of metabolites during preparation of urine samples for metabolomic research: centrifugation, pH, and extraction solvents. Obtained with the use of gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) technique and principal component analysis (PCA), our results indicate that (1) conventional centrifugation causes an apparent loss of some metabolites, indicating that urine samples for metabolomic research should not be centrifuged before procedures are undertaken to recover the metabolites; (2) pH adjustment has a large impact on the recovery of metabolites and is therefore not encouraged; (3) with design of experiment analysis, methanol and water yield the optimal extraction efficiency. Differences between rat and human urine were observed and are discussed. Ninety-nine metabolites identified in rat and human urine are presented. An efficient protocol is proposed for the pretreatment of urine samples.     

Thermospray liquid chromatography/mass spectrometry for the analysis of l-carnitine and its short-chain acyl derivatives

A method utilizing thermospray high-performance liquid chromatography/mass spectrometry for the separation and direct analysis of carnitine, acetylcarnitine, and propionylcarnitine is described. On-column analysis of mixtures of the acylcarnitines with their corresponding stable, isotope-labeled analogs at nanomolar concentrations has indicated that isotope dilution assays can be applied towards the analysis of carnitine and short-chain acylcarnitines present in biological samples.     

利用色谱法快速检测分析啤酒腐败菌的新方法

啤酒在生产过程中很容易染菌,而传统的用于啤酒腐败菌检测的方法耗时长,不能满足实际需求.由于腐败菌污染的啤酒通过色谱检测,相应组分(生物胺、有机酸和风味物质)都会有特征峰的产生,所以本研究通过建立无腐败菌污染的啤酒中各组分的标准色谱图,再使啤酒强制染菌,对其组分进行色谱分析,并与标准色谱图进行比较,从而找出各组分对应的特征峰.未来,此方法可用于实际生产线上快速检测啤酒是否发生微生物污染.     

A capillary gas chromatography/mass spectrometric method for the quantification of hydroxysteroids in human plasma

A specific and sensitive methodology for the quantitative determination of hydroxysteroids dehydroepiandrosterone and pregnenolone and their main metabolites in human plasma is described. Hydroxysteroids were extracted using methanol and steroids were further separated by reverse-phase high-performance liquid chromatography, allowing for minimization of the possible chromatographic interferences. Eluted fractions were collected, pooled, and analyzed by gas chromatography-mass spectrometry as trimethylsilyl ether derivatives. The quantification was performed with single-ion monitoring of the highly abundant m/z 129 or m/z 358 fragments. The combination of the chromatographic characteristics to the specific fragments ensured the selectivity and specificity of the method. Under these conditions the method was linear (typical R2 is superior to 0.98 for all hydroxysteroids studied) over the concentration range of 2 x 10(-9) to 10(-6)M with good precision and accuracy.     

A high-throughput method for microbial metabolome analysis using gas chromatography/mass spectrometry

An analytical high-throughput method based on gas chromatography/mass spectrometry (GC/MS) was developed for fast metabolome investigation. By parallelization and partial automation the time needed for the preanalytical steps could be reduced. In addition a strong decrease of the relative standard deviation of metabolite concentrations from independent samples on the same microtiter plate from 25 to 13% was achieved. Between different plates the relative standard deviation is comparable to the one observed in standard experiments with shaking flasks. Using a fast GC the time need for the full GC/MS-based metabolome analysis could be decreased from 60 to 18 min per run, allowing the measurement of 72 single samples per day and GC/MS machine. In samples of the model organism Corynebacterium glutamicum more than 1000 peaks in the total ion current could be detected in a single fast GC/MS run of which 650 were strong enough to be quantified. Approximately 150 compounds of these were identified using our metabolite MS-library. Correlation analysis of the concentration vectors of independent wild-type samples raised under the same conditions show very high correlations of 0.99+/-0.01 (logs). In conclusion this method allows screenings of large mutant libraries for genetically induced metabolic perturbations.     

Metabolomic analysis of biofluids from rats treated with Aconitum alkaloids using nuclear magnetic resonance and gas chromatography/time-of-flight mass spectrometry

The Aconitum alkaloids aconitine, mesaconitine, and hypaconitine are the main toxic components in a commonly used traditional Chinese herbal medicine Fu Zi. To provide guidelines for the safe use of this medicine, metabolic changes in Wistar rats caused by these compounds were investigated by means of integrated analysis of two metabonomic approaches: ¹H nuclear magnetic resonance (NMR) and gas chromatography/time-of-flight mass spectrometry (GC/TOF–MS). Rats were given a single dose of aconitine, mesaconitine, hypaconitine, or vehicle. The largest metabolic changes were observed 6 h after treatment. Every group receiving a dose had higher urine concentrations of glucose, acetate, dimethylglycine, succinate, and alanine and had lower concentrations of creatinine, citrate, 2-oxoglutarate, N-acetylated metabolites, and trimethylamine-N-oxide (TMAO) than did the control group. These results may reflect the perturbation of renal tubular function within the first 24 h after treatment. The results also revealed a larger perturbation of metabolic profiles in the aconitine group than in the mesaconitine and hypaconitine groups, illustrating how these alkaloids exhibit different toxicities. An analysis of plasma samples collected 7 days postdose showed that there were higher levels of lactate, alanine, and lipids along with lower levels of glucose, β-hydroxybutyrate, and creatine in the plasma of the aconitine and mesaconitine groups than there were in the control and hypaconitine groups. The GC/TOF–MS data from the plasma samples showed that the number of metabolites, with significant changes or with a tendency to change, in the aconitine and mesaconitine groups were dissimilar, suggesting a possible difference in the acute toxicity mechanisms of these alkaloids.