植物细胞凋亡的检测方法

细胞凋亡是一种在动、植物中普遍存在的现象,在动、植物的发育过程中起着非常重要的作用。从形态学、生化及分子生物学、免疫学、生理学等方面介绍了几种检测细胞凋亡的方法,以及各种方法在检测不同植物凋亡时的应用,并对植物细胞凋亡检测技术的前景进行了展望。     

细胞凋亡检测方法探讨

采用图例法和比较法分析评价细胞凋亡检测方法的应用范围和检测结果的相关性,指出只有选择多种适当的方法进行综合检测,才能对结果做出正确而合理的分析判断。流式细胞术简便、快速,从膜上分子、胞内蛋白到DNA水平全方位多角度分析凋亡,可作为检测细胞凋亡的首选方法。     

几种细胞凋亡检测方法的比较

近年来,凋亡细胞的检测方法从以形态学观察发展到生物化学、免疫化学和分子生物学等定性和定量测定技术,并在细胞、染色质到分子水平日趋完善和成熟。这些方法多用于描述DNA的片段化和细胞死亡过程中许多特定成分的检测。细胞凋亡具有其生物复杂性,检测方法要求灵敏度高、特异性强、准确性好、凋亡与坏死的区分明显等。由于每种方法各有优缺点,故应避免使用单一方法检测。     

细胞凋亡检测方法的研究进展

细胞凋亡在生命活动中具有重要的地位,多年以来一直都是生命科学领域的研究热点.为了准确、快速的检测细胞凋亡,人们发明了多种方法,并且随着科技的发展和技术的进步,新的检测方法不断涌现.就近年来关于细胞凋亡的一些检测方法进行综述.     

一种检测早期凋亡细胞的方法

本文介绍了一种定量检测早期凋亡细胞的流式细胞术——Annexin V-PI双染色法,并作了一些改进。     

植物细胞凋亡的ELISA检测

本文利用TUNEL方法在单个细胞水平上对苯胺灵诱导的玉米根尖细胞死亡进行了检测。结果表明,一定浓度的苯胺灵能诱导玉米根尖细胞发生主动性的细胞凋亡,具有细胞凋亡典型的形态和生化特征即细胞核浓缩并形成核碎片、染色质边缘化及DNA特异片段化等（Fig.1)。首次利用ELISA方法在群体水平上对这种细胞凋亡进行了进一步检测。结果表明：动物细胞研究常用的ELISA方法同样也适合用于植物细胞凋亡的检测。在凋亡前期,随着凋亡过程的进行,细胞质中的OD值逐渐上升（Fig.2)。剂量实验表明,0．2mg/mL苯胺灵最适合于诱导细胞凋亡（Fig.3)。     

细胞凋亡检测技术的进展

细胞凋亡是多细胞生物体内一个重要的生命现象,它已成为当前科学和医学领域的研究热点。随着对细胞凋亡的研究不断扩展和深化,许多新的指标逐渐被采纳;同时伴随着一些先进仪器的应用,原有一些指标的灵敏性和准确性也得到了提高,这些都将细胞凋亡的检测水平提升到一个新的高度。     

基于荧光方法的大肠杆菌单管定量检测技术研究

通过研究培养温度、培养基pH值对大肠杆菌生长和培养液荧光强度的影响,确定44℃,培养液pH值7.0～7.5为大肠杆菌MUG酶荧光检测的最佳条件。通过研究培养时间和接种菌浓度与培养液荧光强度的关系,建立起基于荧光方法检测大肠杆菌的单管定量检测技术。通过与平板菌落计数法和最大可能数法比较发现单管定量检测法的相对标准偏差小于这两种常用的方法,而且更为快速、经济,适合用于大肠杆菌的定量检测。     

转基因烟草荧光定量检测方法研究

依据实时定量PCR原理,参照35S启动子、NOS终止子、GUS基因和NPTII基因序列设计TaqMan引物和荧光标记探针。采用美国MJ公司OpticonTM2荧光定量PCR检测系统对烤后烟叶进行转基因定量检测技术研究,从中筛选出扩增效率高,灵敏度好的PCR引物和探针序列,同时通过对扩增体系,扩增条件的梯度实验,优化出荧光定量检测的最佳反应体系和反应条件,从而建立了转基因烟草定量检测方法。该方法经验证其检测灵敏度达到0.05％。在2003年6月参加CORESTA(国际烟草科研与合作中心)组织的国际烟草转基因定量检测合作试验中,该优化转基因烟草定量检测技术获得了较好成绩,对盲检样品检测结果评价(Z-score)列国际12家实验室之首,证明此法灵敏度高、稳定性好。     

应用高通量实时荧光定量PCR方法筛选FL细胞对低剂量微囊藻毒素LR暴露的应答基因

微囊藻毒素是蓝藻的一些属产生的单环七肽,在发生水华的水体中普遍存在[1]。含有微囊藻毒素的水华能引起野生动物、鱼类、家畜、家禽等中毒和死亡,也对人类健康构成严重威胁[2,3]。流行病学调查发现,人群原发性肝癌、大肠癌发病率与饮水源中的微囊藻毒素有关[4]。其中微囊藻毒素LR是微囊藻毒素中最常见的一种,因其高急性毒性,强促癌活性而受到广泛的关注。有研究结果表明微囊藻毒素LR可引起多种细胞发生凋亡[5],本实验室先前的研究也发现微囊藻毒素LR能激活在凋亡过程中起重要作用的酶Caspase-3[6],及引起P53、Bax、Bcl-2等凋亡相关蛋白…     

A high-throughput direct fluorescence resonance energy transfer-based assay for analyzing apoptotic proteases using flow cytometry and fluorescence lifetime measurements

Cytometry is a versatile and powerful method applicable to different fields, particularly pharmacology and biomedical studies. Based on the data obtained, cytometric studies are classified into high-throughput (HTP) or high-content screening (HCS) groups. However, assays combining the advantages of both are required to facilitate research. In this study, we developed a high-throughput system to profile cellular populations in terms of time- or dose-dependent responses to apoptotic stimulations because apoptotic inducers are potent anticancer drugs. We previously established assay systems involving protease to monitor live cells for apoptosis using tunable fluorescence resonance energy transfer (FRET)-based bioprobes. These assays can be used for microscopic analyses or fluorescence-activated cell sorting. In this study, we developed FRET-based bioprobes to detect the activity of the apoptotic markers caspase-3 and caspase-9 via changes in bioprobe fluorescence lifetimes using a flow cytometer for direct estimation of FRET efficiencies. Different patterns of changes in the fluorescence lifetimes of these markers during apoptosis were observed, indicating a relationship between discrete steps in the apoptosis process. The findings demonstrate the feasibility of evaluating collective cellular dynamics during apoptosis.     

Development of a highly sensitive,high-throughput assay for glycosyltransferases using enzyme-coupled fluorescence detection

Glycosyltransferases catalyze transfer of sugar moieties from activated donor molecules to specific acceptor molecules, forming glycosidic bonds. Identification of selective modulators of glycosyltransferases is important both to provide new tools for investigating pathophysiological roles of glycosylation reactions in cells and tissues, and as new leads in drug discovery. Here we describe a universal enzyme-coupled fluorescence assay for glycosyltransferases, based on quantification of nucleotides produced in the glycosyl transfer reaction. GDP, UDP, and CMP are phosphorylated with nucleotide kinase in the presence of excess ATP, generating ADP. Via coupled enzyme reactions involving ADP-hexokinase, glucose-6-phosphate dehydrogenase, and diaphorase, the ADP is utilized for conversion of resazurin to resorufin, which is determined by fluorescence measurement. The method was validated by comparison with an HPLC method, and employed to screen the LOPAC1280 library for inhibitors in a 384-well plate format. The assay performed well, with a Z′-factor of 0.80. We identified 12 hits for human galactosyltransferase B4GALT1 after elimination of false positives that inhibited the enzyme-coupled assay system. The assay components are all commercially available and the reagent cost is only 2 to 10 US cents per well. This method is suitable for low-cost, high-throughput assay of various glycosyltransferases and screening of glycosyltransferase modulators.     

A high-throughput screening fluorescence polarization assay for fatty acid adenylating enzymes in Mycobacterium tuberculosis

Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), encodes for an astonishing 34 fatty acid adenylating enzymes (FadDs), which play key roles in lipid metabolism. FadDs involved in lipid biosynthesis are functionally nonredundant and serve to link fatty acid and polyketide synthesis to produce some of the most architecturally complex natural lipids including the essential mycolic acids as well as the virulence-conferring phthiocerol dimycocerosates, phenolic glycolipids, and mycobactins. Here we describe the systematic development and optimization of a fluorescence polarization assay to identify small molecule inhibitors as potential antitubercular agents. We fluorescently labeled a bisubstrate inhibitor to generate a fluorescent probe/tracer, which bound with a K_D of 245 nM to FadD28. Next, we evaluated assay performance by competitive binding experiments with a series of known ligands and assessed the impact of control parameters including incubation time, stability of the signal, temperature, and DMSO concentration. As a final level of validation the LOPAC1280 library was screened in a 384-well plate format and the assay performed with a Z-factor of 0.75, demonstrating its readiness for high-throughput screening.     

A real-time high-throughput fluorescence assay for sphingosine kinases

Sphingosine kinases (SphKs), of which there are two isoforms, SphK1 and SphK2, have been implicated in regulation of many important cellular processes. We have developed an assay for monitoring SphK1 and SphK2 activity in real time without the need for organic partitioning of products, radioactive materials, or specialized equipment. The assay conveniently follows SphK-dependent changes in 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled sphingosine (Sph) fluorescence and can be easily performed in 384-well plate format with small reaction volumes. We present data showing dose-proportional responses to enzyme, substrate, and inhibitor concentrations. The SphK1 and SphK2 binding affinities for NBD-Sph and the IC₅₀ values of inhibitors determined were consistent with those reported with other methods. Because of the versatility and simplicity of the assay, it should facilitate the routine characterization of inhibitors and SphK mutants and can be readily used for compound library screening in high-throughput format.     

A universal homogeneous assay for high-throughput determination of binding kinetics

There is an increasing demand for assay technologies that enable accurate, cost-effective, and high-throughput measurements of drug–target association and dissociation rates. Here we introduce a universal homogeneous kinetic probe competition assay (kPCA) that meets these requirements. The time-resolved fluorescence energy transfer (TR–FRET) procedure combines the versatility of radioligand binding assays with the advantages of homogeneous nonradioactive techniques while approaching the time resolution of surface plasmon resonance (SPR) and related biosensors. We show application of kPCA for three important target classes: enzymes, protein–protein interactions, and G protein-coupled receptors (GPCRs). This method is capable of supporting early stages of drug discovery with large amounts of kinetic information.     

A high-throughput screen for identification of molecular mimics of Smac/DIABLO utilizing a fluorescence polarization assay

Resistance to apoptosis is afforded by inhibitor of apoptosis proteins (IAPs) which bind to and inhibit the caspases responsible for cleavage of substrates leading to apoptotic cell death. Smac (or DIABLO), a proapoptotic protein released from the mitochondrial intermembrane space into the cytosol, promotes apoptosis by binding to IAPs, thus reversing their inhibitory effects on caspases. We have developed a high-throughput fluorescence polarization assay utilizing a fluorescein-labeled peptide similar to the "IAP binding" domain of Smac N terminus complexed with the BIR3 domain of X-linked IAP (XIAP) to identify small-molecule mimics of the action of Smac. The IC(50)s of peptides and a tetrapeptidomimetic homologous to the N terminus of Smac demonstrated the specificity and utility of this assay. We have screened the National Cancer Institute "Training Set" of 230 compounds, with well-defined biological actions, and the "Diversity Set" of 2000 chemically diverse structures for compounds which significantly reduced fluorescence polarization. Highly fluorescing or fluorescence-quenching compounds (false positives) were distinguished from those which interfered with Smac peptide binding to the XIAP-BIR3 in a dose-dependent manner (true positives). This robust assay offers potential for high-throughput screening discovery of novel compounds simulating the action of Smac/DIABLO.     

Development of a high-throughput fluorescence polarization assay for Bcl-x(L)

Antiapoptotic protein Bcl-x(L) has been demonstrated to play a very important role in a variety of diseases such as cancer. Its biological function can be inhibited by proapoptotic proteins such Bak, Bad, and Bax by forming complexes mediated primarily by the Bcl-2 homology 3 (BH3) domain. To facilitate drug discovery for Bcl-x(L) inhibitors, we have developed and optimized a fluorescence polarization assay based on the interaction between Bcl-x(L) and BH3 domain peptides. We observed that the fluorescein-labeled Bad BH3 peptide [NLWAAQRYGRELRRMSDK(fluorescein)FVD or fluorescent Bad peptide] generates best overall results. Fluorescent Bad peptide interacts strongly with Bcl-x(L) with a K(d) of 21.48nM. The assay is stable over a 24-h period and can tolerate the presence of dimethyl sulfoxide up to 8%. By using a competition assay, several peptides derived from the BH3 region of Bak, Bad, Bax, and Bcl-2 were investigated. Bad and Bak BH3 peptides compete efficiently with IC(50) values of 0.048 and 1.14 microM, respectively, while the peptides from the BH3 region of Bcl-2 and Bax compete weakly. A mutated Bak peptide, which has been shown to be inactive for binding to Bcl-x(L), did not compete. The relative binding order of the peptides (Bad>Bak>Bcl-2>Bax>mutated Bak) correlates well with previously published results. When tested in high-throughput formats, the assay has a signal-to-noise ratio of 15.37 and a Z(') factor of at least 0.73. The plate-to-plate variability for free peptide control and bound peptide control is minimal. This validates the assay not only for investigating the nature of Bcl-x(L)-peptide interaction, but also for high-throughput screening of Bcl-x(L) inhibitors.     

A quantitative stopped-flow fluorescence assay for measuring polymerase elongation rates

The measurement of nucleic acid polymerase elongation rates is often done via a lengthy experimental process involving radiolabeled substrates, quenched elongation experiments, electrophoretic product separation, and band quantitation. In this work, we describe an alternative real-time stopped-flow assay for obtaining kinetic parameters for elongation of extended sequences. The assay builds on our earlier PETE (polymerase elongation template element) assay designed for high-throughput screening purposes [S.P. Mestas, A.J. Sholders, O.B. Peersen, A fluorescence polarization-based screening assay for nucleic acid polymerase elongation activity, Anal. Biochem. 365 (2007) 194-200] and relies on measuring how long it takes a polymerase to reach the end of a defined length template. Using poliovirus polymerase and self-priming hairpin RNA substrates with 6- to 26-nt-long templating regions, we demonstrate that the assay can be used to determine V_max rates for elongation and apparent K_m values for nucleotide triphosphate (NTP) use. Modeling the reaction kinetics as a series of irreversible steps allows us to numerically fit the entire time-based dataset by properly accounting for the temporal distribution of intermediate species. This enables us to determine average elongation rates over heterogeneous templating regions that mimic viral genome substrates. The assay is easily extendable to other RNA and DNA polymerases, can accommodate secondary structures in the template, and can in principle be used for any enzyme traversing along an extended substrate.