Improved methods to detect GTP-binding proteins from plants

Improved methods are described for the detection of G1P-binding proteins (G-proteins) in the protonema of mossFunaria hygrometrica and coleoptiles of corn(Zea mays) and sorghum(Sorghum vulgare). We optimized conditions for the transfer of proteins to nitrocellulose, production of high titer polyclonal anti-Gα (common) antibodies and finally the detection of G-proteins by amplification. In addition to the α-subunit of heterotrimeric G-proteins (M _r 41–43 kDa), a small molecular weight class (< 30 kDa) was also detected by anti-Gα (common) antibodies. An easy, reliable and efficient filter assay is also described to quantify the toxin catalyzed ADP-ribosylation. The apparentK _m of the NAD has been determined to be approximately 1.5μM for the microsomal fraction of moss. Inclusion of G1P stimulated ADP-ribosylation by 2–27-fold. One to three polypeptides representing the α-subunit of heterotrimeric G-proteins of (M_r 37–43 kDa) were ADP-ribosylated in all three plants. The anti-Gβ (C-terminus) antibody cross-reacted strongly with 39 and 34 kDa polypeptide in moss and corn respectively. By employing improved methods two classes of G-proteins have been shown to be present in three plant species.     

Chemical methods to detect S-nitrosation

Nitric oxide (NO) is a cell-signaling molecule involved in a number of physiological and pathophysiological processes. Modification of cysteine residues by NO (or NO metabolites), that is S-nitrosation, changes the function of a broad spectrum of proteins. This reaction represents an important post-translational modification that transduces NO-dependent signals. However, the detection and quantification of S-nitrosation in biological samples remain a challenge mainly because of the lability of S-nitrosation products: S-nitrosothiols (SNO). In this review we summarize recent developments of the methods to detect S-nitrosation. Our focus is on the methods which can be used to directly conjugate the site(s) of S-nitrosation.     

A phage display system designed to detect and study protein–protein interactions

Analysing protein–protein interactions is critical in proteomics and drug discovery. The usage of 2-Hybrid (2λ) systems is limited to an in vivo environment. We describe a bacteriophage 2-Hybrid system for studying protein interactions in vitro . Bait and prey are displayed as fusions to the surface of phage λ that are marked with different selectable drug-resistant markers. An interaction of phages in vitro through displayed proteins allows bacterial infection by two phages resulting in double drug-resistant bacterial colonies at very low multiplicity of infections. We demonstrate interaction of the protein sorting signal Ubiquitin with the Vps9-CUE, a Ubiquitin binding domain, and by the interaction of (Gly-Glu)₄ and (Gly-Arg)₄ peptides. Interruptions of the phage interactions by non-fused (free) bait or prey molecules show how robust and unique our approach is. We also demonstrate the use of Ubiquitin and CUE display phages to find binding partners in a λ-display library. The unique usefulness to 2λ is also described.     

The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability

Differential scanning fluorimetry (DSF) is a rapid and inexpensive screening method to identify low-molecular-weight ligands that bind and stabilize purified proteins. The temperature at which a protein unfolds is measured by an increase in the fluorescence of a dye with affinity for hydrophobic parts of the protein, which are exposed as the protein unfolds. A simple fitting procedure allows quick calculation of the transition midpoint; the difference in the temperature of this midpoint in the presence and absence of ligand is related to the binding affinity of the small molecule, which can be a low-molecular-weight compound, a peptide or a nucleic acid. DSF is best performed using a conventional real-time PCR instrument. Ligand solutions from a storage plate are added to a solution of protein and dye, distributed into the wells of the PCR plate and fluorescence intensity measured as the temperature is raised gradually. Results can be obtained in a single day.     

Real-time molecular methods to detect infectious viruses

Waterborne transmitted viruses pose a public health threat due to their stability in aquatic environment and the easy transmission with high morbidity rates at low infectious doses. Two major challenge of virus analysis include a lack of adequate information in infectivity and the inability to cultivate certain epidemiologically important viruses in vitro. The use of fluorescent probes in conjunction with fluorescence microscopy allows us to reveal dynamic interactions of the viruses with different cellular structures in living cells that are impossible to detect by immunological or PCR-based experiments. Real-time viral detection in vivo provides sufficient information regarding multiple steps in infection process at molecular level, which will be valuable for the prevention and control of viral infection.     

预测蛋白质间相互作用的生物信息学方法

后基因组时代的研究模式,已从原来的序列-结构-功能转向基因表达-系统动力学-生理功能。建立蛋白质间相互作用的完全网络,即蛋白质相互作用组(interactome),将有助于从系统角度加深对细胞结构和功能的认识,并为新药靶点的发现和药物设计提供理论基础。一系列系统分析蛋白质相互作用的实验方法已经建立,近年来,出现了多种预测蛋白质相互作用的生物信息学方法,这些方法不仅是对传统实验方法的有价值的补充,而且能够扩展实验方法的预测范围;同时,在开发这些方法的过程中建立了一些重要的分子进化和分子生物学慨念。本文综述了9种生物信息学方法的原理、方法评估、存在的问题．并分析了这个领域的发展前景。     

A yeast one-hybrid system to detect methylation-dependent DNA-protein interactions

We developed a method for site-selective CpG methylation of the budding yeast genome. The method recruits LexA-fused M.SssI DNA methyltransferase to LexA operator sequences integrated adjacent to the target site. Microarray analysis of methylated DNAs indicated that the tethered enzyme selectively methylates the region around the target site. Exploiting this method to methylate bait DNA in the one-hybrid system, we demonstrated methylation-dependent DNA binding of methyl-CpG binding proteins, MBD1 and Kaiso, in vivo. This methylation-dependent one-hybrid system would provide a versatile tool for the search and analysis of proteins that recognize methylated DNA to participate in epigenetic regulation.     

The use of FRET imaging microscopy to detect protein-protein interactions and protein conformational changes in vivo.

Intermolecular and intramolecular FRET between two spectrally overlapping green fluorescent protein variants fused to two different host proteins or at two different sites within the same protein offers a unique opportunity to monitor real-time protein-protein interactions or protein conformational changes. By using fluorescence digital imaging microscopy, one can visualize the location of green fluorescent proteins within a living cell and follow the time course of the changes in FRET corresponding to cellular events at a millisecond time resolution. The observation of such dynamic molecular events in vivo provides vital insight into the action of biological molecules.     

Improved photo-CIDNP methods for studying protein structure and folding

Two new techniques offering considerable improvements in the quality of ¹H photo-CIDNP spectra of proteins are demonstrated. Both focus on the problem of progressive photo-degradation of the flavin dye used to generate polarization in exposed tryptophan, tyrosine and histidine side-chains. One approach uses rapid addition and removal of protein/flavin solution between light flashes to mix the NMR sample and introduce fresh dye into the laser-irradiated region. The other involves chemical oxidation of photo-reduced flavin by the addition of hydrogen peroxide. In both cases a larger number of scans can be accumulated before the flavin is exhausted than would otherwise be possible. The techniques are demonstrated by 600 MHz CIDNP-NOESY spectroscopy of bovine holo--lactalbumin, and by real-time CIDNP observation of the refolding of bovine apo--lactalbumin following rapid dilution from a high concentration of chemical denaturant.     

Chemiluminescent-based methods to detect subpicomole levels of c-type cytochromes

A variety of luminol-based substrates and either an autoradiographic film or a charge-coupled device (CCD) imaging system were used for chemiluminescence detection of c-type cytochromes. The Pierce Femto peroxidase substrate was at least 10 times more sensitive when using film than the highly cited 3,3('),5,5(')-tetramethylbenzidine (benzidine derivative) staining method and 50 times more sensitive when using a CCD imaging system. Film or CCD imaging result in highly sensitive and quantitative signals. The quantitative detection of c-type cytochromes from single colonies or from less than 1ml of a bacterial culture is possible.     

Comparison of methods to detect resistance of Penicillium expansum to thiabendazole

Aims:  Because of the lack of a standard method, the aim of this work is to evaluate the suitability of the broth microdilution method CLSI M38-A in determining the resistance level of some Penicillium expansum isolates to thiabendazole (TBZ). The ability of the isolates to produce patulin (PAT) and citrinin (CIT) has been also assessed.
Methods and Results:  Penicillium expansum isolates (128) were assayed (apples, pears, grapes and five reference strains). It was observed that 69·4% of the strains isolated from apples and pears were resistant to TBZ. Sensitive isolates were inhibited at 0·25–0·5 μg ml⁻¹ whilst resistant isolates still grew at 512 μg ml⁻¹. PAT was produced by all P. expansum isolates. CIT was detected in 98·8% of TBZ-resistant isolates and in 89·1% of the TBZ-sensitive isolates.
Conclusions:  The preliminary screening method combined with the adaptation of the method CLSI M38-A, can be a good strategy to be used in assessing the in vitro activity of TBZ against a large number of isolates.
Significance and Impact of the Study:  The proposed methodology can be a contribution to the standardization of susceptibility tests to fungicides against P. expansum.     

评估蛋白质相互作用可信度的生物信息学方法

随着基因组规模的高通量实验鉴定技术和计算预测方法的发展,出现了大量蛋白质相互作用数据,但大规模蛋白质相互作用数据中的较高比例的假阳性影响了相互作用数据的质量。生物信息学方法能够从已有的数据和知识出发,通过计算方法系统评估大规模蛋白质相互作用的可信度。本文从过程模型设计、数据集构建、特征选择与综合属性抽取、一些算法使用、实例概述等方面介绍了生物信息学方法评估蛋白质相互作用可信度的研究特点与进展。     

Computational methods to detect conserved non-genic elements in phylogenetically isolated genomes: application to zebrafish

Many important model organisms for biomedical and evolutionary research have sequenced genomes, but occupy a phylogenetically isolated position, evolutionarily distant from other sequenced genomes. This phylogenetic isolation is exemplified for zebrafish, a vertebrate model for cis-regulation, development and human disease, whose evolutionary distance to all other currently sequenced fish exceeds the distance between human and chicken. Such large distances make it difficult to align genomes and use them for comparative analysis beyond gene-focused questions. In particular, detecting conserved non-genic elements (CNEs) as promising cis-regulatory elements with biological importance is challenging. Here, we develop a general comparative genomics framework to align isolated genomes and to comprehensively detect CNEs. Our approach integrates highly sensitive and quality-controlled local alignments and uses alignment transitivity and ancestral reconstruction to bridge large evolutionary distances. We apply our framework to zebrafish and demonstrate substantially improved CNE detection and quality compared with previous sets. Our zebrafish CNE set comprises 54 533 CNEs, of which 11 792 (22%) are conserved to human or mouse. Our zebrafish CNEs (http://zebrafish.stanford.edu) are highly enriched in known enhancers and extend existing experimental (ChIP-Seq) sets. The same framework can now be applied to the isolated genomes of frog, amphioxus, Caenorhabditis elegans and many others.     

Log-linear model-based multifactor dimensionality reduction method to detect gene gene interactions

MOTIVATION: The identification and characterization of susceptibility genes that influence the risk of common and complex diseases remains a statistical and computational challenge in genetic association studies. This is partly because the effect of any single genetic variant for a common and complex disease may be dependent on other genetic variants (gene-gene interaction) and environmental factors (gene-environment interaction). To address this problem, the multifactor dimensionality reduction (MDR) method has been proposed by Ritchie et al. to detect gene-gene interactions or gene-environment interactions. The MDR method identifies polymorphism combinations associated with the common and complex multifactorial diseases by collapsing high-dimensional genetic factors into a single dimension. That is, the MDR method classifies the combination of multilocus genotypes into high-risk and low-risk groups based on a comparison of the ratios of the numbers of cases and controls. When a high-order interaction model is considered with multi-dimensional factors, however, there may be many sparse or empty cells in the contingency tables. The MDR method cannot classify an empty cell as high risk or low risk and leaves it as undetermined. RESULTS: In this article, we propose the log-linear model-based multifactor dimensionality reduction (LM MDR) method to improve the MDR in classifying sparse or empty cells. The LM MDR method estimates frequencies for empty cells from a parsimonious log-linear model so that they can be assigned to high-and low-risk groups. In addition, LM MDR includes MDR as a special case when the saturated log-linear model is fitted. Simulation studies show that the LM MDR method has greater power and smaller error rates than the MDR method. The LM MDR method is also compared with the MDR method using as an example sporadic Alzheimer's disease.