Methods in molecular medicine最新文献

Allergy and asthma are characterized by airway hyperresponsiveness and chronic mucosal inflammation mediated by CD4+ Th2 lymphocytes and their cytokines. It is unclear why allergic individuals make a Th2-type T-cell response whereas other (non-allergic) individuals do not. Recently, attention has focused on regulatory mechanisms, such as T-regulatory cells, preventing IgE responses to allergens in nonatopic individuals. Regulatory CD4+CD25+ T cells have been described in both mice and humans. The suppressive phenotype of these cells has been associated with the expression of the forkhead transcription factor, Foxp3. It has been suggested that allergic disease may arise from an inappropriate balance between allergen activation of regulatory CD4+CD25+ T cells and effector Th2 cells or from the impairment in the suppressive activity of these so-called T-regulatory cells. The isolation of these T-regulatory cells is described in order to further our understanding of the role of these cells in allergic disease and asthma and allow us to design novel therapies.

RNA polymerase is essential to the viability of bacteria in all phases of growth and development and is a proven chemotherapeutic target as the cellular target of the rifamycin class of antibiotics. However, despite the characterization of multiple different classes of natural products that selectively target bacterial RNA polymerase, and the identification of a limited number of synthetic compound inhibitors, only agents of the rifamycin class have been developed and approved for human clinical use as antibiotics. Herein we describe a scintillation proximity assay (SPA) for identifying and characterizing inhibitors of bacterial RNA polymerases and that is applicable to de novo drug discovery programs through application of automated high-throughput screening methods. In addition, we describe gel electrophoresis-based methods that are applicable to the detailed characterization of inhibitors of transcriptional initiation or elongation by bacterial RNA polymerases.

Advancements in our understanding of variation in the human genome and rapid improvements in high-throughput genotyping technology have made it feasible to study most of the human genetic diversity that is due to common variations in relation to observable phenotypes. Over the past few years, public SNP databases have matured and empirical genome-wide SNP data, such as that generated by the International HapMap Project, have shown the utility and efficiency of selecting and testing informative markers ("tag SNPs") that exploit redundancies among nearby polymorphisms due to linkage disequilibrium (LD). In this chapter, we will demonstrate how to use the HapMap resource and the Haploview program to process and analyze genetic data from HapMap, to evaluate LD relations between SNPs, and to select tagging SNPs to be examined in disease association studies.

Standardized methods for the solubilization of proteins prior to proteomics analyses incorporating two-dimensional gel electrophoresis (2-DE) are essential for providing reproducible data that can be subjected to rigorous statistical interrogation for comparative studies investigating disease-genesis. In this chapter, we discuss the imaging and image analysis of proteins separated by 2-DE, in the context of determining protein abundance alterations related to a change in biochemical or biophysical conditions. We then describe the principles behind 2-DE gel statistical analysis, including subtraction of background noise, spot detection, gel matching, spot quantitation for data comparison, and statistical requirements to create meaningful gel data sets. We also emphasize the need to develop reproducible and robust protocols for protein sample preparation and 2-DE itself.

Immunoelectrophoresis can be used for analysis of individual proteins in complex mixtures. The conditions involved in immunoelectrophoresis are mild, avoiding the risk of denaturation, and it is possible to perform relative quantification of individual components. The principle disadvantage is the dependence on rabbit antisera as reagents. The usefulness of immunoelectrophoresis in allergy research is greatly enhanced by the possibility of identification of allergens to which the individual in question has IgE. The common principle is characterized by two independent electrophoreses having direction of current perpendicular to each other, i.e., crossed immunoelectrophoresis (CIE). This ultimately results in the formation of characteristic bell-shaped precipitates, each precipitate representing one antigen. There is a linear relationship between the amount of antigen and size of precipitate for a given antibody concentration for each precipitate and so relative quantification can be performed. The sensitivity and resolution power of CIE is very high and there are multiple variations of the technique, some of which will be illustrated in this chapter.

There is currently considerable interest in the role of specific IgG antibodies in allergy. Several studies suggest that specific IgG antibodies may play a protective role in allergy. Successful immunotherapy is associated with increases in allergen-specific IgG antibodies which correlate with clinical outcome. Other studies have identified an inverse relationship between exposure to cat and sensitization, which was associated with high titer specific IgG and IgG(4). This immune response was described as a modified Th2 response, because both IgE and IgG(4) require Th2 cytokine IL-4 for their production. A modified Th2 response was described with laboratory animal allergy, where there was almost a twofold reduction in the risk of developing work-related chest symptoms.In this chapter, we review the major factors to be considered in the development of an ELISA for the determination of specific IgG and IgG(4) antibodies.

The formation of peptide bonds is the central chemical reaction during protein synthesis and is catalyzed by the peptidyl transferase center residing in the large ribosomal subunit. This active site is composed of universally conserved rRNA nucleosides. The peptidyl transferase center is by far the most frequently used target site of natural antibiotics in the cell. Here we describe a novel, simple, and convenient method to assess peptide bond formation which we named SPARK. The basic principle of SPARK is the use of two reaction substrates that closely resemble the natural tRNA substrates (one is biotinylated and the other carries a tritium label) that become covalently connected during transpeptidation. Formation of this peptide bond then allows capture and direct quantification of the radiolabled product, now joined to the biotin group, using the scintillation proximity assay technology. Binding of the tritiated radioligand to streptavidin-coated beads causes the excitation of the bead-embedded scintillant, thus resulting in the detection of radioactivity. Since no product purification step is required, SPARK is amenable to simple automation, which makes it useful in high-throughput screens of natural or synthetic compound libraries in the search for novel antibiotics.

Perturbations in genes play a key role in the pathogenesis of cancer. Microarray-based technology is an ideal way in which to study the effects and interactions of multiple genes in cancer. There are many technologic challenges in running a microarray study, including annotation of genes likely to be involved, designing the appropriate experiment, and ensuring adequate quality assurance steps are implemented. Once data are normalized, they need to be analyzed; and for this, there are numerous software packages and approaches.

DNA gyrase and DNA topoisomerase (topo) IV are the bacterial targets of coumarin and quinolone antimicrobial agents. Widespread resistance to clinically important antibiotics such as beta-lactams and macrolides has stimulated the development of novel gyrase and topo IV inhibitors especially against Streptococcus pneumoniae and other Gram-positive pathogens. Here, we describe how gyrase and topo IV activities are measured and how inhibitors of these enzymes may be assayed, focusing as a paradigm on DNA supercoiling by S. pneumoniae gyrase, DNA decatenation by S. pneumoniae topo IV, and DNA cleavage by both enzymes. These approaches provide mechanistic insight on inhibitor action and allow identification of dual gyrase/topo IV targeting agents that can minimize the emergence of bacterial resistance.

This chapter describes reliable flow cytometric methods for assessment of two important physiologic characteristics of bacteria, membrane potential and membrane permeability, which can provide indications of the effects of antimicrobial agents on microorganisms.