Methods in molecular medicine最新文献

DNA sequencing is increasingly used in a range of medical activities involving DNA diagnostics and research. This is the result of improving technology and cheaper costs. Paradoxically, a greater demand for DNA sequencing has placed additional work on the laboratory because sequencing profiles must be checked visually despite the availability of informatics-based tools in interpreting DNA sequence traces. In this environment it is essential to have more sophisticated software that will allow the sites of known and unknown DNA variants to be quickly identified, as well as providing an objective assessment of quality for the DNA sequence generated. This chapter describes the Applied Biosystems SeqScape software program (version 2.5) and how it has assisted in the interpretation of DNA sequencing in a DNA diagnostic laboratory.

The inhibition of bacterial ribosomal subunit formation is a novel target for translational inhibitors. Inhibition of subunit biogenesis has been shown to be equivalent to the inhibition of protein biosynthesis for many antibiotics. This chapter describes three methods for examining the inhibition of subunit formation in growing bacterial cells. The first method permits the determination of the IC50 value for inhibition of assembly and protein synthesis. The second is a pulse and chase labeling procedure to measure the kinetics of subunit formation. The third procedure allows an examination of ribosome reformation after antibiotic removal as a part of the post-antibiotic effect. Together these procedures give a description of the relative inhibitory effects of an antibiotic on translation and subunit formation.

While bacterial protein synthesis is the target of about half of the known antibiotics, the great structural-functional complexity of the translational machinery still offers remarkable opportunities for identifying novel and specific inhibitors of unexploited targets. We designed a knowledge-based in vitro translation assay to identify inhibitors selectively targeting the bacterial or the yeast translational apparatus, preferentially blocking the early steps of protein synthesis. Using a natural-like, "universal" model mRNA and cell-free extracts prepared from Eschericha coli, Saccharomyces cerevisiae, and HeLa cells, we were able to translate, with comparable yields in the three systems, the immunogenic peptide encoded by this "universal" mRNA. The immuno-enzymatic quantification of the translated peptide in the presence of a potential inhibitor can identify a selective bacterial or fungal inhibitor inactive in the human system. When applied to the high-throughput screening (HTS) of a library of approximately 25,000 natural products, this assay led to the identification of two novel and specific inhibitors of bacterial translation.

Hybridization is the formation of hybrid nucleic acid molecules with complementary nucleotide sequences in DNA:DNA, DNA:RNA, or RNA:RNA forms. In situ hybridization is a highly sensitive technique that allows detection and localization of specific DNA or RNA molecules in morphologically preserved isolated cells, histological tissue sections, or chromosome preparations. In situ hybridization has broad range of applications and has been used to (a) localize viral infection, (b) identify sites of gene expression, (c) analyze mRNA transcription and tissue distribution, and (d) map gene sequences in chromosomes. There are several advantages of the use of in situ hybridization including the fact that it can be applied to archival materials and frozen tissues and can be combined with immunohistochemistry to detect protein as well as mRNA of interest or phenotype of cells expressing the target genome, detecting more than one nucleic acid sequences using different labeling methods.The major steps involved in in situ hybridization are as follows: probe preparation and labeling, tissue fixation, permeabilization, hybridization, and signal detection and these are described in detail in this chapter.

ELISAs offer excellent specificity and, once fully optimized, sensitivity that rivals that of bioassays. The major variables that need to be experimentally determined when developing an ELISA are the optimal number of fresh cells required per well, the optimal antigen concentrations for stimulation, period of culture, and the anticipated intensity of the response. In this chapter, we review the major factors to be considered in the development and application of ultrasensitive ELISAs to the analysis of human immune responses. We specify the conditions we have found to be optimal for quantifying a number of cytokines of demonstrated relevance to human immune regulation and discuss the major pitfalls inherent in this approach.

Chemokines are primarily low molecular mass proteins that are produced and usually released by a wide variety of cell types. Differential chemokine responses can be excellent early markers of immune dysfunction, allowing clinical intervention prior to expression of full blown undesirable effector responses. Thus, assessment of the nature and intensity of Ag-dependent chemokine production provides a valuable tool for probing human immune regulation.Here, we provide detailed instructions on approaches we have developed to assess the nature and intensity of recall responses to a wide variety of exogenous and endogenous antigens capable of consistently stimulating chemokine responses by PBMC from adult and pediatric populations. This chapter is divided into two sections. The first is focused on culture techniques for eliciting antigen-driven chemokine responses for a panel of chemokines that are relevant to immune function. The second section details assay systems for their quantitative analysis.

Mast cells generate mediators of inflammation which are stored in granules and secreted on activation either by allergen crosslinking of membrane-bound IgE or through other stimuli. Most methods for mast cell identification rely on the histochemical detection of constituents of the secretory granules. Although staining for mast cells with histochemical stains can be rapid and relatively inexpensive, it is not always possible to distinguish reliably between mast cells and basophils in tissues. A further problem with the staining of mast cells with commonly used basic dyes is that the reagents employed to fix the tissues can influence the results, leading to confusion regarding the numbers of mast cells present in various tissues. Recognition that there is considerable heterogeneity between mast cell populations in the degree to which staining properties are lost with formalin fixation has led to mast cell subsets being defined on this basis. The development and application of procedures for identifying mast cell proteases has led to important advances in our understanding of the role of mast cells and in the nature of heterogeneity in man. The techniques described here should allow the reliable detection of mast cells and mast cell subsets in a range of tissues and cell preparations. There will be a continuing need for validation, for consideration of potential sources of error, and for the development of new and more reliable techniques for mast cell identification.

Mast cells are key effector cells of the allergic response. When stimulated by specific allergen through the high-affinity IgE receptors or through other stimuli, these cells release a number of potent mediators of inflammation. Amongst these are the serine proteases tryptase and chymase. In humans, tryptase is the most abundant mediator stored in mast cells. Chymase is present in more moderate amounts in a subpopulation of mast cells (MC(TC)). This subtype of mast cells predominates in connective tissue, whereas the other major subtype, the MC(T), predominates in mucosal tissue. Both proteases have been shown to act on specific extracellular proteins and peptides, as well as to alter the behavior of various cell types. Inhibitors of tryptase have been found to be efficacious in animal and human models of asthma, and both proteases are currently being investigated as potential targets for therapeutic intervention. Such pharmacological, physiological, and biochemical studies require the availability of purified tryptase and chymase. In this chapter, we shall describe procedures for the purification of tryptase and chymase from human tissues and provide protocols for monitoring purification and characterization of the final product. The preparation of recombinant proteases will not be covered, though some of the procedures described may be readily adapted for their purification from recombinant expression systems. The procedures described here have been developed for the purification of the human proteases and will require some modification if applied to purify mast cell proteases from the tissues of other species.

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.