Methods in molecular medicine最新文献

Aminoglycoside antibiotics are highly potent, wide-spectrum bactericidals (1, 2). Bacterial resistance to aminoglycosides, however, is a major problem in the clinical use of aminoglycosides. Enzymatic modification of aminoglycosides is the most frequent resistance mode among several resistance mechanisms employed by resistant pathogens (1,3). Three families of aminoglycoside modifying enzymes, O-phosphotransferases, N-acetyltransferases, and N-nucleotidyltransferases, are known to have more than 50 enzymes (1,3,4). In this chapter, determination of enzymatic activity of a single enzyme from each family in the presence and absence of an inhibitor is described.

eConsulting, in all its contexts, can promote and improve the amount and quality of services and knowledge transferred to and among the community of health care providers and consumers. It can also improve the efficiency and effectiveness of the specialist and generalist workforce and accessibility to the services provided. This chapter defines eConsulting, provides the context, and introduces a conceptual framework to describe its current practice and future possibilities. A clinical scenario of a patient with a breast lump is used to ground the molecular, clinical, organizational, and social, legal, and ethical issues in real world practice. The approach/method used is based on the clinical process, evidence-based practice, and appraising the quality, validity, relevance, and usefulness of the information. The practicalities and utility of current eConsulting tools are discussed with a view to future ubiquitous use. Working through this chapter should assist readers to understand and describe (1) how eConsultations can link and translate scientific research into clinical practice, (2) the current implications of eConsultations, (3) the future potential of eConsultations.

Increasingly, the molecular genetics laboratory has to assess the biological significance of changes (variants) in a DNA sequence. Using the large genes BRCA1 and BRCA2 as examples, some approaches used to determine the biological significance of DNA variants are described. These include the characterization of the variant through a review of the literature and the various databases to assess if it has previously been described. Potential difficulties with the various databases that are available are described. Other considerations include the co-inheritance of the variant with other DNA changes, and its evolutionary conservation. Determining the possible effect of the variant on protein function is described in terms of the Grantham assessment as well as identifying functional domains. Studies looking at the distribution of the variant in both the population and the family can also help in assessing its significance. Loss of the variant in a tumor sample would imply that it is not deleterious. Ultimately, it is not any single parameter that helps determine the DNA variants biological significance. Usually this requires multiple lines of evidence.

Disease- and locus-specific variant databases have been a valuable resource to clinical and research geneticists. With the recent rapid developments in technologies, the number of DNA variants detected in a typical molecular genetics laboratory easily exceeds 1,000. To keep track of the growing inventory of DNA variants, many laboratories employ information technology to store the data as well as distributing the data and its associated information to clinicians and researchers via the Web. While it is a valuable resource, the hosting of a web-accessible database requires collaboration between bioinformaticians and biologists and careful planning to ensure its usability and availability. In this chapter, a series of tutorials on building a local DNA variant database out of a sample dataset will be provided. However, this tutorial will not include programming details on building a web interface and on constructing the web application necessary for web hosting. Instead, an introduction to the two commonly used methods for hosting web-accessible variant databases will be described. Apart from the tutorials, this chapter will also consider the resources and planning required for making a variant database project successful.

Under normal conditions, airborne allergens are present at very low concentrations. Allergens may be carried on relatively large identifiable particles such as grains of pollen and mould spores or smaller amorphous particles or both. The methods that have been applied to quantify animal airborne allergens will be described in this chapter. By careful selection of the air sampling equipment and conditions, samples can be collected which quantify, for example, the personal exposure of an individual when performing a specific task or changes in exposure when allergen control methods are implemented. If as with animal allergens, an airborne allergen is not comprised of identifiable microscopic fragments, it is necessary to extract the soluble allergen for quantification in a specific immunoassay. The basic methods used for the elution of animal allergen from polytetrafluoroethylene (PTFE) filters will be described. The optimization of this method to suit different allergens and the influence of the buffer on extraction efficiency and stability of the allergen during storage will also be discussed.

T-cell receptors (TcR) recognize short linear peptides (9-15 amino acid long), which have been processed by an 'antigen-presenting cell' and complexed to products of the major histocompatibility complex (MHC). Peptides of the appropriate shape and charge are able to bind within the groove of the MHC molecule and it is this complex which is recognized by the TcR. The MHC molecules are highly polymorphic, but each individual will only express one maternal and one paternal allele of HLA Class 1 molecule A, B, and C and HLA Class II DR, DP, and DQ. As a result of TcR specificity and MHC restriction, a clone of T cells will bear a specific receptor which has a very limited repertoire of targets (peptide-MHC complexes). When sufficient numbers of TcR are engaged on a T-cell surface, a cascade of signal transduction events is initiated which results in cellular activation. When investigating the immune response, it may be advantageous to be able to identify which MHC molecules bind a particular peptide well and give rise to a vigorous T-cell response. Such information may be useful in generating effective vaccines. In this chapter, we provide examples of Epstein-Barr virus-transformed lymphoid cell lines and fibroblast cell lines to determine MHC restriction elements.

The emergence of bacterial pathogens resistant to current antibiotics has caused an urgent demand for new treatments. Peptide deformylase (PDF) has become an exciting target for designing novel antibiotics. To facilitate the screening of PDF inhibitors, three robust, coupled assays have been developed. The first method couples the PDF reaction with that of formate dehydrogenase. Formate dehydrogenase oxidizes formate into CO2 with a concomitant reduction of NAD+ to NADH, which can be monitored spectrophotometrically. The second method involves Aeromonas aminopeptidase (AAP) as the coupling enzyme and an artificial substrate, f-Met-Leu-p-nitroanilide. The sequential action of PDF and AAP releases p-nitroanilide as a highly chromogenic product. In the third method, f-Met-Lys-7-amino-4-methylcoumarin is used as the substrate. Deformylation by PDF gives an excellent substrate for dipeptidyl peptidase I, which releases the dipeptide Met-Lys and fluorogenic 7-amino-4-methylcoumarin. The combination of these assay methods should meet the needs of most laboratories.

Widespread resistance to antibiotics in current clinical use is increasing at an alarming rate. Novel approaches in antimicrobial therapy will be required in the near future to maintain control of infectious diseases. An enormous array of small cationic peptides exists in nature as part of the innate defense systems of organisms ranging from bacteria to humans. For most naturally occurring linear peptides, such as magainins and cecropins, a common feature is their capacity to form an amphipathic alpha-helix (with polar and nonpolar groups on opposite faces of the helix), a structural feature believed to be important in their antimicrobial function as membrane-lytic agents. A massive effort over the past two decades has resulted in a better understanding of the molecular mechanism of antimicrobial peptides and the production of more potent analogues. To date, however, few of these peptides have been shown to have clinical efficacy, especially for systemic use, in large part due to insufficient selectivity between target and host cells. Recently, we developed a new strategy in the design of antimicrobial peptides. These linear cationic peptides, which form amphipathic beta-sheets rather than alpha-helices, demonstrated superior selectivity in binding to the lipids contained in bacterial vs. mammalian plasma membranes. Here we describe methods to evaluate the structure and function of cationic antimicrobial peptides.

Bacterial signal transduction systems can be used as drug targets. The signal transduction targets fall into two groups--sensor kinases and response regulators. Previously reported studies describe hits that were thought to inactivate sensor kinases but on closer examination were found to act elsewhere instead; a possible reason for this is that full-length sensor kinases are integral membrane proteins whose activity might reflect interaction with the cell membrane or with membrane components. We describe a model system that instead is based on the interaction between a test compound and a response regulator in a homogeneous phase reaction. In this system, response regulator-DNA complex formation and its inhibition by a test compound are measured by fluorescence polarization. The model system should be readily adaptable to drug discovery based on other bacterial two-component s transduction systems.