Methods in molecular medicine最新文献

Dendritic cells (DC) are widely considered to be the major antigen-presenting cell (APC) type in immune responses. These cells are obtained from adherent cells or are purified CD14(+) monocytes from peripheral blood mononuclear cells (PBMC) by in vitro stimulation with granulocyte, macrophage-colony-stimulating factor (GM-CSF) plus interleukin (IL)-4. They express high levels of MHC class II and costimulatory molecules, internalize Ag rapidly via Fc receptors and mannose receptors, and, by macropinocytosis, produce large amounts of IL-12 on CD40 ligation, and are potent in presenting soluble Ag and in stimulating allogeneic mixed-leukocyte reactions. To study primary T-cell responses and cytokine production in allergy patients, we have developed an in vitro system by using highly purified T cells as responder cells and monocyte-derived DC (MDC) as the APC. MDC provide a convenient and potent APC source for T-cell response studies.

In Escherichia coli, the molecular chaperone HSP70 (DnaK) is necessary for 30S and 50S ribosomal subunit assembly at temperatures above 37 degrees C. Inhibitors of DnaK should therefore hinder ribosome biogenesis, in addition to all of the other DnaK-dependent cellular functions. An easily testable phenotype of DnaK is described here based on alpha-complementation of beta-galactosidase. This protein fragment complementation requires a functional DnaK in vivo, offering a suitable method for screening for DnaK inhibitors. Subsequently, it will be of great importance to check whether inhibitors of bacterial DnaK selected in this way have an effect (inhibitory or stimulatory) on the activities of eukaryotic HSP70 and HSC70 chaperones, because of the universal conservation in all biota of these chaperones in both their structural and functional properties. This question is important due to their implication in many pathways in immunology, cancer biology, and neurodegenerative disorders.

Key enzymes that assemble the bacterial cell wall are also the target of the Beta-lactam class of antibiotics. The covalent binding of labeled penicillin to these proteins has been used in numerous studies in drug discovery, antibiotic mechanisms of action and resistance, and cell wall physiology. Methods to label and measure penicillin binding proteins in two prototypical organisms, a Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus), are described. The methods discussed include identifying penicillin-binding proteins in both intact cells (in vivo measurements) and isolated cell membranes.

Infections caused by multidrug-resistant Gram-negative pathogens play a major role in the morbidity and mortality of hospitalized patients. The rise of resistance to current antibiotic therapies has made the discovery of new agents urgent. One of the major antibiotic resistance mechanisms utilized by more than 15 species of Gram-negative bacterial cells is the Resistance Nodulation Division (RND) efflux pump, which eliminates several classes of antibiotics such as penicillins and cephalosporin macrolides aminoglycosides, fluoroquinolonesx and tetracyclines. Here we describe a multistep process to identify compounds that inhibit the RND-type efflux pumps. This involves measuring the inhibition of accumulation of ethidium bromide in E. coli or Haemophilus influenzae cells and confirming that the inhibition is specific for the efflux pumps by using genetic constructs and biochemical methods to measure nonspecific inhibition due to e.g. intrinsic antibacterial activity or membrane disruption. In whole bacterial cells synergism antagonism or indifference of the combination of an antibiotic with the putative inhibitor is determined and this is then confirmed by quantitating viable bacterial cells in liquid culture over 24 h.

Fatty acid biosynthesis is one of the relatively newer targets in antibacterial drug discovery. The presence of distinct fatty acid synthases (FAS) in mammals and bacteria and the fact that most bacterial FAS enzymes are essential for viability make this a very attractive antimicrobial drug target. The enzyme beta-ketoacyl ACP synthase (KASIII or FabH) is the key enzyme that initiates fatty acid biosynthesis in a type II dissociated FAS. This enzyme catalyzes the condensation of acyl CoA and malonyl ACP (acyl carrier protein) to form a beta-ketoacyl ACP product, which is further processed to form mature fatty acids that are involved in various essential cellular processes and structures like phospholipid biosynthesis, cell wall formation, etc. Herein we describe a new assay for the Mycobacterium tuberculosis FabH (mtFabH) enzyme involved in a key initiation step in the synthesis of mycolic acids, which are an integral component of the cell wall. The assay eliminates the need for the cumbersome washing steps or specialty scintillation proximity assay beads and the preparation of acyl carrier proteins required in other assay formats. This discontinuous assay involves the reduction of radiolabled long-chain beta-ketoacyl CoA product to its dihydroxy derivative, which partitions into a nonpolar phase for quantitation, while the reduced radiolabeled substrate derivative remains in the aqueous phase.

The enteric adenoviruses of subgroup F (Ad40 and Ad41) pose some special problems of cultivation, as they cannot be readily passaged in many of the cell types used to propagate the more commonly used subgroup C serotypes (Ad2 and Ad5) and there is no standard plaque assay. Methods to propagate Ad40 in complementing cell lines and to evaluate infectivity and particle number are presented in this chapter.

The adenovirus major late transcription unit (MLTU) encodes the main structural capsid proteins. Expression from the MLTU is accomplished through alternative mRNA processing and use of a terminal exon coding strategy. The capsid proteins hexon, penton, and fiber contribute to efficient infection by adenovirus, and each contributes in some manner to the antiviral immune response against adenovirus infection. The ability to manipulate these genes affords one the opportunity to "detarget" adenovirus, to retarget adenovirus, and to alter immune recognition. In this chapter, we are presenting a terminal exon-replacement strategy that can be used to genetically manipulate capsid proteins expressed from the MLTU. An emphasis will be placed on manipulations of fiber as an intact terminal exon.

Gene expression profiling using microarrays is a powerful method for studying the biology of hematopoietic stem cells (HSCs). Here, we present methods for activating HSCs with the chemotherapeutic drug 5-Fluorouracil, isolating HSCs from whole bone marrow, and performing microarray analysis. We also discuss quality control criteria for identifying good arrays and bioinformatics strategies for analyzing them. Using these methods, we have characterized the gene expression signatures of HSC quiescence and proliferation and have constructed a molecular model of HSC activation and self-renewal.

For donor selection in hematopoetic stem cell transplantation, two-digit sequencespecific oligonucleotide (SSO) typing may be sufficient in the related sibling transplant setting. However, SSO typing is not sensitive enough to differentiate between the alleles that share the same cis-trans linkeage sequence casettes. In unrelated donor selection, PCR using sequence-specific primers, a flexible and widely used method known to cause less ambiguious results, may be preferable. However, this technique is limited by the number of the samples that can be processed at one time and also by the number of the primer mixes that can be utilized.

Stem cell migration/trafficking is a field of interest that is shared by pathologists, histologists, clinical transplantation teams, cardiologists, neurologists, and many other members of different disciplines. Until the findings of a successful combination of in situ methods, the origin of chimeric parenchymal cells was a dilemma. These doublelabeling techniques have brought insight to our new concept of stem cell biology. It has been extremely helpful in the detection of the origin of terminally differentiated, including hematopoietic and nonhematopoietic, cells appearing following allogeneic stem cell transplantation. It has also become a standard approach for evaluation of repopulation following tissue injury in solid organ transplant patients or experimental models. Although very useful, this technique has its advantages and pitfalls. It requires expertise in application and interpretation. Suitable selection of specific markers against parenchymal cells and preferably a cocktail of antibodies targeting infiltrating inflammatory cells are mandatory. One pitfall of this method is its restriction to sex-mismatched pairs. The spectrum of labels for X and Y chromosomes are suitable for combination. To prevent misinterpretation, the precautions needed are defined in this chapter.