Springer protocols feed by ImmunologyThis is an RSS file. You can use it to subscribe to this data in your favourite RSS reader, such as GoogleReader, or to display this data on your own website or blog. Subscribe to this data using MyMedWorm.Subscribe to this data using GoogleReader.Subscribe to this data using Bloglines.Subscribe to this data using MyYahoo.

This page shows you the latest items in this publication.

Analysis of Frequency and Phenotype of Antigen-Specific T CellsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Over the last decade, our understanding of the cellular immune system has been greatly advanced through the development of methods to identify antigen-specific T cells directly ex vivo. The major reagents and techniques used for this purpose are (i) tetramerised MHC:peptide complexes (tetramers) which bind to specific T-cell receptors (TCR) and (ii) assays that detect T cells which synthesise cytokines in response to cognate stimulation (intracellular cytokine staining (ICS)). Here, we provide a detailed description of the procedure for generating and using class I MHC:peptide tetramers to label peptide-specific T cells an...
Source: Springer protocols feed by Immunology - July 1, 2008 Category: Allergy & Immunology Source Type: info

Purification of the T Cell Antigen Receptor and Analysis by Blue-Native PAGEEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We present here four methods to isolate the TCR in a native form and details to analyse it by BN-PAGE.
Source: Springer protocols feed by Immunology - July 1, 2008 Category: Allergy & Immunology Source Type: info

Non-Replicating Recombinant Vaccinia Virus Expressing CD80 to Enhance T-Cell StimulationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The following method describes the generation of a recombinant vaccinia virus expressing a costimulatory molecule (human CD80 or B7.1).
Source: Springer protocols feed by Immunology - July 1, 2008 Category: Allergy & Immunology Source Type: info

B Cell Helper AssaysEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Activation, proliferation and differentiation of naïve B lymphocytes into memory B cells and plasma cells requires engagement of the B cell receptor (BCR) coupled to T-cell help (1, 2). T cells deliver help in cognate fashion when they are activated upon recognition of specific MHC–peptide complexes presented by B cells. T cells can also deliver help in a non-cognate or bystander fashion, when they do not find specific MHC–peptide complexes on B cells and are activated by alternative mechanisms. T-cell dependent activation of B cells can be studied in vitro by experimental models called “B cell helpe...
Source: Springer protocols feed by Immunology - July 1, 2008 Category: Allergy & Immunology Source Type: info

transkingdom RNA Interference (tkRNAi): A Novel Method to Induce Therapeutic Gene SilencingEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
RNA interference is a phenomenon in which specific, endogenous genes are silenced by mRNA degradation. This technology is highly regarded as a potential therapeutic due to its high efficacy and low toxicity. However, the difficulty of delivering RNAi to target cells has impeded the development of RNAi-based therapies. One method to overcome this barrier is the use of a nonpathogenic bacteria vector, Escherichia coli, to deliver RNAi to target cells with high efficacy. In transkingdom interference RNAi (tkRNAi) delivery, E. coli were engineered to transcribe short RNA (shRNA) from a plasmid (TRIP) containing the invasin gen...
Source: Springer protocols feed by Immunology - July 1, 2008 Category: Allergy & Immunology Source Type: info

Flow Cytometry and Cell ActivationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Flow cytometry is combined with highly specific fluorophore-conjugated antibodies that will only bind to the activated forms of molecules. The advances in flow cytometry enable to perform quantitative multiplexed analysis of single cells within heterogeneous populations stained with specific antibodies for phenotyping in conjunction with antibodies to phosphorylated, i.e., activated molecules within signaling pathways. By reactivating signaling pathways in vitro it is possible to collect data on the responsive state of complex cell populations such as immune cells. In this protocol, peripheral blood mononuclear cells (PBMC...
Source: Springer protocols feed by Immunology - July 1, 2008 Category: Allergy & Immunology Source Type: info

Investigating T Cells by Polychromatic Flow CytometryEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Since its development, flow cytometry gave a relevant contribution to the field of Immunology. Its unique potential to analyse multiple parameters at the single cell level allowed the identification of unknown cell subsets with specific roles in immunoregulation as well as in the pathogenesis of several diseases. More recently, with the advent of new equipments and fluorochromes, the possibility exists to analyse simultaneously a large number (up to 19) of parameters in a single cell. This strategy, defined polychromatic flow cytometry (PFC), has been widely utilised in the last years for the fine analysis of immune cell p...
Source: Springer protocols feed by Immunology - July 1, 2008 Category: Allergy & Immunology Source Type: info

Generation of Human T Cell ClonesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Peripheral blood T lymphocytes are a pool of cells with extremely different characteristics and, therefore, it may be difficult to obtain clear-cut results and to attribute a certain function to a defined T cell population in several experimental settings. The availability of a population of human T lymphocytes deriving from the same progenitor with a unique phenotype and function (clone) may therefore be of help.
Source: Springer protocols feed by Immunology - July 1, 2008 Category: Allergy & Immunology Source Type: info

Limiting Dilution Analysis of Antigen-Specific T CellsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe here an example of LDA experiment assessing antigen-specific T cell proliferation of microcultures in the presence or absence of adjuvant and illustrate how to estimate the frequencies of precursor T cells using an online tool that we made publicly available.
Source: Springer protocols feed by Immunology - July 1, 2008 Category: Allergy & Immunology Source Type: info

T Cell Epitope-Mapping by Cytokine Gene Expression AssayEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The following method describes the identification of candidate immunogenic peptides through their ability to recall an immune T-cell activation from peripheral blood mononuclear cells (PBMCs) of individuals with defined HLA–peptide restrictions that have been previously exposed to the antigen. Isolated PBMCs are plated out at a concentration of 1×106 cells/ml in a 200 μl medium and incubated overnight to reduce cytokine gene expression due to cell manipulation. After starving, cells are either directly stimulated with individual peptides or not stimulated and incubated from 3 to 12 h according to e...
Source: Springer protocols feed by Immunology - July 1, 2008 Category: Allergy & Immunology Source Type: info

Cytokine Multiplex Immunoassay: Methodology and (Clinical) ApplicationsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Subsets of T cells can be distinguished on basis of their cytokine production and secretion profile.
Source: Springer protocols feed by Immunology - July 1, 2008 Category: Allergy & Immunology Source Type: info

0.0Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Over the last decade, our understanding of the cellular immune system has been greatly advanced through the development of methods to identify antigen-specific T cells directly ex vivo. The major reagents and techniques used for this purpose are (i) tetramerised MHC:peptide complexes (tetramers) which bind to specific T-cell receptors (TCR) and (ii) assays that detect T cells which synthesise cytokines in response to cognate stimulation (intracellular cytokine staining (ICS)). Here, we provide a detailed description of the procedure for generating and using class I MHC:peptide tetramers to label peptide-specific T cells an...
Source: Springer protocols feed by Immunology - July 1, 2008 Category: Allergy & Immunology Source Type: info

0.0Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We present here four methods to isolate the TCR in a native form and details to analyse it by BN-PAGE.
Source: Springer protocols feed by Immunology - July 1, 2008 Category: Allergy & Immunology Source Type: info

0.0Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The following method describes the generation of a recombinant vaccinia virus expressing a costimulatory molecule (human CD80 or B7.1).
Source: Springer protocols feed by Immunology - July 1, 2008 Category: Allergy & Immunology Source Type: info

0.0Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Activation, proliferation and differentiation of naïve B lymphocytes into memory B cells and plasma cells requires engagement of the B cell receptor (BCR) coupled to T-cell help (1, 2). T cells deliver help in cognate fashion when they are activated upon recognition of specific MHC–peptide complexes presented by B cells. T cells can also deliver help in a non-cognate or bystander fashion, when they do not find specific MHC–peptide complexes on B cells and are activated by alternative mechanisms. T-cell dependent activation of B cells can be studied in vitro by experimental models called “B cell helpe...
Source: Springer protocols feed by Immunology - July 1, 2008 Category: Allergy & Immunology Source Type: info

0.0Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
RNA interference is a phenomenon in which specific, endogenous genes are silenced by mRNA degradation. This technology is highly regarded as a potential therapeutic due to its high efficacy and low toxicity. However, the difficulty of delivering RNAi to target cells has impeded the development of RNAi-based therapies. One method to overcome this barrier is the use of a nonpathogenic bacteria vector, Escherichia coli, to deliver RNAi to target cells with high efficacy. In transkingdom interference RNAi (tkRNAi) delivery, E. coli were engineered to transcribe short RNA (shRNA) from a plasmid (TRIP) containing the invasin gen...
Source: Springer protocols feed by Immunology - July 1, 2008 Category: Allergy & Immunology Source Type: info

0.0Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Flow cytometry is combined with highly specific fluorophore-conjugated antibodies that will only bind to the activated forms of molecules. The advances in flow cytometry enable to perform quantitative multiplexed analysis of single cells within heterogeneous populations stained with specific antibodies for phenotyping in conjunction with antibodies to phosphorylated, i.e., activated molecules within signaling pathways. By reactivating signaling pathways in vitro it is possible to collect data on the responsive state of complex cell populations such as immune cells. In this protocol, peripheral blood mononuclear cells (PBMC...
Source: Springer protocols feed by Immunology - July 1, 2008 Category: Allergy & Immunology Source Type: info

0.0Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Since its development, flow cytometry gave a relevant contribution to the field of Immunology. Its unique potential to analyse multiple parameters at the single cell level allowed the identification of unknown cell subsets with specific roles in immunoregulation as well as in the pathogenesis of several diseases. More recently, with the advent of new equipments and fluorochromes, the possibility exists to analyse simultaneously a large number (up to 19) of parameters in a single cell. This strategy, defined polychromatic flow cytometry (PFC), has been widely utilised in the last years for the fine analysis of immune cell p...
Source: Springer protocols feed by Immunology - July 1, 2008 Category: Allergy & Immunology Source Type: info

0.0Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Peripheral blood T lymphocytes are a pool of cells with extremely different characteristics and, therefore, it may be difficult to obtain clear-cut results and to attribute a certain function to a defined T cell population in several experimental settings. The availability of a population of human T lymphocytes deriving from the same progenitor with a unique phenotype and function (clone) may therefore be of help.
Source: Springer protocols feed by Immunology - July 1, 2008 Category: Allergy & Immunology Source Type: info

0.0Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe here an example of LDA experiment assessing antigen-specific T cell proliferation of microcultures in the presence or absence of adjuvant and illustrate how to estimate the frequencies of precursor T cells using an online tool that we made publicly available.
Source: Springer protocols feed by Immunology - July 1, 2008 Category: Allergy & Immunology Source Type: info

0.0Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The following method describes the identification of candidate immunogenic peptides through their ability to recall an immune T-cell activation from peripheral blood mononuclear cells (PBMCs) of individuals with defined HLA–peptide restrictions that have been previously exposed to the antigen. Isolated PBMCs are plated out at a concentration of 1×106 cells/ml in a 200 μl medium and incubated overnight to reduce cytokine gene expression due to cell manipulation. After starving, cells are either directly stimulated with individual peptides or not stimulated and incubated from 3 to 12 h according to e...
Source: Springer protocols feed by Immunology - July 1, 2008 Category: Allergy & Immunology Source Type: info

0.0Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Subsets of T cells can be distinguished on basis of their cytokine production and secretion profile.
Source: Springer protocols feed by Immunology - July 1, 2008 Category: Allergy & Immunology Source Type: info

Non-Replicating Recombinant Vaccinia Virus Expressing CD8 to Enhance T-Cell StimulationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The following method describes the generation of a recombinant vaccinia virus expressing a costimulatory molecule (human CD80 or B7.1).
Source: Springer protocols feed by Immunology - July 1, 2008 Category: Allergy & Immunology Source Type: info

High-Throughput Screening of Peptide Deformylase InhibitorsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 substrat...
Source: Springer protocols feed by Immunology - December 1, 2007 Category: Allergy & Immunology Source Type: info

Mycobacterium tuberculosis β-Ketoacyl Acyl Carrier Protein Synthase III (mtFabH) Assay: Principles and MethodEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 β-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 β-ketoacyl ACP product, which is further processed to form mature fatty acids tha...
Source: Springer protocols feed by Immunology - December 1, 2007 Category: Allergy & Immunology Source Type: info

Methods for Assessing the Structure and Function of Cationic Antimicrobial PeptidesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 α-helix (with polar and nonpolar groups on opposite faces of the helix), a structural feature believed to be important in t...
Source: Springer protocols feed by Immunology - December 1, 2007 Category: Allergy & Immunology Source Type: info

Assays for Β-Lactamase Activity and InhibitionEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The ability, either innate or acquired, to produce β-lactamases, enzymes capable of hydrolyzing the endocyclic peptide bond in β-lactam antibiotics, would appear to be a primary contributor to the ever-increasing incidences of resistance to this class of antibiotics. To date, four distinct classes, A, B, C, and D, of β-lactamases have been identified. Of these, enzymes in classes A, C, and D utilize a serine residue as a nucleophile in their catalytic mechanism while class B members are Zn+2-dependent for their function. Efforts have been and still continue to be made toward the development of potent inhibit...
Source: Springer protocols feed by Immunology - December 1, 2007 Category: Allergy & Immunology Source Type: info

Methods to Assay Inhibitors of tRNA Synthetase ActivityEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Aminoacyl-tRNA synthetases (aa-RS) attracted interest as potential targets for new antibacterial compounds. Most organisms express 20 aa-RSs: one for each amino acid. Aa-RSs are essential proteins in all living organisms. When one aa-RS is inhibited, the corresponding tRNA is not charged and is therefore unavailable for translation. This leads to protein synthesis inhibition, which in turn causes cell growth arrest. Consequently, each compound that inhibits any of the aa-RS could be a potential antibacterial agent. Only one aa-RS inhibitor, the Ile-RS inhibitor mupirocin, is currently marketed as an antibacterial agent. We...
Source: Springer protocols feed by Immunology - December 1, 2007 Category: Allergy & Immunology Source Type: info

Flow Cytometry of Bacterial Membrane Potential and PermeabilityEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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.
Source: Springer protocols feed by Immunology - December 1, 2007 Category: Allergy & Immunology Source Type: info

Assays for the Identification of Inhibitors Targeting Specific Translational StepsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 HeL...
Source: Springer protocols feed by Immunology - December 1, 2007 Category: Allergy & Immunology Source Type: info

Methods to Assay Inhibitors of DNA Gyrase and Topoisomerase IV ActivitiesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 ap...
Source: Springer protocols feed by Immunology - December 1, 2007 Category: Allergy & Immunology Source Type: info

A Method to Assay Penicillin-Binding ProteinsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Key enzymes that assemble the bacterial cell wall are also the target of the Β-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.
Source: Springer protocols feed by Immunology - December 1, 2007 Category: Allergy & Immunology Source Type: info

SPARK: A New Peptidyl Transferase Activity AssayEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 othe...
Source: Springer protocols feed by Immunology - December 1, 2007 Category: Allergy & Immunology Source Type: info

Screening for Compounds That Affect the Interaction Between Bacterial Two-Component Signal Transduction Response Regulator Protein and Cognate Promoter DNAEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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.
Source: Springer protocols feed by Immunology - December 1, 2007 Category: Allergy & Immunology Source Type: info

Biocomputational Strategies for Microbial Drug Target IdentificationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In conclusion, the results of such a strategy underscore the utility of large genomic databases for in silico systematic drug target identification in the post-genomic era.
Source: Springer protocols feed by Immunology - December 1, 2007 Category: Allergy & Immunology Source Type: info

A Method to Assay Inhibitors of DNA Polymerase IIIC ActivityEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The need for new drugs to treat infections caused by antibiotic-resistant bacterial strains has prompted many studies to identify novel targets in pathogenic bacteria. Among the three DNA polymerases expressed by bacteria, one of these, designated pol III, is responsible for DNA replication and growth of bacteria and, therefore, warrants consideration as a drug target. However, the pol III enzymes of Gram-positive and Gram-negative species are quite different, and the Gram-positive enzyme pol IIIC has been more extensively studied as a drug target than the Gram-negative enzyme pol IIIE.
Source: Springer protocols feed by Immunology - December 1, 2007 Category: Allergy & Immunology Source Type: info

Three Methods to Assay Inhibitors of Ribosomal Subunit AssemblyEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 an...
Source: Springer protocols feed by Immunology - December 1, 2007 Category: Allergy & Immunology Source Type: info

Studies of Enzymes That Cause Resistance to Aminoglycosides AntibioticsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 ...
Source: Springer protocols feed by Immunology - December 1, 2007 Category: Allergy & Immunology Source Type: info

Methods to Identify and Characterize Inhibitors of Bacterial RNA PolymeraseEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 bac...
Source: Springer protocols feed by Immunology - December 1, 2007 Category: Allergy & Immunology Source Type: info

Inhibition ofChaperone-Dependent Bacterial Ribosome BiogenesisEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In Escherichia coli, the molecular chaperone HSP70 (DnaK) is necessary for 30S and 50S ribosomal subunit assembly at temperatures above 37°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 α-complementation of β-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 t...
Source: Springer protocols feed by Immunology - December 1, 2007 Category: Allergy & Immunology Source Type: info

Bacterial Efflux Pump InhibitorsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 in...
Source: Springer protocols feed by Immunology - December 1, 2007 Category: Allergy & Immunology Source Type: info

A Method to Assay Inhibitors of Lipopolysaccharide SynthesisEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
This chapter is orgTreatment of Gram-negative bacterial infections is complicated by innate and acquired drug resistance resulting in a limited number of effective antibiotics. Several Gram-negative bacteria, for which current therapies are ineffective, have recently been identified as potential bioterror agents. These findings highlight the need for new antibiotics, specifically antibiotics that act on new drug targets to circumvent drug resistance. Potential targets in Gram-negative bacteria include enzymes involved in the biosynthesis of lipopolysaccharides (LPS) that form outer membranes of these organisms. UDP-3-O-(R-...
Source: Springer protocols feed by Immunology - December 1, 2007 Category: Allergy & Immunology Source Type: info

The Activity of rRNA Resistance Methyltransferases Assessed by MALDI Mass SpectrometryEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe here a method using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to determine the location and number of methyl groups added at any site in the rRNA. The method is particularly suited to studying in vitro methylation of RNA transcripts by resistance methyltransferases such as Erm.
Source: Springer protocols feed by Immunology - December 1, 2007 Category: Allergy & Immunology Source Type: info

Drosophila Immunity: Methods for Monitoring the Activity of Toll and Imd Signaling PathwaysEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Invertebrates lack an adaptive immune system and rely on innate immunity to resist pathogens. The response of Drosophila melanogaster to bacterial and fungal infections involves two signaling pathways, Toll and Imd, both of which activate members of the nuclear factor (NF)-κB family of transcription factors, leading to antimicrobial peptide (AMP) gene expression. In this chapter, we present the current methods used in our laboratory to monitor the activity of both signaling pathways.
Source: Springer protocols feed by Immunology - November 19, 2007 Category: Allergy & Immunology Source Type: info

Ex Vivo and In Vitro Primary Mast CellsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe here (1) fractionation methods suitable for purifying mouse or rat peritoneal mast cells and for purifying human mast cells of various origins, and (2) conditions for generating pure cultured mast cell populations from mouse, rat, and human tissues.
Source: Springer protocols feed by Immunology - November 19, 2007 Category: Allergy & Immunology Source Type: info

Dissection of the Antiviral NK Cell Response by MCMV MutantsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Our understanding of virus control by natural killer (NK) cells relies mainly on in vitro observations. The significance of these findings for virus control in vivo is not yet fully understood. Complexity is added by the fact that many viruses, particularly herpesviruses, are equipped with sets of genes that, dependent on the genetic background of the host, modify the NK cell response. The advent of recombinant DNA technology and mutagenesis procedures for BAC-cloned viral genomes has made it possible not only to screen for viral proteins with such functions but also to assess their biological relevance. Mutant viruses wit...
Source: Springer protocols feed by Immunology - November 19, 2007 Category: Allergy & Immunology Source Type: info

Genetic Analysis of Caenorhabditis elegans Innate ImmunityEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Innate immunity is an ancient and conserved defense mechanism. The worm Caenorhabditis elegans provides a useful tool for studying the function of the innate immune system at the molecular and cellular levels within the context of a whole organism. The powerful genetics of the worm, combined with efficacy of gene knockdown by RNA interference (RNAi), offer complementary tools for analyzing the contribution of individual genes to innate immunity. It is important, however, to exclude pleiotropic effects that confound results. In this chapter, we will describe the procedures for performing both forward and reverse genetic scr...
Source: Springer protocols feed by Immunology - November 19, 2007 Category: Allergy & Immunology Source Type: info

ENU Mutagenesis in MiceEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Forward genetics has led to many “breakthrough” discoveries, and with the mouse genome almost fully sequenced, the creation of phenotypes through random germline mutagenesis has become an efficient means by which to find the function of yet undescribed genes. In this chapter, we will provide a practical guideline for performing germline mutagenesis in mice. In particular, we will focus on the application of this technology to identify genes that are essential to innate immune defense.
Source: Springer protocols feed by Immunology - November 19, 2007 Category: Allergy & Immunology Source Type: info