T cell integrin overexpression as a model of murine autoimmunity

Integrin adhesion molecules have important adhesion and signaling functions. They also play a central role in the pathogenesis of many autoimmune diseases. Over the past few years we have described a T cell adoptive transfer model to investigate the role of T cell integrin adhesion molecules in the development of autoimmunity. This report summarizes the methods we used in establishing this murine model. By treating murine CD4+ T cells with DNA hypomethylating agents and by transfection we were able to test thein vitro effects of integrin overexpression on T cell autoreactive proliferation, cytotoxicity, adhesion and trafficking. Furthermore, we showed that the ability to inducein vivo autoimmunity may be unique to the integrin lymphocyte function associated antigen-1 (LFA-1). Published: October 24, 2003     

Screening of compounds toxicity against human Monocytic cell line-THP-1 by flow cytometry

The worldwide rapid increase in bacterial resistance to numerous antibiotics requires on-going development of new drugs to enter the market. As the development of new antibiotics is lengthy and costly, early monitoring of compound’s toxicity is essential in the development of novel agents. Our interest is in a rapid, simple, high throughput screening method to assess cytotoxicity induced by potential agents. Some intracellular pathogens, such as Mycobacterium tuberculosis primary site of infection is human alveolar macrophages. Thus, evaluation of candidate drugs for macrophage toxicity is crucial. Protocols for high throughput drug toxicity screening of macrophages using flow cytometry are lacking in the literature. For this application we modified a preexisting technique, propidium iodide (PI) exclusion staining and utilized it for rapid toxicity tests. Samples were prepared in 96 well plates and analyzed by flow cytometry, which allowed for rapid, inexpensive and precise assessment of compound’s toxicity associated with cell death. Published: October 1, 2004.     

Combining genetic and biochemical approaches to identify functional molecular contact points

Protein-protein interactions are required for many viral and cellular functions and are potential targets for novel therapies. Here we detail a series of genetic and biochemical techniques used in combination to find an essential molecular contact point on the duck hepatitis B virus polymerase. These techniques include differential immunoprecipitation, mutagenesis and peptide competition. The strength of these techniques is their ability to identify contact points on intact proteins or protein complexes employing functional assays. This approach can be used to aid identification of putative binding sites on proteins and protein complexes which are resistant to characterization by other methods.     

The use of microscopy and three-dimensional visualization to evaluate the structure of microbial biofilms cultivated in the calgary biofilm device

Microbes frequently live within multicellular, solid surface-attached assemblages termed biofilms. These microbial communities have architectural features that contribute to population heterogeneity and consequently to emergent cell functions. Therefore, three-dimensional (3D) features of biofilm structure are important for understanding the physiology and ecology of these microbial systems. This paper details several protocols for scanning electron microscopy and confocal laser scanning microscopy (CLSM) of biofilms grown on polystyrene pegs in the Calgary Biofilm Device (CBD). Furthermore, a procedure is described for image processing of CLSM data stacks using amira™, a virtual reality tool, to create surface and/or volume rendered 3D visualizations of biofilm microorganisms. The combination of microscopy with microbial cultivation in the CBD — an apparatus that was designed for highthroughput susceptibility testing — allows for structure-function analysis of biofilms under multivariate growth and exposure conditions.     

Analysis of among-site variation in substitution patterns

Substitution patterns among nucleotides are often assumed to be constant in phylogenetic analyses. Although variation in the average rate of substitution among sites is commonly accounted for, variation in the relative rates of specific types of substitution is not. Here, we review details of methodologies used for detecting and analyzing differences in substitution processes among predefined groups of sites. We describe how such analyses can be performed using existing phylogenetic tools, and discuss how new phylogenetic analysis tools we have recently developed can be used to provide more detailed and sensitive analyses, including study of the evolution of mutation and substitution processes. As an example we consider the mitochondrial genome, for which two types of transition deaminations (C⇒T and A⇒G) are strongly affected by single-strandedness during replication, resulting in a strand asymmetric mutation process. Since time spent single-stranded varies along the mitochondrial genome, their differential mutational response results in very different substitution patterns in different regions of the genome. Published: September 2, 2004.     

Effector cell mediated cytotoxicity measured by intracellular Granzyme B release in HIV infected subjects

CD8+ cytotoxic T lymphocyte (CTL) activity is currently believed to be one of the key immunologic mechanisms responsible for the prevention or attenuation of HIV-1 infection. The induction of CD8+ T cell activation may also result in the production of soluble or non-classical lytic factors that are associated with protection from infection or slower disease progression. Traditionally, CD8+ CTL responses have been measured by the classic chromium release assay, monitoring the ability of T cells (Effector cells) to lyse radiolabelled HLA — matched “target cells“ that express the appropriate antigen-MHC complex. This method is not only labor intensive, semi quantitative assay at best, but also needs fresh, non-cryopreserved cells. Recently, cytokine specific ELISPOT assays or tetrameric MHC-I/ peptide complexes have utilized to directly quantitate circulating CD8+ effector cells, and these assays are more sensitive, quantitative and reproducible than the traditional CTL lysis assay and can also be performed on cryopreserved cells. Although these are reproducible assays for the assessment of soluble antiviral activity secreted by activated T cell populations they can be extremely expensive to perform. We have used FACS Analysis to measure Granzyme B release as a function of cell mediated cytotoxicity. This method helps quantitate the CTL activity and also identifies the phenotype of the cells elucidating this immune response. The method described not only monitors immunological response but also is also simple to perform, precise and extremely time efficient and is ideal for screening a large number of samples. Published: September 5, 2003     

Cell culture-based analysis of postsynaptic membrane assembly in muscle cells

We report a method for studying postsynaptic membrane assembly utilizing the replating of aneural cultures of differentiated skeletal muscle cells onto laminin-coated surfaces. A significant limitation to the current cell culturebased approaches has been their inability to recapitulate the multistage surface acetylcholine receptor (AChR) redistribution events that produce complex AChR clusters found at the intact neuromuscular junction (NMJ). By taking advantage of the ability of substrate laminin to induce advanced maturation of AChR aggregates on the surface of myotubes, we have developed a secondary-plating method that allows more precise analysis of the signaling events connecting substrate laminin stimulation to complex AChR cluster formation. We validate the utility of this method for biochemical and microscopy studies by demonstrating the roles of RhoGTPases in substrate laminin-induced complex cluster assembly.     

Measuring solution viscosity and its effect on enzyme activity

In proteins, some processes require conformational changes involving structural domain diffusion. Among these processes are protein folding, unfolding and enzyme catalysis. During catalysis some enzymes undergo large conformational changes as they progress through the catalytic cycle. According to Kramers theory, solvent viscosity results in friction against proteins in solution, and this should result in decreased motion, inhibiting catalysis in motile enzymes. Solution viscosity was increased by adding increasing concentrations of glycerol, sucrose and trehalose, resulting in a decrease in the reaction rate of the H⁺-ATPase from the plasma membrane ofKluyveromyces lactis. A direct correlation was found between viscosity (η) and the inhibition of the maximum rate of catalysis (V _max). The protocol used to measure viscosity by means of a falling ball type viscometer is described, together with the determination of enzyme kinetics and the application of Kramers’ equation to evaluate the effect of viscosity on the rate of ATP hydrolysis by the H⁺-ATPase. Published: May 1, 2003     

A method for detecting functional activity related expression in gross brain regions,specific brain nuclei and individual neuronal cell bodies and their projections

We have developed a system to visualize functionally activated neurons and their projections in the brain. This system utilizes a transgenic mouse, fos-tau-lacZ (FTL), which expresses the marker gene, lacZ, in neurons and their processes after activation by many different stimuli. This system allows the imaging of activation from the level of the entire brain surface, through to individual neurons and their projections. The use of this system involves detection of neuronal activation by histochemical or immunohistochemical detection of β-galactosidase (βgal), the product of the lacZ gene. Furthermore, the underlying brain state of the FTL mice determines the basal levels of expression of βgal. Here we describe in detail our protocols for detection of FTL expression in these mice and discuss the main variables which need to be considered in the use of these mice for the detection and mapping of functionally activated neurons, circuits and regions in the brain.     

Application of a colorimetric assay to identify putative ribofuranosylaminobenzene 5′-phosphate synthase genes expressed with activity in<Emphasis Type="Italic">Escherichia coli</Emphasis>

Tetrahydromethanopterin (H₄MPT) is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H4MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H4MPT and tetrahydrofolate is ribofuranosylaminobenzene 5′-phosphate synthase (RFAP synthase). Given the importance of RFAP synthase in H₄MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H4MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies inEscherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase fromArchaeoglobus fulgidus was produced inE. coli and purified to homogeneity. The production of active RFAP synthase fromMethanothermobacter thermautotrophicus was achieved by coexpression of the geneMTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase. Florida Agricultural Experiment Station Journal Series no. R-09353 Published: March 4, 2003     

A sensitive quantification of HHV-6B by real-time PCR

Human herpesvirus (HHV)-6B is a pathogen causing latent infection in virtually all humans. Nevertheless, the interaction of HHV-6B with its host cells is poorly understood. Although HHV-6B is approximately 90% homologous to HHV-6A, it expresses certain B-specific genes. In order to quantify the amount of expressed viral mRNA we have developed a method using real-time PCR on a LightCycler instrument. Here we describe an assay for the detection of the HHV-6B B6 mRNA, but our approach can easily be extended to involve other mRNAs. This method is useful during the study of HHV-6B biology and offers reliable and reproducible, quantitative detection of viral mRNA below the attomol range. Published: December 9, 2002     

Serum-free cryopreservation of five mammalian cell lines in either a pelleted or suspended state

Herein we have explored two practical aspects of cryopreserving cultured mammalian cells during routine laboratory maintenance. First, we have examined the possibility of using a serum-free, hence more affordable, cryopreservative. Using five mammalian lines (Crandell Feline Kidney, MCF7, A72, WI 38 and NB324K), we found that the serum-free alternative preserves nearly as efficiently as the serum-containing preservatives. Second, we compared cryostorage of those cells in suspended versus a pellet form using both aforementioned cryopreservatives. Under our conditions, cells were in general recovered equally well in a suspended versus a pellet form. Published: April 7, 2004.     

A proteomic approach based on peptide affinity chromatography, 2-dimensional electrophoresis and mass spectrometry to identify multiprotein complexes interacting with membrane-bound receptors

There is accumulating evidence that membrane-bound receptors interact with many intracellular proteins. Multiprotein complexes associated with ionotropic receptors have been extensively characterized, but the identification of proteins interacting with G protein-coupled receptors (GPCRs) has so far only been achieved in a piecemeal fashion, focusing on one or two protein species. We describe a method based on peptide affinity chromatography, two-dimensional electrophoresis, mass spectrometry and immunoblotting to identify the components of multiprotein complexes interacting directly or indirectly with intracellular domains of GPCRs or, more generally, any other membrane-bound receptor. Using this global approach, we have characterized multiprotein complexes that bind to the carboxy-terminal tail of the 5-hydroxytryptamine type 2C receptor and are important for its subcellular localization in CNS cells (Bécamel et al., EMBO J., 21(10): 2332, 2002). Published: December 9, 2002     

Dynamic force microscopy for imaging of viruses under physiological conditions

Dynamic force microscopy (DFM) allows imaging of the structure and the assessment of the function of biological specimens in their physiological environment. In DFM, the cantilever is oscillated at a given frequency and touches the sample only at the end of its downward movement. Accordingly, the problem of lateral forces displacing or even destroying bio-molecules is virtually inexistent as the contact time and friction forces are reduced. Here, we describe the use of DFM in studies of human rhinovirus serotype 2 (HRV2) weakly adhering to mica surfaces. The capsid of HRV2 was reproducibly imaged without any displacement of the virus. Release of the genomic RNA from the virions was initiated by exposure to low pH buffer and snapshots of the extrusion process were obtained. In the following, the technical details of previous DFM investigations of HRV2 are summarized. Published: June 29, 2004.     

PCR-based detection of a rare linear DNA in cell culture

The described method allows for detection of rare linear DNA fragments generated during genomic deletions. The predicted limit of the detection is one DNA molecule per 10⁷ or more cells. The method is based on anchor PCR and involves gel separation of the linear DNA fragment and chromosomal DNA before amplification. The detailed chemical structure of the ends of the linear DNA can be defined with the use of additional PCR-based protocols. The method was applied to study the short-lived linear DNA generated during programmed genomic deletions in a ciliate. It can be useful in studies of spontaneous DNA deletions in cell culture or for tracking intracellular modifications at the ends of transfected DNA during gene therapy trials. Published: November 11, 2002