Direct application of plasmid DNA containing type I interferon transgenes to vaginal mucosa inhibits HSV-2 mediated mortality

The application of naked DNA containing type I interferon (IFN) transgenes is a promising potential therapeutic approach for controlling chronic viral infections. Herein, we detail the application of this approach that has been extensively used to restrain ocular HSV-1 infection, for antagonizing vaginal HSV-2 infection. We show that application of IFN-α1, -α 5, and -β transgenes to vaginal mouse lumen 24 hours prior to HSV-2 infection reduces HSV-2 mediated mortality by 2.5 to 3-fold. However, other type I IFN transgenes (IFN- α 4, -α 5, -α 6, and -α 9) are non effectual against HSV-2. We further show that the efficacy of IFN-1 transgene treatment is independent of CD4+ T lymphocytes. However, in mice depleted of CD8+ T lymphocytes, the ability of IFN-α 1 transgene treatment to antagonize HSV-2 was lost.     

Methods for analyzing the role of DNA methylation and chromatin structure in regulating T lymphocyte gene expression

Chromatin structure, determined in part by DNA methylation, is established during differentiation and prevents expression of genes unnecessary for the function of a given cell type. We reported that DNA methylation and chromatin structure contributes to lymphoidspecific ITGAL (CD11a) and PRF1 (perforin) expression. We used bisulfite sequencing to compare methylation patterns in the ITGAL promoter and 5′ flanking region of T cells and fibroblasts, and in the PRF1 promoter and upstream enhancer of CD4+ and CD8+ T cells with fibroblasts. The effects of methylation on promoter function were tested using regional methylation of reporter constructs, and confirmed by DNA methyltransferase inhibition. The relationship between DNA methylation and chromatin structure was analyzed by DNaseI hypersensitivity. Herein we described the methods and results in greater detail. Published: September 16, 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     

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.     

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     

An improved method for constructing and selectively silanizing double-barreled,neutral liquid-carrier,ion-selective microelectrodes

We describe an improved, efficient and reliable method for the vapour-phase silanization of multi-barreled, ion-selective microelectrodes of which the silanized barrel(s) are to be filled with neutral liquid ion-exchanger (LIX). The technique employs a metal manifold to exclusively and simultaneously deliver dimethyldichlorosilane to only the ion-selective barrels of several multi-barreled microelectrodes. Compared to previously published methods the technique requires fewer procedural steps, less handling of individual microelectrodes, improved reproducibility of silanization of the selected microelectrode barrels and employs standard borosilicate tubing rather than the less-conventional theta-type glass. The electrodes remain stable for up to 3 weeks after the silanization procedure. The efficacy of a double-barreled electrode containing a proton ionophore in the ion-selective barrel is demonstrated in situ in the leaf apoplasm of pea (Pisum) and sunflower (Helianthus). Individual leaves were penetrated to depth of ∼150 μm through the abaxial surface. Microelectrode readings remained stable after multiple impalements without the need for a stabilizing PVC matrix.     

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     

Evaluation of intra- and interspecific divergence of satellite DNA sequences by nucleotide frequency calculation and pairwise sequence comparison

Satellite DNA sequences are known to be highly variable and to have been subjected to concerted evolution that homogenizes member sequences within species. We have analyzed the mode of evolution of satellite DNA sequences in four fishes from the genusDiplodus by calculating the nucleotide frequency of the sequence array and the phylogenetic distances between member sequences. Calculation of nucleotide frequency and pairwise sequence comparison enabled us to characterize the divergence among member sequences in this satellite DNA family. The results suggest that the evolutionary rate of satellite DNA inD. bellottii is about two-fold greater than the average of the other three fishes, and that the sequence homogenization event occurred inD. puntazzo more recently than in the others. The procedures described here are effective to characterize mode of evolution of satellite DNA. Published: March 4, 2003     

Methodology for detecting trace amounts of microchimeric DNA from peripheral murine white blood cells by real-time PCR

Real-time PCR methodology can successfully quantitate microchimeric cell populations at a concentration of 100 microchimeric cells/100,000 host cells; however, it has not been successful in quantitating DNA from trace numbers of microchimeric white blood cells which we reported are present in murine peripheral blood at a concentration as low as 2/100,000 host cells. We report methodology using primers for a portion of the H2-k^b murine histocompatibility sequence, specific for the C57BL/6J mouse. When these primers were used in the presence of 11,000 μM primer, a 20-fold increase in the median manufacturer’s recommended concentration, the assay could be optimized to detect 34 pg of C57BL/6J DNA in a background of 2.5 μg of carrier BALB/cJ DNA (1/100,000). These conditions resulted in a detection limit half as sensitive as that found when no carrier DNA was present. Published: April 7, 2003     

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     

In vivo and in vitro techniques for comparative study of antiviral T-cell responses in the amphibian Xenopus

Activation of lymphocytes in mammals is often quantified by measuring the amount of proliferation during the expansion phase of an immune response. Bromodeoxyuridine (BrdU) incorporation and carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution assays are some of the techniques widely used in mammalian studies of pathogeninduced proliferation and provide a convenient way of quantifying the cellular response. We have extended the use of these proliferation assays to the amphibian Xenopus laevis. We have developed this species as a valuable comparative model to study immunity against a wellknown amphibian pathogen, Frog Virus 3 (FV3). Fluorescence activated cell sorting was used to assess the level of BrdU incorporation of lymphocytes in vivo and CFSE dilution in an in vitro activation assay. Both techniques have shown that splenic lymphocytes proliferate specifically upon FV3 challenge. This indicates that common methods for detection of proliferation upon immunologic challenge are easily applied to other vertebrate species, as it highlights the evolutionary conservation of the proliferative nature of immune responses throughout vertebrate phyla.     

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.     

A method to identify p62’s UBA domain interacting proteins

The UBA domain is a conserved sequence motif among polyubiquitin binding proteins. For the first time, we demonstrate a systematic, high throughput approach to identification of UBA domain-interacting proteins from a proteome-wide perspective. Using the rabbit reticulocyte lysatein vitro expression cloning system, we have successfully identified eleven proteins that interact with p62’s UBA domain, and the majority of the eleven proteins are associated with neurodegenerative disorders, such as Alzheimer’s disease. Therefore, p62 may play a novel regulatory role through its UBA domain. Our approach provides an easy route to the characterization of UBA domain interacting proteins and its application will unfold the important roles that the UBA domain plays. Published: December 12, 2003.     

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.     

Use of ion-channel modulating agents to study cyanobacterial Na<Superscript>+</Superscript>-K<Superscript>+</Superscript> fluxes

Here we describe an experimental design aimed to investigate changes in total cellular levels of Na⁺ and K⁺ ions in cultures of freshwater filamentous cyanobacteria. Ion concentrations were measured in whole cells by flame photometry. Cellular Na⁺ levels increased exponentially with rising alkalinity, with K⁺ levels being maximal for optimal growth pH (∼8). At standardized pH conditions, the increase in cellular Na⁺, as induced by NaCl at 10 mM, was coupled by the two sodium channel-modulating agents lidocaine hydrochloride at 1 μM and veratridine at 100 μM. Both the channel-blockers amiloride (1 mM) and saxitoxin (1 μM), decreased cell-bound Na⁺ and K⁺ levels. Results presented demonstrate the robustness of well-defined channel blockers and channel-activators in the study of cyanobacterial Na⁺- K⁺ fluxes. Published: June 29, 2004.     

Inactivation of sortilin (a novel lysosomal sorting receptor) by dominant negative competition and RNA interference

To assess the role of sortilin in the sorting and trafficking of sphingolipid activator proteins (SAPs) the function of sortilin was abolished by a dominant-negative mutant and by the use of RNAi. Mutant sortilin lacking the carboxyl-terminal region that contains the sorting signal abolished the trafficking of SAPs to the lysosomes. Both sortilin and SAPs were retained in the Golgi apparatus. The use of chemically synthesized siRNA effectively blocked the trafficking of SAPs to the lysosomes as well. Additionally, we created a stable COS-7 cell line transfected with the pSilencer 3.1 H1 neo vector containing a selected siRNA template oligonucleotide (small hairpin interference RNA) where the levels of sortilin were greatly suppressed. The elimination of sortilin by this method will permit to determine whether or not sortilin is involved in a general mechanism of lysosomal sorting that involves the trafficking of various soluble lysosomal proteins other than SAPs.