Characterization of SCAR markers of Eimeria spp. of domestic fowl and construction of a public relational database (The Eimeria SCARdb)

This study reports the development and characterization of 151 sequence characterized amplified region (SCAR) markers for the seven Eimeria species that infect the domestic fowl. From this set, 84 markers are species-specific and 67 present partial specificity. The complete nucleotide sequence was derived for all markers, revealing the presence of micro- and minisatellite repetitive units in 22 SCARs, with up to five distinct repeat units being observed per marker. Only 15 markers showed significant hits in similarity searches against public sequence databases, thus confirming their anonymous and non-coding character. Finally, a relational database of the markers (the Eimeria SCARdb) was developed and made available on the Internet, providing a valuable resource of SCAR markers that can be useful for molecular diagnosis, and also for epizootiological, genetic variability and genome mapping studies.     

Development of recombinant vesicular stomatitis viruses that exploit defects in host defense to augment specific oncolytic activity

Vesicular stomatitis virus (VSV) is a negative-stranded RNA virus normally sensitive to the antiviral actions of alpha/beta interferon (IFN-alpha/beta). Recently, we reported that VSV replicates to high levels in many transformed cells due, in part, to susceptible cells harboring defects in the IFN system. These observations were exploited to demonstrate that VSV can be used as a viral oncolytic agent to eradicate malignant cells in vivo while leaving normal tissue relatively unaffected. To attempt to improve the specificity and efficacy of this system as a potential tool in gene therapy and against malignant disease, we have genetically engineered VSV that expresses the murine IFN-beta gene. The resultant virus (VSV-IFNbeta) was successfully propagated in cells not receptive to murine IFN-alpha/beta and expressed high levels of functional heterologous IFN-beta. In normal murine embryonic fibroblasts (MEFs), the growth of VSV-IFNbeta was greatly reduced and diminished cytopathic effect was observed due to the production of recombinant IFN-beta, which by functioning in a manner involving autocrine and paracrine mechanisms induced an antiviral effect, preventing virus growth. However, VSV-IFNbeta grew to high levels and induced the rapid apoptosis of transformed cells due to defective IFN pathways being prevalent and thus unable to initiate proficient IFN-mediated host defense. Importantly, VSV expressing the human IFN-beta gene (VSV-hIFNbeta) behaved comparably and, while nonlytic to normal human cells, readily killed their malignant counterparts. Similar to our in vitro observations, following intravenous and intranasal inoculation in mice, recombinant VSV (rVSV)-IFNbeta was also significantly attenuated compared to wild-type VSV or rVSV expressing green fluorescent protein. However, VSV-IFNbeta retained propitious oncolytic activity against metastatic lung disease in immunocompetent animals and was able to generate robust antitumor T-cell responses. Our data indicate that rVSV designed to exploit defects in mechanisms of host defense can provide the basis for new generations of effective, specific, and safer viral vectors for the treatment of malignant and other disease.     

Identification of differentially expressed genes like cofilin2 in growing collateral arteries

Arteriogenesis, the growth of pre-existing collateral arteries, can be induced in rabbits by occlusion of the femoral artery. In order to analyze the differential gene expression in arteriogenesis, cDNA of collateral arteries 24h after femoral occlusion or sham operation was subjected to suppression subtractive hybridization (SSH). We demonstrated an upregulation of the U6 snRNA binding protein Lsm5, cytochrome b, an expressed sequence tag, and the actin-depolymerizing factor cofilin2 mRNA in collateral arteries 24h after femoral ligation. For cofilin2, we also detected an increase in the protein level and a localization predominantly in smooth muscle cells of collaterals. Simultaneously with the upregulation of cofilin2 we found a downregulation of the alpha-smooth muscle actin mRNA in growing collateral arteries. In summary, our data showed an augmented expression level of genes contributing to different fundamental processes of arteriogenesis.     

Hydrogen exchange-mass spectrometry analysis of beta-amyloid peptide structure

beta-Amyloid peptide (A beta) is the primary protein component of senile plaques in Alzheimer's disease and is believed to be responsible for the neurodegeneration associated with the disease. A beta has proven to be toxic only when aggregated; however, the structure of the aggregated species associated with toxicity is unknown. In the present study, we use hydrogen-deuterium isotope exchange (HX)-electrospray ionization mass spectrometry (MS) along with enzymatic digestion as a tool to examine at near residue level, the changes in A beta structure associated with aggregation to a fibril form. Our results show that the structure of A beta intermediate species formed early in the course of fibrillogenesis is dependent upon solvent conditions. Additionally, the HX-MS data of peptic A beta fragments suggest that the C-terminal segment of the peptide is approximately 35% protected from exchange in fibril-containing samples, relative to monomeric A beta species prepared in DMSO/H(2)O. The N-terminus (residues 1-4) is completely unprotected from exchange, and the fragment containing residues 5-19 is over 50% protected from exchange in the fibril-containing samples. This work contributes to our understanding of A beta structure associated with aggregation and toxicity and further application of this approach may aid in the design of agents that intervene in the A beta aggregation processes associated with neurotoxicity.     

Identification,purification, and characterization of an eukaryotic-like phosphopantetheine adenylyltransferase (coenzyme A biosynthetic pathway) in the hyperthermophilic archaeon Pyrococcus abyssi

Although coenzymeA (CoA) is essential in numerous metabolic pathways in all living cells, molecular characterization of the CoA biosynthetic pathway in Archaea remains undocumented. Archaeal genomes contain detectable homologues for only three of the five steps of the CoA biosynthetic pathway characterized in Eukarya and Bacteria. In case of phosphopantetheine adenylyltransferase (PPAT) (EC 2.7.7.3), the putative archaeal enzyme exhibits significant sequence similarity only with its eukaryotic homologs, an unusual situation for a protein involved in a central metabolic pathway. We have overexpressed in Escherichia coli, purified, and characterized this putative PPAT from the hyperthermophilic archaeon Pyrococcus abyssi (PAB0944). Matrix-assisted laser desorption ionization-time of flight mass spectrometry and high performance liquid chromatography measurements are consistent with the presence of a dephospho-CoA (dPCoA) molecule tightly bound to the polypeptide. The protein indeed catalyzes the synthesis of dPCoA from 4'-phosphopantetheine and ATP, as well as the reverse reaction. The presence of dPCoA stabilizes PAB0944, as it induces a shift from 76 to 82 degrees C of the apparent Tm measured by differential scanning microcalorimetry. Potassium glutamate was found to stabilize the protein at 400 mm. The enzyme behaves as a monomeric protein. Although only distantly related, secondary structure prediction indicates that archaeal and eukaryal PPAT belong to the same nucleotidyltransferase superfamily of bacterial PPAT. The existence of operational proteins highly conserved between Archaea and Eukarya involved in a central metabolic pathway challenge evolutionary scenarios in which eukaryal operational proteins are strictly of bacterial origin.     

Diversity and specificity of Frankia strains in nodules of sympatric Myrica gale, Alnus incana, and Shepherdia canadensis determined by rrs gene polymorphism

The identity of Frankia strains from nodules of Myrica gale, Alnus incana subsp. rugosa, and Shepherdia canadensis was determined for a natural stand on a lake shore sand dune in Wisconsin, where the three actinorhizal plant species were growing in close proximity, and from two additional stands with M. gale as the sole actinorhizal component. Unisolated strains were compared by their 16S ribosomal DNA (rDNA) restriction patterns using a direct PCR amplification protocol on nodules. Phylogenetic relationships among nodular Frankia strains were analyzed by comparing complete 16S rDNA sequences of study and reference strains. Where the three actinorhizal species occurred together, each host species was nodulated by a different phylogenetic group of Frankia strains. M. gale strains from all three sites belonged to an Alnus-Casuarina group, closely related to Frankia alni representative strains, and were low in diversity for a host genus considered promiscuous with respect to Frankia microsymbiont genotype. Frankia strains from A. incana nodules were also within the Alnus-Casuarina cluster, distinct from Frankia strains of M. gale nodules at the mixed actinorhizal site but not from Frankia strains from two M. gale nodules at a second site in Wisconsin. Frankia strains from nodules of S. canadensis belonged to a divergent subset of a cluster of Elaeagnaceae-infective strains and exhibited a high degree of diversity. The three closely related local Frankia populations in Myrica nodules could be distinguished from one another using our approach. In addition to geographic separation and host selectivity for Frankia microsymbionts, edaphic factors such as soil moisture and organic matter content, which varied among locales, may account for differences in Frankia populations found in Myrica nodules.     

A systematic approach to the complete study of a signaling molecule: ribosomal p90rsk as an example

Ribosomal p90rsk is a kinase of central importance in transducing mitogenic signals from an activated receptor to the cell nucleus and for protein synthesis. Here, we analyze the optimal steps to fully describe this kinase in both normal neutrophils and leukemic cell lines. These are: (i) immunological analyses (immunoblotting and immunoprecipitation); (ii) enzyme activity assays (in vitro and "in-gel"); and (iii) immunobiochemical combination methods (immunoprecipitation/kinase assay, immunoprecipitation/"in-gel" assay and ion exchange chromatography/immunoblotting). For the enzyme assays, we describe a novel method to measure ribosomal p90rsk kinase activity "in-gel", based on a renatured-protein method that allows for the direct quantitation of enzyme activity. Finally, we present an algorithm that can be readily implemented to the quantification of the extent of stimulation of a kinase in response to a particular extracellular stimuli. In our case, it was found that activation of p90rsk was higher in proliferating leukemic cells than in mature neutrophils, indicating that a suppression of key signal transduction links could contribute to the maturational arrest typical of acute leukemia. All the techniques and strategies described here for p90rsk could be easily extrapolated to the study of any signal transduction molecule, provided it has a phosphotransferase activity.     

Two-dimensional display and whole genome comparison of bacterial pathogen genomes of high G+C DNA content

Three highly mutable loci of the wall-less pathogens Mycoplasma bovis, Mycoplasma pulmonis and Mycoplasma agalactiae undergo high-frequency genomic rearrangements and generate extensive antigenic variation of major surface lipoproteins. Adjacent to each locus, an open reading frame exists as a single chromosomal copy and is predicted to encode a site-specific DNA recombinase exhibiting high homology to the recombinases XerD of Escherichia coli and CodV of Bacillus subtilis. Each of the mycoplasmal proteins are members of the lambda integrase family of tyrosine site-specific recombinases and likely mediates site-specific DNA inversions observed within the adjacent, variable loci.