Project description:fRSL1 is a jumbo myovirus stably and lytically infecting the phytopathogenic bacterium Ralstonia solanacearum. In this study, we investigate the infection cycle of fRSL1 and provide a genomic, proteomic and transcriptomic view of this phage. Its 231-kbp genome sequence showed many genes lacking detectable homologs in the current databases and was vastly different from previously studied phage genomes. In addition to these orphan proteins, fRSL1 was found to encode several enzymes that are unique among known viruses. These include enzymes for the salvage pathway of NAD+ and for the biosynthetic pathways of lipid, carbohydrate and homospermidine. A chitinase-like protein was found to be a potential lysis enzyme. Our proteomics analysis suggests that fRSL1 virions contain at least 25 distinct proteins. We identified six of them including a tail sheath protein and a topoisomerase IB by N-terminal sequencing. Based on a DNA microarray analysis, we identified two transcription patterns. Total RNA was isolated from 3 ml of fRSL1-infected MAFF730138 cells (containing 1 x 108 CFU infected at moi 0.5 ~ 4) at 0, 10, 30, 90 min, and 30 h post infection (p.i.) using an RNAprotect Bacteria Reagent kit (Qiagen K.K., Tokyo, Japan) according to the manufacturer’s protocol. The integrity and concentration of total RNA were measured using a bioanalysis unit (Agilent 2100 Bioanalyser, Aligent Tech. Palo Alto, CA). Fluorescence-labeled antisense RNA was synthesized by direct incorporation of Cy5-UTP (GE Healthcare Bio-Science Corp., Piscataway, NJ), using each RNA sample and an RNA Transcript SureLABEL Core kit (Takara Bio Inc.). The labeled antisense RNAs were hybridized simultaneously to the microarray chip. DNA microarray preparation, hybridization, processing, scanning, and analyses were performed by Filgen Inc. (Nagoya Japan). For each fRSL1 ORF, 1 to 5 coding sequences of 35 to 40 nucleotide-long were synthesized. A total of 1,100 sequences were put in duplicate on a 40 x 55 array chip. The fluorescence images of hybridized microarrays were obtained with a GenePix 4000B scanner (Molecular Devices, Sunnyvale, CA). The Array-Pro Analyzer Ver4.5 (Media Cybernetics, Inc., Silver Spring, MD) was used to determine the signal intensity of each spot and its local background. Scan data images were alalyzed using Microarray Data Analysis Tool Ver3.0 software (Filgen Inc.)

Project description:Background: Minimal change nephrotic syndrome (MCNS) is considered to be associated with T cell dysfunction, via unknown mechanisms. Experimental observations suggest that some humoral factors alter the permeability of glomerular filtration barrier. However, the nature of such factors remains still uncertain. Methods: Using cDNA microarrays, we performed gene expression profiling of peripheral blood mononuclear cells (PBMC) from three patients with MCNS during nephrosis and remission phases. To confirm the cDNA microarray results, we performed quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses in nephrosis and remission samples from 20 MCNS patients and six patients with nephrotic syndrome due to membranous nephropathy. Results: Out of 24,446 genes screened, 33 genes were up-regulated (at least 1.5-fold) in PBMC from these MCNS patients during the nephrosis phase. Up-regulated genes mainly encoded proteins involved in signal transduction and cytokine response. For further examination, we selected two genes encoding provable secretary proteins, chemokine (C-C) ligand 13 (also known as monocyte chemotactic protein-4) (CCL13) and a novel galectin-related protein (HSPC159). The results of RT-PCR showed that expressions of CCL13 and HSPC159 mRNA in nephrosis PBMC samples are higher than those in remission PBMC samples from all 20 MCNS patients examined. On the other hand, these mRNA expression patterns were variable among six patients with membranous nephropathy. Conclusions: We conclude that CCL13 and HSPC159 mRNA expressions in PBMC is up-regulated in MCNS patients during the nephrosis phase. These expression changes in PBMC might be involved in the pathophysiologic processes of MCNS. Using cDNA microarrays, we performed gene expression profiling of peripheral blood mononuclear cells (PBMC) from three patients with MCNS during nephrosis and remission phases. To confirm the cDNA microarray results, we performed quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses in nephrosis and remission samples from 20 MCNS patients and six patients with nephrotic syndrome due to membranous nephropathy.

Project description:The induction of genetic competence is a strategy used by bacteria to increase their genetic repertoire under stressful environmental conditions. Recently, Streptococcus pneumoniae has been shown to co-ordinate the uptake of transforming DNA with fratricide via increased expression of the peptide pheromone responsible for competence induction. Here, we document that environmental stress-induced expression of the peptide pheromone competence-stimulating peptide (CSP) in the oral pathogen Streptococcus mutans. We showed that CSP is involved in the stress response and determined the CSP-induced regulon in S. mutans by microarray analysis. Contrary to pneumococcus, S. mutans responds to increased concentrations of CSP by cell lysis in only a fraction of the population. We have focused on the mechanism of cell lysis and have identified a novel bacteriocin as the M-bM-^@M-^Xdeath effectorM-bM-^@M-^Y. Most importantly, we showed that this bacteriocin causes cell death via a novel mechanism of action: intracellular action against self. We have also identified the cognate bacteriocin immunity protein, which resides in a separate unlinked genetic locus to allow its differential regulation. The role of the lytic response in S. mutans competence is also discussed. Together, these findings reveal a novel autolytic pathway in S. mutans which may be involved in the dissemination of fitness-enhancing genes in the oral biofilm. Streptococcus mutans UA159 were grown with 2 uM CSP or without (uninduced control) to mid-log phase. Total RNA was extracted as described above. The cDNAs were prepared for hybridization using the PFGRC protocol. Microarray chips were scanned using a Gene Pix 4000B (Axon) and analyzed using the TM4 Microarray Software Suite (http://www.tm4.org/). Transcript levels were measured by cDNA hybridized to a fourfold redundant S. mutans microarray and averaged for three replicated hybridizations. Differential gene expression was based on a post-normalization cut-off of M-BM-1> twofold.

Project description:Perfluorooctane sulfonate (PFOS) has been manufactured for over 50 years in increasing quantities and has been used for several industrial and commercial aims. Due to the persistence and the bioaccumulation of this pollutant, it can be found worldwide in wildlife and humans. Biochemical effects of PFOS exposure are mainly studied in mammalian model species and information about effects on fish species remain largely scarce. This lack of toxicity data points out that there is an urgent need for the mechanistic molecular understanding of the mode of action of this pollutant. In the present study, common carp (Cyprinus carpio) was exposed through water for 14 days at concentrations of 0.1; 0.5 and 1 mg/l PFOS. Liver was selected as target tissue. Custom microarrays were constructed from cDNA libraries obtained with Suppression Subtractive Hybridization-Polymerase chain reaction (SSH-PCR) experiments. Microarray data revealed that the expression of several genes in the liver was influenced by PFOS exposure and real-time PCR was used to confirm these gene expression changes. The affected genes were mainly involved in energy metabolism, reproduction and stress response. Furthermore, the relative condition factor and the hepatosomatic index of the exposed fish were significantly lower after 14 days of exposure as well as the available glycogen reserves. At all levels of biological organization, indications of a trade-off between the metabolic cost of toxicant exposure on one hand and processes vital to the survival of the organism on the other hand were seen. Our results support the prediction that increases in energy expenditure negatively affects processes vital to the survival of an organism, such as growth. Keywords: PFOS, common carp, microarray, condition factor, energy reserves, metabolic cost There were 3 biological replicates for each exposure concentration. For each biological replicate control versus exposed hybridizations were carried out. The mean of the biological replicates was calculated for the differentially expressed genes.

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