Project description:The general expression profile in human lymphoblastoid cell lines treated with 9 uM, 90 uM or 900 uM bolus of hydrogen peroxide at 0, 4,12,24, and 48 hours. Four cell lines (GM07055, GM14569, GM14467, and 12057) were treated separately and total RNA was pooled in equal amounts prior to labeling and hybridization to Affymetrix Human Gene 1.0 ST microarray. Four lymphoblastoid cell lines (GM07055, GM12057, GM14569, and GM14467) were obtained from the Coriell Institute for Medical Research (Camden, NJ). 24 hours before bolus treatments, cells were cultured at a density of 1 x10^6 cells/mL to create 17 cultures for each cell line (a total of 68 samples). At the beginning of the experiment, one culture was collected from each cell line and total RNA prepared, this was equally pooled and prepared for microarray analysis (time zero, treatment zero). Each cell line was then treated with a 9 (4 cultures), 90 (4 cultures), or 900 uM hydrogen peroxide bolus (4 cultures) or water (4 cultures). Four, 12, 24 or 48 hours following the hydrogen peroxide bolus, cells were collected for each cell line of each treatment and total RNA prepared. RNA from the four cell lines were equalling pooled for each time and each hydrogen peroxide (or water) bolus amount resulting in a total of 16 additional samples which were also labeled and hybridized to Affymetrix Human Gene 1.0 ST microarrays.
Project description:The general expression profile in human lymphoblastoid cell lines treated with 9 uM, 90 uM or 900 uM bolus of hydrogen peroxide at 0, 4,12,24, and 48 hours. Four cell lines (GM07055, GM14569, GM14467, and 12057) were treated separately and total RNA was pooled in equal amounts prior to labeling and hybridization to Affymetrix Human Gene 1.0 ST microarray.
Project description:The goal of this study is to characterize a gshF::cam L. plantarum strain NZ7608. From phenotype characterization we have observed that the gene gshF plays a role in hydrogen peroxide resistance in L. plantarum and therefore we wanted to analyze the global transcriptome response of strain NZ7608 towards peroxide and compared this to wild type response. In this experiment we compared two L. plantarum strains: WCFS1 (wild type) and NZ7618 (gshF::cam). Both strains were grown -in duplo- on CDM containing 0.5% glucose at 37 degrees celsius. When cell density reached OD600=1.0 a hydrogen peroxide stress was given to the cultures to a final concentration of 10mM. Samples were taken at times (0 10 and 30 minutes) upon hydrogen peroxide. All withdrawn samples were used for this microarray experiment. Keywords: peroxide, gshF mutant
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes
Project description:RNA-seq for prostate cancer cell lines DU145 and 22RV1 with or without PAGE4 overexpression with hydrogen peroxide or PBS treatment
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.