Project description:MCF7 cells at 80% confluency were treated with 5 micromolar azacytidine with gene expression profiling determined at 0, 1, 2, 3, 4, 6 and 12 hours of exposure to compound. Keywords: time course
Project description:MCF7 cells at 80% confluency were treated with 5 micromolar azacytidine with gene expression profiling determined at 0, 1, 2, 3, 4, 6 and 12 hours of exposure to compound. Keywords: time course Approximately 5,000,000 million cells were plated into 10 cm plates from a common pool. Cells were grown in DMEM supplemented with 10% foetal calf serum until 80% confluency was attained. Cells were then exposed to 5 micromolar azacytidine prepared as a 10 millimolar stock in DMSO. Each time point was represented by three biological replicates. Total RNA was extracted from the cells using the trizol method and RNA samples were validated for integrity throughout the isolation and extraction procedure. All gene expression profiles showed similar distributions with similar raw median intensities.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
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.