Project description:We evaluated the transcriptome changes induced by infection of Hela 229 cells with Shigella flexneri. The sample set consists of a control (mock), total population of infected sample and infected sample sorted into Shigella positive and Shigella negative population.
Project description:Question Addressed: What is the host response (specifically apoptosis pathways) to Shigella infection and what is the contribution of MxiE to the host response? The arrays are apoptosis Exon hit arrays, thus splice variation can be determined. Array data from HeLa cells that remained Uninfected or were infected with wildtype S. flexneri serotype 2a strain 2457T or an isogenic mixE mutant. These groupings are further divided such that Uninfected or Infected cells remained untreated or were treated with the apopotosis inducer staurosporine (STS). The design of the experiment was a time course and samples were harvested at the indicated time points (hr). All hybridizations were conducted against a common reference sample that was made from pooled samples of normal uninfected healthy HeLa cells. Infection: HeLa cells were infected with wt Shigella (wt), mxiE mutant (mixE) or were left uninfected (uninfected) Compound Based Treatment: Cultures were treated with Staurosporin (STS) or were left untreated (untreated) Infection time: Cells were harvested at the indicated time after treatment
Project description:Question Addressed: What is the host response (specifically apoptosis pathways) to Shigella infection and what is the contribution of MxiE to the host response? The arrays are apoptosis Exon hit arrays, thus splice variation can be determined. Array data from HeLa cells that remained Uninfected or were infected with wildtype S. flexneri serotype 2a strain 2457T or an isogenic mixE mutant. These groupings are further divided such that Uninfected or Infected cells remained untreated or were treated with the apopotosis inducer staurosporine (STS). The design of the experiment was a time course and samples were harvested at the indicated time points (hr). All hybridizations were conducted against a common reference sample that was made from pooled samples of normal uninfected healthy HeLa cells. Infection: HeLa cells were infected with wt Shigella (wt), mxiE mutant (mixE) or were left uninfected (uninfected) Compound Based Treatment: Cultures were treated with Staurosporin (STS) or were left untreated (untreated) Infection time: Cells were harvested at the indicated time after treatment 20 Samples
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.
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.