Project description:MX1 is a well-characterized interferon-induced antiviral gene. MX1 is activated by viral infection due to interferon production in cells. We treated non-permissive Huh7 cells and permissive HRP4 cells with interferon. We compared the expression of genes induced by interferon to determine host factors affecting HCV replication.
Project description:MX1 is a well-characterized interferon-induced antiviral gene. MX1 is activated by viral infection due to interferon production in cells. We treated non-permissive Huh7 cells and permissive HRP4 cells with interferon. We compared the expression of genes induced by interferon to determine host factors affecting HCV replication. Huh7 cells and HRP4 cells were treated with 40U/ml interferon-α for 6h. RNA was extracted and hybridized on Affymetrix microarrays
Project description:Drugs directly targeting Hepatitis C (HCV) are often rendered useless by the high mutation rate of the virus. Thus, we deduce that targeting of host factor that affect HCV replication may provide enhanced therapy fort HCV infection. Hepatocyte cell line Huh7 is known to be non-permissive for Hepatits C (HCV) replication. Through a method developed by the Rice laboratory (Blight, K.J., et al., J Virol, 2002), selection of a small subset of permissive hepatocytes is possible. The Rice laboratory generated the first permissive cell line, Huh7.5, using this method. We generated another permissive cell line, HRP1, using the same method. With microarray, we compared the expression of host mRNAs in non-permissive Huh7 to both Huh7.5 and HRP1 searching for host factors lost in the cell lines permisive for HCV replication.
Project description:Drugs directly targeting Hepatitis C (HCV) are often rendered useless by the high mutation rate of the virus. Thus, we deduce that targeting of host factor that affect HCV replication may provide enhanced therapy fort HCV infection. Hepatocyte cell line Huh7 is known to be non-permissive for Hepatits C (HCV) replication. Through a method developed by the Rice laboratory (Blight, K.J., et al., J Virol, 2002), selection of a small subset of permissive hepatocytes is possible. The Rice laboratory generated the first permissive cell line, Huh7.5, using this method. We generated another permissive cell line, HRP1, using the same method. With microarray, we compared the expression of host mRNAs in non-permissive Huh7 to both Huh7.5 and HRP1 searching for host factors lost in the cell lines permisive for HCV replication. Non-permissive cell line Huh7 and permissive cell lines Huh7.5 and HRP1 were harvested for RNA extraction and hybridization on Affymetrix microarrays.
Project description:A powerful approach to study innate antiviral response is to compare the difference between wild type Huh7 cells, which do not support robust replication of hepatitis C virus (HCV)2, versus certain subclones of Huh7 cells that are permissive for HCV replication. We generated two permissive cell lines and two independent non-permissive subclone from Huh7 cells. We compared the global methylation pattern of these different cells and find that Huh7 cells exist as a heterogeneous population of cells with distinct patterns of gene methylation.
Project description:A powerful approach to study innate antiviral response is to compare the difference between wild type Huh7 cells, which do not support robust replication of hepatitis C virus (HCV)2, versus certain subclones of Huh7 cells that are permissive for HCV replication. We generated two permissive cell lines and two independent non-permissive subclone from Huh7 cells. We compared the global methylation pattern of these different cells and find that Huh7 cells exist as a heterogeneous population of cells with distinct patterns of gene methylation. Comparison of Huh7, HRP1, HRP4, Huh7-pNeo1 and Huh7-pNeo2 cells.
Project description:Comparing the gene expression profiling of HDGF-silenced RD-ES cells and control RD-ES cells to identify genes regulated by HDGF in RD-ES cells. Keywords: expression analysis
Project description:Comparing the gene expression profiling of HDGF-silenced RD-ES cells and control RD-ES cells to identify genes regulated by HDGF in RD-ES cells. Keywords: expression analysis Control RD-ES cells and HDGF-silenced RD-ES cells were profiled on 22K Human Genome Array
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.