Project description:Gene expression profiling of elutriated monocytes cultured for 48 hours in RPMI-1640, 10% AB plamsa and one of the following: GMCSF (56IU/ml), IL-4 (2000 IU/ml), 15 kDa Granulysin (10 nM) Sources tested: monocytes form 3 donors tested at time 0, 4 hours, 12 hours, 24 hours, 48 hours
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:Human primary keratinocytes were collected at 0, 1, 3, 6, 12, 24 and 48 hours after addition of 1.8mM Calcium and RNA was extracted.
Project description:Transcriptional profiling of MeJA-treated pearl millet seedlings over time [0, 12, 24 and 48 hours post treatment (hpt)]. Keywords: Time course, Stress response
