Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA92779 | ENA |
REPOSITORIES: ENA
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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.
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Project description:The effect on mucosal gene expression of potent acid inhibition with proton pump inhibitors (PPI’s) given to humans in ordinary, therapeutic doses is virtually unknown. Eight patients suffering from gastro-oesophageal reflux disease were included in this study. Endoscopic biopsies were taken from the corpus mucosa before and towards the end of a three-month treatment with the PPI esomeprazole 40 mg daily. Total RNA was extracted and samples (2 microgram total RNA) were labeled for microarray analysis. For each patient, RNA from both time points were labeled and hybridized to a single array. The gene expression patterns for 7700 genes were analyzed and differentially expressed genes were identified using one-sample Student t test. The aim of this study was, by use of cDNA microarray, to get an overview of the gene expression pattern in gastric corpus mucosa in patients after three months’ treatment with the proton pump inhibitor esomeprazole. Further characterization of the functional roles of genes whose expression is modulated by potent acid inhibition may give new insight into the molecular responses to this therapeutic intervention, including the mucosal response to the moderately increased gastrin levels encountered in clinical practice. Keywords: Repeat sample group, treatment analysis
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