Project description:cGMP is well-known secound messanger involved in vascular homeostasis. However little is known the effect of cGMP inducer on mRNA expression in cancer cells. We performed microarrays to revealed the transcriptional change of a human pancreatic ductal adenocarcinoma (PDCA) cell line (Panc -1) to cGMP inducer Bay41-2272 in order to provide insight into impact of cGMP induction on Panc-1 cells.
Project description:cGMP is well-known secound messanger involved in vascular homeostasis. However little is known the effect of cGMP inducer on mRNA expression in cancer cells. We performed microarrays to revealed the transcriptional change of a human pancreatic ductal adenocarcinoma (PDCA) cell line (Panc -1) to cGMP inducer Bay41-2272 in order to provide insight into impact of cGMP induction on Panc-1 cells. Panc-1 Human PDCA cells were treated with vehicle (DMSO) or 5 μM Bay41-2272 for 48 h and total RNA was extracted by using TRIzol Reagent. The microarray analysis was conducted on the Panc-1 cells expressing by using Agilent Microarray.
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:Expression analysis of human pancreatic cancer cell lines (Capan-1, Panc-1, Panc-1+ve and Panc-1-ve) and pancreatic ductal cell line (HPDE)