Project description:ASCL1 is known to act as transcriptional activator of notch signaling pathway. We have found that ASCL1 is over expressed in secondary glioblastoma. In order to delineate its functional role in gliomagensis we have used gene silencing approach to identify genes differentially regulated upon ASCL1 down regulation in U87MG glioma cell line.
Project description:This experiment is designed to evaluate gene expression alteration following AEG-1 (MTDH) silencing in glioma cells. AEG-1 is found to be a potential regulator through interaction with other transcription factor. We intended to investigate the enriched transcription factor dependent downstream gene sets regulated by AEG-1 form the differential expressed genes we obtained from AEG-1 silencing. Total RNA were extracted from U87MG glioma cells stably silencing AEG-1 and correspondent pSuper Retro vector cells respectively. Passage 5 of the indicated cells after infection and selection were harvested for RNA extraction.
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.
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:Both established glioma cells lines U87MG and U373 were used for studying their interactions in the indirect co-cultures. Eventhough both being derived from the glioma tissue, those two cell lines prove morphologically and physiologically disctinct. Therefore their intre-cellular interactions were examined on gene exprexssion level in vitro to observe whether those co-cultures could make for a suitable in vitro cell model mimicking the in vivo glioma tumour heterogeneity. Three biological replicates of four cell set-ups were performed: U87MG monoculture, U373 monoculture, U87MG co-cultured with U373 (in Boyden chambers) and U373 co-cultured with U87MG (in Boyden chambers).
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.
Project description:Phosphatase EYA1 was overexpressed in glioma cell line U87MG and phospho-proteomic analysis was performed to identify novel EYA1 substrates in glioma cells.