Project description:Both benign nevi and melanomas exhibit high rates of activating mutations in BRAF kinase, suggesting that this event is important for melanocyte proliferation and melanoma initiation in vivo; however, the precise mechanism of BRAF kinase action in these lesions remains to be elucidated. We have used comprehensive gene expression profiling to evaluate the downstream effectors of activated BRAFV600E kinase in primary human melanocytes and identified a dominant proliferative target gene signature for BRAF kinase in these cells. We used microarrays to detail the global gene expression pattern change induced by the activation of RAS/BRAF/MEK/ERK signaling pathway and identified distinct classes of up-regulated genes during this process. Keywords: Genetic modification
Project description:Both benign nevi and melanomas exhibit high rates of activating mutations in BRAF kinase, suggesting that this event is important for melanocyte proliferation and melanoma initiation in vivo; however, the precise mechanism of BRAF kinase action in these lesions remains to be elucidated. We have used comprehensive gene expression profiling to evaluate the downstream effectors of activated BRAFV600E kinase in primary human melanocytes and identified a dominant proliferative target gene signature for BRAF kinase in these cells. We used microarrays to detail the global gene expression pattern change induced by the activation of RAS/BRAF/MEK/ERK signaling pathway and identified distinct classes of up-regulated genes during this process. Experiment Overall Design: Primary human melanocytes were isolated from neonatal foreskins. Melanocytes isolated from five individual donors were pooled together in order to reduce genetic variations and transduced by BRAFV600E expressing lentivirus and control virus with MOI 10 respectively. Cells were harvested 5 days following infection and total RNA was extracted and purified by RNeasy kit by following manufacturerâs protocol. RNA quality check, double strand complementry DNA synthesis, hybridization with Human Genome U133 Plus 2.0 Array Chips (Affymetrix Inc. Santa Clara, CA), initial data extraction, normalization using RMAExpress software.
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: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: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. One-condition experment, gene expression of 3A6
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes
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.