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Target Gene Expression (target + gene_expression)
Selected AbstractsTulp3 is a critical repressor of mouse hedgehog signalingDEVELOPMENTAL DYNAMICS, Issue 5 2009Don A. Cameron Abstract Precise regulation of the morphogen sonic hedgehog (Shh) and modulation of the Shh signaling pathway is required for proper specification of cell fate within the developing limbs and neural tube, and resultant tissue morphogenesis. Tulp3 (tubby-like protein 3) is a protein of unknown function which has been implicated in nervous system development through gene knockout studies. We demonstrate here that mice lacking the Tulp3 gene develop abnormalities of both the neural tube and limbs consistent with improper regulation of Shh signaling. Tulp3,/, embryos show expansion of Shh target gene expression and display a ventralization of neural progenitor cells in the caudal neural tube. We further show that Tulp3,/,/Shh,/, compound mutant embryos resemble Tulp3 mutants, and express Shh target genes in the neural tube and limbs which are not expressed in Shh,/, embryos. This work uncovers a novel role for Tulp3 as a negative regulatory factor in the Hh pathway. Developmental Dynamics 238:1140,1149, 2009. © 2009 Wiley-Liss, Inc. [source] Stage-dependent craniofacial defects resulting from Sprouty2 overexpressionDEVELOPMENTAL DYNAMICS, Issue 7 2007L. Henry Goodnough Abstract Sprouty genes encode intracellular regulators of receptor tyrosine kinases that function in a variety of developmental events. Although mice carrying null mutations in Sprouty genes exhibit craniofacial anomalies, the precise role of these regulatory proteins in facial development remains unclear. Here, we show that overexpression of spry2 at the initiation of craniofacial development results in a dramatic arrest in outgrowth of the facial prominences. Although endogenous spry2 and fibroblast growth factor 8 (fgf8) are coexpressed throughout much of craniofacial development, overexpression of spry2 did not alter the spatiotemporal patterns of fgf target gene expression. The morphological consequences of spry2 overexpression were specific: all of the facial prominences were truncated, but despite this gross malformation, the programs of osteogenesis and chondrogenesis were not impaired. Collectively, these data suggest that Sprouty2 plays a role in the outgrowth of facial prominences independent of canonical Fgf signaling. Developmental Dynamics 236:1918,1928, 2007. © 2007 Wiley-Liss, Inc. [source] The dynamics of developmental system drift in the gene network underlying wing polyphenism in ants: a mathematical modelEVOLUTION AND DEVELOPMENT, Issue 3 2008Marcos Nahmad SUMMARY Understanding the complex interaction between genotype and phenotype is a major challenge of Evolutionary Developmental Biology. One important facet of this complex interaction has been called "Developmental System Drift" (DSD). DSD occurs when a similar phenotype, which is homologous across a group of related species, is produced by different genes or gene expression patterns in each of these related species. We constructed a mathematical model to explore the developmental and evolutionary dynamics of DSD in the gene network underlying wing polyphenism in ants. Wing polyphenism in ants is the ability of an embryo to develop into a winged queen or a wingless worker in response to an environmental cue. Although wing polyphenism is homologous across all ants, the gene network that underlies wing polyphenism has evolved. In winged ant castes, our simulations reproduced the conserved gene expression patterns observed in the network that controls wing development in holometabolous insects. In wingless ant castes, we simulated the suppression of wings by interrupting (up- or downregulating) the expression of genes in the network. Our simulations uncovered the existence of four groups of genes that have similar effects on target gene expression and growth. Although each group is comprised of genes occupying different positions in the network, their interruption produces vestigial discs that are similar in size and shape. The implications of our results for understanding the origin, evolution, and dissociation of the gene network underlying wing polyphenism in ants are discussed. [source] Interferon regulatory factor-3 activation, hepatic interferon-stimulated gene expression, and immune cell infiltration in hepatitis C virus patients,HEPATOLOGY, Issue 3 2008Daryl T.-Y. Interferon regulatory factor-3 (IRF-3) activation directs ,/, interferon production and interferon-stimulated gene (ISG) expression, which limits virus infection. Here, we examined the distribution of hepatitis C virus (HCV) nonstructural 3 protein, the status of IRF-3 activation, and expression of IRF-3 target genes and ISGs during asynchronous HCV infection in vitro and in liver biopsies from patients with chronic HCV infection, using confocal microscopy and functional genomics approaches. In general, asynchronous infection with HCV stimulated a low-frequency and transient IRF-3 activation within responsive cells in vitro that was associated with cell-to-cell virus spread. Similarly, a subset of HCV patients exhibited the nuclear, active form of IRF-3 in hepatocytes and an associated increase in IRF-3 target gene expression in hepatic tissue. Moreover, ISG expression profiles formed disease-specific clusters for HCV and control nonalcoholic fatty liver disease patients, with increased ISG expression among the HCV patients. We identified the presence of T cell and plasmacytoid dendritic cell infiltrates within all biopsy specimens, suggesting they could be a source of hepatic interferon in the setting of hepatitis C and chronic inflammatory condition. Conclusion: These results indicate that HCV can transiently trigger IRF-3 activation during virus spread and that in chronic HCV, IRF-3 activation within infected hepatocytes occurs but is limited. (HEPATOLOGY 2007.) [source] Heterogeneous expression of Wnt/,-catenin target genes within colorectal cancerINTERNATIONAL JOURNAL OF CANCER, Issue 9 2007Falk Hlubek Abstract Invasion of common colorectal adenocarcinomas is coupled with a transient loss of epithelial differentiation of tumor cells. Previously, we have shown that dedifferentiated tumor cells at the invasive front (IF) accumulate the transcriptional activator ,-catenin in the nucleus, in contrast to cells of the tumor center. To characterize the cells of these two morphogenic tumor areas, gene expression profiling was performed. Our study demonstrates that intratumorous heterogeneity in colorectal cancer correlates with differential expression of 510 genes between the central tumor region (TC) and the IF. Many genes differentially expressed at the IF are involved in cellular invasion processes like cell motility, cell adhesion and extracellular matrix interaction. This in vivo analysis shows overexpression of known Wnt/,-catenin target genes either in the entire tumor tissue (compared to normal mucosa) or specifically at the IF. Thus, even though all tumor cells overexpress ,-catenin, the existence of at least 2 groups of Wnt/,-catenin target genes selectively activated in different tumor regions is suggested. The concomitant high expression of inflammation- and tissue repair-related genes at the IF supports the hypothesis that an inflammation-activated microenvironment may trigger selective Wnt/,-catenin target gene expression and contribute to the malignant progression of colorectal cancer. © 2007 Wiley-Liss, Inc. [source] Long-term RNA interference and its application to hepatitis B virusJOURNAL OF DIGESTIVE DISEASES, Issue 3 2009Jin Shui PAN RNA interference (RNAi) is an ancient defensive mechanism in eukaryotes to control gene expressing and defend their genomes from foreign invaders. It refers to the phenomenon that double-stranded RNA results in the sequence-specific silencing of target gene expression. Although it was documented in a relatively short time ago, intensive research has facilitated making its mechanism clear. Researchers have found that it was a powerful tool for analyzing the functions of genes and treating tumors, infectious diseases and genetic abnormalities that are associated with a dominant gene defect. However, delivery in vivo, low blood stability and poor intracellular uptake present significant challenges for the development of RNAi reagents in clinical use. Thus, long-term inducible RNAi was designed. There are hundreds of millions of hepatitis B virus (HBV) carriers in the world at present, a portion of whom will lose their lives after several years due to chronic complications such as cirrhosis, hepatocellular carcinomas or both. Although a preventive vaccine is now available, the present therapeutic options for chronically infected patients are limited and of low efficiency. Admittedly, to date most RNAi experiments have been done in vitro, but it is hoped that they may be developed into a therapeutic strategy for HBV in the near future. In this article the principles and construction of long-term RNA are discussed. Its therapeutic potentiality and attention to the potential hazards will also outlined. We conclude that this ancient defensive mechanism can be recruited as a powerful weapon in the fight against HBV. [source] In vivo molecular imaging of adenoviral versus lentiviral gene therapy in two bone formation modelsJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 8 2006Brian T. Feeley Abstract Regional gene therapy techniques are promising methods to enhance bone formation in large bone defects that would be difficult to treat with allograft or autograft bone stock. In this study, we compared in vivo temporal expression patterns of adenoviral- and lentiviral-mediated gene therapy in two bone formation models. Primary rat bone marrow cells (RBMC) were transduced with lentiviral or adenoviral vectors containing luciferase (Luc) or BMP-2 cDNA, or cotransduced with vectors containing Luc and bone morphogenetic protein 2 (BMP-2). In vitro protein production was determined with luciferase assay or ELISA (for BMP-2 production) weekly for 12 weeks. Two bone formation models were used,a hind limb muscle pouch or radial defect,in SCID mice. A cooled charged-coupled device (CCD) camera was used to image in vivo luciferase expression weekly for 12 weeks. In vitro, adenoviral expression of BMP-2 and luciferase was detected by ELISA or luciferase assay, respectively, for 4 weeks. Lentiviral expression of BMP-2 and luciferase was sustained in culture for 3 months. Using the CCD camera, we found that adenoviral vectors expressed luciferase expression for up to 21 days, but lentiviral vectors expressed target gene expression for 3 months in vivo in both bone formation models. There was no detectable difference in the amount of bone formed between the adenoviral and lentiviral groups. Lentiviral-mediated delivery of BMP-2 can induce long term in vitro and in vivo gene expression, which may be beneficial when developing tissue engineering strategies to heal large bone defects or defects with a compromised biologic environment. © 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 24:1709,1721, 2006 [source] Gene Knockdown: A Powerful Tool for Gene Function Study in FishJOURNAL OF THE WORLD AQUACULTURE SOCIETY, Issue 3 2008Surintorn Boonanuntanasarn So far, there are a number of fish genome projects, including experimental and economically important fish that provide available DNA sequence information. However, the function of a gene cannot be deduced only by its DNA sequence. Therefore, a technique with which to investigate the function of the fish gene is needed. Gene knockdown (GKD), or antisense technology, is now being used as a powerful technique to study gene functions in living organisms. GKD effects result from the introduction of an antisense molecule into living cells. The antisense agents bind to target messenger RNA, thus inactivating the target gene expression. The appropriately spatial inhibitory effects on protein production from corresponding gene resulted in the phenotypic change. Therefore, the function of the gene can be understood. To date, there are a number of antisense molecules that can affect efficient GKD in fish. These include antisense oligonucleotides, small interfering RNA, and ribozyme. These antisense molecules cause specific gene inhibitor effects with different mechanisms. The various antisense mechanism types facilitate a number of GKD applications with various approaches in animals. In this review, we demonstrate the characteristics of each antisense molecule, its mechanism, and its application, especially for gene functional analysis in fish. [source] The application of Tet repressor in prokaryotic gene regulation and expressionMICROBIAL BIOTECHNOLOGY, Issue 1 2008Ralph Bertram Summary Inducible gene expression based upon Tet repressor (tet regulation) is a broadly applied tool in molecular genetics. In its original environment, Tet repressor (TetR) negatively controls tetracycline (tc) resistance in bacteria. In the presence of tc, TetR is induced and detaches from its cognate DNA sequence tetO, so that a tc antiporter protein is expressed. In this article, we provide a comprehensive overview about tet regulation in bacteria and illustrate the parameters of different regulatory architectures. While some of these set-ups rely on natural tet -control regions like those found on transposon Tn10, highly efficient variations of this system have recently been adapted to different Gram-negative and Gram-positive bacteria. Novel tet -controllable artificial or hybrid promoters were employed for target gene expression. They are controlled by regulators expressed at different levels either in a constitutive or in an autoregulated manner. The resulting tet systems have been used for various purposes. We discuss integrative elements vested with tc-sensitive promoters, as well as tet regulation in Gram-negative and Gram-positive bacteria for analytical purposes and for protein overproduction. Also the use of TetR as an in vivo biosensor for tetracyclines or as a regulatory device in synthetic biology constructs is outlined. Technical specifications underlying different regulatory set-ups are highlighted, and finally recent developments concerning variations of TetR are presented, which may expand the use of prokaryotic tet systems in the future. [source] Review: The role of microRNAs in kidney diseaseNEPHROLOGY, Issue 6 2010JORDAN YZ LI ABSTRACT MicroRNAs (miRNAs) are short non-coding RNAs that modulate physiological and pathological processes by inhibiting target gene expression via blockade of protein translation or by inducing mRNA degradation. These miRNAs potentially regulate the expression of thousands of proteins. As a result, miRNAs have emerged rapidly as a major new area of biomedical research with relevance to kidney disease. MiRNA expression has been shown to differ between the kidney and other organs as well as between different kidney regions. Furthermore, miRNAs have been found to be functionally important in models of podocyte development, diabetic nephropathy and polycystic kidney disease. Of particular interest, podocyte-specific deletion of Dicer, a key enzyme in the biogenesis of miRNA, results in proteinuria and severe renal impairment in mice. One miRNA (miR-192) can also act as an effector of transforming growth factor-, activity in the high-glucose environment of diabetic nephropathy. Differential expression of miRNAs has been reported in kidney allograft rejection. It is anticipated that future studies involving miRNAs will generate new insights into the complex pathophysiology underlying various kidney diseases, generate diagnostic biomarkers and might be of value as therapeutic targets for progressive kidney diseases. The purpose of this review is to highlight key miRNA developments in kidney diseases and how this might influence the diagnosis and management of patients with kidney disease in the future. [source] Characterization of the RSL1-dependent conditional expression system in LNCaP prostate cancer cells and development of a single vector formatTHE PROSTATE, Issue 8 2007Julie Lessard Abstract Background Conditional expression systems are useful tools for the study of gene function but the use of these systems in prostate cancer cells is limited by the undesired biological effects of the inducing ligands. The RheoSwitch system employs RheoSwitch Ligand 1 (RSL1), a non-steroidal analog of the insect hormone ecdysone, to activate a modified nuclear receptor heterodimer that controls target gene expression via GAL4 response elements. This system has not been tested in prostate cancer cells. Methods We established LNCaP human prostate cancer cell lines that constitutively express RheoSwitch transcription factors to quantify RSL1-dependent expression and assess the effects of RSL1 on cell proliferation and endogenous gene expression. Potential RSL1-responsive genes were identified using Affymetrix microarrays and validated by Northern blot hybridization. A single-vector format was developed to establish cell lines that conditionally produce a recombinant protein. Results Stable cell lines displayed tight and potent (over several orders of magnitude) RSL1-dependent regulation of a transiently transfected luciferase reporter gene. RSL1 did not affect basal or androgen-stimulated cell proliferation and exerted minimal effects on the expression of endogenous genes. Cell lines established using the single-vector system also displayed strictly RSL1-dependent production of the recombinant protein encoded by the stably integrated RSL1-responsive expression cassette. Conclusions The RheoSwitch system is well suited for conditional gene expression in prostate cancer cells. The single-vector format should facilitate the production of stable cell lines. This system should be useful for the study of proteins involved in prostate cancer in both cell and animal models of the disease. Prostate 67: 808,819, 2007. © 2007 Wiley-Liss, Inc. [source] Androgen receptor corepressors: An overviewTHE PROSTATE, Issue 2 2005Liang Wang Androgens play pivotal roles in sex differentiation and development, in reproductive functions, and sexual behavior. The actions of androgens are mediated through the intracellular androgen receptor (AR), a member of the nuclear receptor (NR) superfamily, which regulates a wide range of target gene expression. Recent studies indicate that the proper transcriptional activity of AR is modulated by AR coregulators, including coactivators that can enhance AR transactivation and corepressors that can suppress AR transactivation. Here, we summarize the recent discoveries relating to AR corepressor function with the following different mechanisms: (1) corepressors that inhibit the DNA binding or nuclear translocation of AR; (2) corepressors that recruit histone deacetylases; (3) corepressors that interrupt the interaction between AR and its coactivators; (4) corepressors that interrupt the interaction between the N-terminus and C-terminus of AR; (5) corepressors that function as scaffolds for other AR coregulators; (6) corepressors that target the basal transcriptional machinery; (7) other mechanisms. The potential impact and future directions of AR corepressors are also discussed. © 2004 Wiley-Liss, Inc. [source] MicroRNA-146a contributes to abnormal activation of the type I interferon pathway in human lupus by targeting the key signaling proteinsARTHRITIS & RHEUMATISM, Issue 4 2009Yuanjia Tang Objective MicroRNA have recently been identified as regulators that modulate target gene expression and are involved in shaping the immune response. This study was undertaken to investigate the contribution of microRNA-146a (miR-146a), which was identified in the pilot expression profiling step, to the pathogenesis of systemic lupus erythematosus (SLE). Methods TaqMan microRNA assays of peripheral blood leukocytes were used for comparison of expression levels of microRNA between SLE patients and controls. Transfection and stimulation of cultured cells were conducted to determine the biologic function of miR-146a. Bioinformatics prediction and validation by reporter gene assay and Western blotting were performed to identify miR-146a targets. Results Profiling of 156 miRNA in SLE patients revealed the differential expression of multiple microRNA, including miR-146a, a negative regulator of innate immunity. Further analysis showed that underexpression of miR-146a negatively correlated with clinical disease activity and with interferon (IFN) scores in patients with SLE. Of note, overexpression of miR-146a reduced, while inhibition of endogenous miR-146a increased, the induction of type I IFNs in peripheral blood mononuclear cells (PBMCs). Furthermore, miR-146a directly repressed the transactivation downstream of type I IFN. At the molecular level, miR-146a could target IFN regulatory factor 5 and STAT-1. More importantly, introduction of miR-146a into the patients' PBMCs alleviated the coordinate activation of the type I IFN pathway. Conclusion The microRNA miR-146a is a negative regulator of the IFN pathway. Underexpression of miR-146a contributes to alterations in the type I IFN pathway in lupus patients by targeting the key signaling proteins. The findings provide potential novel strategies for therapeutic intervention. [source] HLA,B27 up-regulation causes accumulation of misfolded heavy chains and correlates with the magnitude of the unfolded protein response in transgenic rats: Implications for the pathogenesis of spondylarthritis-like diseaseARTHRITIS & RHEUMATISM, Issue 1 2007Matthew J. Turner Objective HLA,B27 is implicated in the pathogenesis of spondylarthritis (SpA), yet the molecular mechanisms are incompletely defined. HLA,B27 misfolding has been associated with endoplasmic reticulum stress and activation of the unfolded protein response (UPR) in macrophages from HLA,B27/human ,2 -microglobulin,transgenic (B27-transgenic) rats. This study was performed to assess the mechanisms that drive activation of the HLA,B27,induced UPR and to determine whether splenocytes respond in a similar manner. Methods Splenocytes were isolated and bone marrow macrophages were derived from B27-transgenic and wild-type rats. Cells were treated for up to 24 hours with cytokines that induce class I major histocompatibility complex expression. HLA,B27 expression and misfolding were assessed by real-time reverse transcription,polymerase chain reaction, flow cytometry, and immunoblotting. Activation of the UPR was measured by quantifying UPR target gene expression and X-box binding protein 1 messenger RNA (mRNA) splicing. Results HLA,B27 mRNA up-regulation was accompanied by a dramatic increase in the accumulation of misfolded heavy chains and preceded robust activation of the UPR in macrophages. When macrophages were treated with various cytokines, the magnitude of the UPR correlated strongly with the degree of HLA,B27 up-regulation. In contrast, B27-transgenic splenocytes exhibited only low-level differences in the expression of UPR target genes after exposure to interferon-, or concanavalin A, which resulted in minimal HLA,B27 up-regulation. Conclusion These results suggest that HLA,B27,associated activation of the UPR in macrophages is attributable to the accumulation of misfolded heavy chains, and that certain cell types may be more susceptible to the effects of HLA,B27 misfolding. Strategies that eliminate HLA,B27 up-regulation and/or the accumulation of misfolded heavy chains may be useful in evaluating the role of these events in the pathogenesis of SpA. [source] Auraptene, a citrus fruit compound, regulates gene expression as a PPAR, agonist in HepG2 hepatocytesBIOFACTORS, Issue 1 2008Nobuyuki Takahashi Abstract Citrus fruit compounds have various activities that improve pathological conditions in many tissues. In this study, we examined the effect of auraptene contained mainly in the peel of citrus on peroxisome proliferator-activated receptor-, (PPAR,) activation. To examine effects of auraptene on the PPAR, activation in hepatocytes, PPAR ligand assay system was developed using HepG2 hepatocytes, in which the endogenous PPAR, expression level is very low. In the PPAR ligand assay, the addition of auraptene showed significant effects on the transactivation of GAL4/PPAR, chimera proteins in a dose-dependent manner. Actually, treatment with auraptene induced the up-regulation of PPAR target genes, such as acyl-CoA oxidase (ACO), carnitine-palmitoyl transferase 1A (CPT1A) and acyl-CoA synthetase (ACS), in PPAR,-expressing HepG2 hepatocytes. The regulation of gene expression was dependent on PPAR, because mock-transfected HepG2 hepatocytes showed no regulation. The up-regulation of PPAR target gene expression by auraptene was sufficient to enhance oleic acid uptake into PPAR,-expressing HepG2 hepatocytes. These results indicate that auraptene acts as a PPAR, agonist in hepatocytes and that auraptene may improve lipid abnormality through PPAR, activation in the liver. [source] Mechanisms of constitutive activation of Janus kinase 2-V617F revealed at the atomic level through molecular dynamics simulationsCANCER, Issue 8 2009Tai-Sung Lee PhD Abstract BACKGROUND: The tyrosine kinase Janus kinase 2 (JAK2) is important in triggering nuclear translocation and regulation of target genes expression through signal transducer and activator of transcription pathways. The valine-to-phenylalanine mutation at amino acid 617 (V617F), which results in the deregulation of JAK2, has been implicated in the oncogenesis of chronic myeloproliferative disease. However, both the mechanism of JAK2 autoinhibition and the mechanism of V617F constitutive activation remain unclear. METHOD: In this work, the authors used molecular dynamics simulation techniques to establish plausible mechanisms of JAK2 autoinhibition and V617F constitutive activation at the atomic level. RESULTS: In wild-type JAK2, the activation loop of JAK2-homology domain 1 (JH1) is pulled toward the JH1/JH2 interface through interactions with key residues of JH2, especially S591, F595, and V617, and stabilizes the inactivated form of JH1. In the case of V617F, through the aromatic ring-ring stacking interaction, F617 blocks the interaction of JH1 the activation loop, S591, and F595, thus causing the JH1 activation loop to move back to its activated form. CONCLUSIONS: The current results indicated that this simulation-derived mechanism of JAK2 autoregulation is consistent with current available experimental evidence and may lead to a deeper understanding of JAK2 and other kinase systems that are regulated by pseudokinases. Cancer 2009. © 2009 American Cancer Society. [source] Growth and phenotype of potato plants expressing an antisense gene of P-protein of glycine decarboxylase under control of a promoter with preference for the mesophyllANNALS OF APPLIED BIOLOGY, Issue 1 2001T WINZER Summary A cDNA encoding P-protein of glycine decarboxylase was expressed in antisense orientation in leaves of potato (Solanum tuberosum cv. Solara) under control of the promoter of a P-protein gene of glycine decarboxylase from Flaveria pringlei. This promoter targets gene expression preferentially to the leaf mesophyll cells. In two of the transgenic lines, mitochondria oxidise glycine only with extremely low rates. Phenotypically, these transgenic lines were only marginally different from wild type plants under ambient carbon dioxide concentrations and indistinguishable from wild type plants when grown under 800 ppm carbon dioxide. When grown in ambient carbon dioxide, transgenic plants accumulated high amounts of glycine during the light period followed by nearly complete degradation in the following night. [source] | |||||||||||||