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Continuous Expression (continuous + expression)
Selected AbstractsThe E8 repression domain can replace the E2 transactivation domain for growth inhibition of HeLa cells by papillomavirus E2 proteinsINTERNATIONAL JOURNAL OF CANCER, Issue 10 2007Frank Stubenrauch Abstract Continuous expression of the human papillomavirus (HPV) oncoproteins E6 and E7 is required for the growth of cervical cancer cell lines. So far, only the overexpression of the wild type papillomavirus E2 protein has been shown to induce growth arrest in HPV18-positive HeLa cells by repressing E6/E7 transcription. Growth arrest by E2 requires the aminoterminal transcription activation domain in addition to the carboxyterminal DNA-binding domain. Several papillomaviruses such as the carcinogenic HPV31 express in addition to E2 an E8,E2C fusion protein in which the E8 domain, which is required for repression of replication and transcription, replaces the E2 activation domain. In this report, we demonstrate that the HPV31 E8,E2C protein is able to inhibit the growth of HeLa cells but not of HPV-negative C33A cervical cancer cells. Growth repression by E8,E2C correlates with repression of the endogenous HPV18 E6/E7 promoter and the reappearance of E6- and E7-regulated p53, pRb and p21 proteins, suggesting that E8,E2C inhibits growth by reactivating dormant tumor suppressor pathways. Growth inhibition requires an intact E8 repression domain in addition to the carboxyterminal E2C DNA binding domain. Chromatin immunoprecipitation experiments suggest that the E8 repression domain enhances binding to the HPV18 promoter sequence in vivo. In summary, our results demonstrate that the small E8 repression domain can functionally replace the large E2 transactivation domain for growth inhibition of HeLa cervical cancer cells. © 2007 Wiley-Liss, Inc. [source] A role for protein kinase CK2 in plant development: evidence obtained using a dominant-negative mutantTHE PLANT JOURNAL, Issue 1 2008Jordi Moreno-Romero Summary Protein kinase CK2 is an evolutionary conserved Ser/Thr phosphotransferase composed of two distinct subunits, , (catalytic) and , (regulatory), that combine to form a tetrameric complex. Plant genomes contain multiple genes for each subunit, the expression of which gives rise to different active holoenzymes. In order to study the effects of loss of function of CK2 on plant development, we have undertaken a dominant-negative mutant approach. We generated an inactive catalytic subunit by site-directed mutagenesis of an essential lysine residue. The mutated open reading frame was cloned downstream of an inducible promoter, and stably transformed Arabidopsis thaliana plants and tobacco BY2 cells were isolated. Continuous expression of the CK2 kinase-inactive subunit did not prevent seed germination, but seedlings exhibited a strong phenotype, affecting chloroplast development, cotyledon expansion, and root and shoot growth. Prolonged induction of the transgene was lethal. Moreover, dark-germinated seedlings exhibited an apparent de-etiolated phenotype that was not caused by disruption of the light-signalling pathways. Short-term induction of the CK2 kinase-inactive subunit allowed plant survival, but root growth and lateral root formation were significantly affected. The expression pattern of CYCB1;1::GFP in the root meristems of mutant plants demonstrated an important decrease of mitotic activity, and expression of the CK2 kinase-inactive subunit in stably transformed BY2 cells provoked perturbation of the G1/S and G2 phases of the cell cycle. Our results are consistent with a model in which CK2 plays a key role in cell division and cell expansion, with compelling effects on Arabidopsis development. [source] Continuous expression in tobacco leaves of a Brassica napus PEND homologue blocks differentiation of plastids and development of palisade cellsTHE PLANT JOURNAL, Issue 1 2005Paul Wycliffe Summary Brassica napus complementary deoxyribonucleic acid (cDNA) clones encoding a DNA-binding protein, BnPEND, were isolated by Southwestern screening. A distinctive feature of the protein was a bZIP-like sequence in the amino-terminal portion, which, after expression in Escherichia coli, bound DNA. BnPEND transcripts were present in B. napus roots and flower buds, and to a lesser extent in stems, flowers and young leaves. Treatment in the dark for 72 h markedly increased the amount of BnPEND transcript in leaves of all ages. Sequence comparison showed that BnPEND was similar to a presumed transcription factor from B. napus, GSBF1, a protein deduced from an Arabidopsis thaliana cDNA (BX825084) and the PEND protein from Pisum sativum, believed to anchor the plastid DNA to the envelope early during plastid development. Homology to expressed sequence tag (EST) sequences from additional species suggested that BnPEND homologues are widespread among the angiosperms. Transient expression of BnPEND fused with green fluorescent protein (GFP) in Nicotiana benthamiana epidermal cells showed that BnPEND is a plastid protein, and that the 15 amino acids at the amino-terminal contain information about plastid targeting. Expression of BnPEND in Nicotiana tabacum from the Cauliflower Mosaic Virus 35S promoter gave stable transformants with different extents of white to light-green areas in the leaves, and even albino plants. In the white areas, but not in adjacent green tissue, the development of palisade cells and chloroplasts was disrupted. Our data demonstrate that the BnPEND protein, when over-expressed at an inappropriate stage, functionally blocks the development of plastids and leads to altered leaf anatomy, possibly by preventing the release of plastid DNA from the envelope. [source] Fast set-up of doxycycline-inducible protein expression in human cell lines with a single plasmid based on Epstein,Barr virus replication and the simple tetracycline repressorFEBS JOURNAL, Issue 3 2007Markus Bach We have developed a novel plasmid vector, pEBTetD, for full establishment of doxycycline-inducible protein expression by just a single transfection. pEBTetD contains an Epstein,Barr virus origin of replication for stable and efficient episomal propagation in human cell lines, a cassette for continuous expression of the simple tetracycline repressor, and a cytomegalovirus-type 2 tetracycline operator (tetO2)-tetO2 promoter. As there is no integration of vector into the genome, clonal isolation of transfected cells is not necessary. Cells are thus ready for use 1 week after transfection; this contrasts with 3,12 weeks for other systems. Adequate regulation of protein expression was accomplished by abrogation of mRNA polyadenylation. In northern analysis of seven cDNAs coding for transport proteins, pools of transfected human embryonic kidney 293 cells showed on/off mRNA ratios in the order of 100 : 1. Cell pools were also analyzed for regulation of protein function. With two transport proteins of the plasma membrane, the on/off activity ratios were 24 : 1 and 34 : 1, respectively. With enhanced green fluorescent protein, a 23 : 1 ratio was observed based on fluorescence intensity data from flow cytometry. The unique advantage of our system rests on the unmodified tetracycline repressor, which is less likely, by relocation upon binding of doxycycline, to cause cellular disturbances than chimera of tetracycline repressor and eukaryotic transactivation domains. Thus, in a comprehensive comparison of on- and off-states, a steady cellular background is provided. Finally, in contrast to a system based on Flp recombinase, the set-up of our system is inherently reliable. [source] Ultrastructural and immunocytochemical characterization of immortalized odontoblast MO6-G3INTERNATIONAL ENDODONTIC JOURNAL, Issue 6 2006C. Mesgouez Abstract Aim, To investigate an immortalized murine odontoblast cell line as a potential alternative for experimental studies on dentinogenesis. Methodology, The MO6-G3 cell line was investigated morphologically over 3, 7, 11 and 42 days of culture, using histochemical localization of dentine sialoprotein (DSP), alkaline phosphatase (AP), type I collagen and actin filaments, histoenzymatic staining and biochemical investigation of AP and finally, transmission and scanning electron microscopy. Results, Scanning electron micrographs showed elongated cells. Accordingly, a polarized organization of odontoblasts was observed by transmission electron microscopy, identifying distinct subcellular compartments as described in vivo. The secretion apparatus, which includes cisternae of rough endoplasmic reticulum, Golgi apparatus saccules and secretion vesicles and granules, was longitudinally organized in the supranuclear compartment ending distally in the secretory pole. A cellular process was observed. The investigation of the cytoskeleton network revealed that actin microfilaments were organized in parallel stress fibre oriented depending on the longitudinal axis of the cytoplasm. Immunofluorescent labelling showed a continuous expression of type I collagen, DSP and AP. A unipolar distribution characterized intracellular DSP immunoreactivity. Histoenzymology revealed AP active sites increasing from 3 to 11 days albeit with a moderate level of activity comparatively to the in vivo situation in dental cells. Conclusion, This cell line MO6-G3 not only showed the criteria of odontoblast phenotype as previously reported but also the characteristic morphodifferentiation pattern of polarized odontoblasts at the cellular level but with an apparent random distribution. [source] Targeted Expression of SHH Affects Chondrocyte Differentiation, Growth Plate Organization, and Sox9 Expression,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2004Sara Tavella Abstract The role of Hedgehogs (Hh) in murine skeletal development was studied by overexpressing human Sonic Hedgehog (SHH) in chondrocytes of transgenic mice using the collagen II promoter/enhancer. Overexpression caused a lethal craniorachischisis with major alterations in long bones because of defects in chondrocyte differentiation. Introduction: Hedgehogs (Hhs) are a family of secreted polypeptides that play important roles in vertebrate development, controlling many critical steps of cell differentiation and patterning. Skeletal development is affected in many different ways by Hhs. Genetic defects and anomalies of Hhs signaling pathways cause severe abnormalities in the appendicular, axial, and cranial skeleton in man and other vertebrates. Materials and Methods: Genetic manipulation of mouse embryos was used to study in vivo the function of SHH in skeletal development. By DNA microinjection into pronuclei of fertilized oocytes, we have generated transgenic mice that express SHH specifically in chondrocytes using the cartilage-specific collagen II promoter/enhancer. Transgenic skeletal development was studied at different embryonic stages by histology. The expression pattern of specific chondrocyte molecules was studied by immunohistochemistry and in situ hybridization. Results: Transgenic mice died at birth with severe craniorachischisis and other skeletal defects in ribs, sternum, and long bones. Detailed analysis of long bones showed that chondrocyte differentiation was blocked at prehypertrophic stages, hindering endochondral ossification and trabecular bone formation, with specific defects in different limb segments. The growth plate was highly disorganized in the tibia and was completely absent in the femur and humerus, leading to skeletal elements entirely made of cartilage surrounded by a thin layer of bone. In this cartilage, chondrocytes maintained a columnar organization that was perpendicular to the bone longitudinal axis and directed toward its outer surface. The expression of SHH receptor, Patched-1 (Ptc1), was greatly increased in all cartilage, as well as the expression of parathyroid hormone-related protein (PTHrP) at the articular surface; while the expression of Indian Hedgehog (Ihh), another member of Hh family that controls the rate of chondrocyte maturation, was greatly reduced and restricted to the displaced chondrocyte columns. Transgenic mice also revealed the ability of SHH to upregulate the expression of Sox9, a major transcription factor implicated in chondrocyte-specific gene expression, in vivo and in vitro, acting through the proximal 6.8-kb-long Sox9 promoter. Conclusion: Transgenic mice show that continuous expression of SHH in chondrocytes interferes with cell differentiation and growth plate organization and induces high levels and diffuse expression of Sox9 in cartilaginous bones. [source] BH3-only proteins Bid and BimEL are differentially involved in neuronal dysfunction in mouse models of Huntington's diseaseJOURNAL OF NEUROSCIENCE RESEARCH, Issue 12 2007Juan M. García-Martínez Abstract Apoptosis, a cell death mechanism regulated by Bcl-2 family members, has been proposed as one of the mechanisms leading to neuronal loss in Huntington's disease (HD). Here we examined the regulation of Bcl-2 family proteins in three different mouse models of HD with exon 1 mutant huntingtin: the R6/1, the R6/1:BDNF+/,, and the Tet/HD94 in which the huntingtin transgene is controlled by the tetracycline-inducible system. Our results disclosed an increase in the levels of the BH3-only proteins Bid and BimEL in the striatum of HD mouse models that was different depending on the stage of the disease. At 16 weeks of age, Bid was similarly enhanced in the striatum of R6/1 and R6/1:BDNF+/, mice, whereas BimEL protein levels were enhanced only in R6/1:BDNF+/, mice. In contrast, at later stages of the disease, both genotypes displayed increased levels of Bid and BimEL proteins. Furthermore, Bax, Bak, Bad, Bcl-2, and Bcl-xL proteins were not modified in any of the points analyzed. We next explored the potential reversibility of this phenomenon by analyzing conditional Tet/HD94 mice. Constitutive expression of the transgene resulted in increased levels of Bid and BimEL proteins, and only the Bid protein returned to wild-type levels 5 months after mutant huntingtin shutdown. In conclusion, our results show that enhanced Bid protein levels represent an early mechanism linked to the continuous expression of mutant huntingtin that, together with enhanced BimEL, may be a reporter of the progress and severity of neuronal dysfunction. © 2007 Wiley-Liss, Inc. [source] Molecular targeted therapies for diffuse large B-cell lymphoma based on apoptosis profiles,THE JOURNAL OF PATHOLOGY, Issue 5 2010Saskia AGM Cillessen Abstract Diffuse large B-cell lymphoma (DLBCL) is the most common type of adult non-Hodgkin lymphoma and is treated with chemotherapy in combination with rituximab. Despite this aggressive therapy, the disease is fatal in 30,40% of patients. Inhibition of the apoptosis signalling pathways is strongly related to response to chemotherapy and eventual clinical outcome. In order to survive, lymphoma cells depend on disruption of the apoptosis pathway by mutations in apoptosis inducing genes or by continuous expression of anti-apoptotic proteins. The development of molecules targeting these apoptosis inhibitors provides a very promising opportunity to specifically target tumour cells without toxicity to non-malignant cells in DLBCL patients. Sensitivity for most of these antagonists can be predicted based on biological markers, suggesting the possibility of pre-defining patients who will most likely benefit from these targeted therapies. Experimental therapies aimed at restoring the upstream apoptosis pathway or targeting apoptosis inhibitors are currently being tested in clinical trials and are expected to be effective particularly in chemotherapy-refractory DLBCL, providing hope for patients who are refractory to current therapies. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source] Combined analysis of intracellular signalling and immunophenotype of human peripheral blood basophils by flow cytometry: a proof of conceptCLINICAL & EXPERIMENTAL ALLERGY, Issue 11 2007D. G. Ebo Summary Background The signal transduction pathways and control mechanisms involved in IgE-mediated basophil activation remain incompletely understood. Objectives To investigate whether basophilic intracellular signal transduction and immunophenotype can be analysed simultaneously by flow cytometry. Methods Basophils in whole blood were stimulated with anti-IgE and latex antigen at various concentrations and during different time courses. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) as a representative of the intracellular signal transduction pathway and surface expression of CD63 was assessed simultaneously flow cytometrically. The effect of pre-incubation with IL-3 was assessed. Results Stimulation of the basophils with anti-IgE and allergen induces a rapid phosphorylation of p38 MAPK that peaks between 1 and 5 min and returns to baseline levels after 60 min. In contrast, CD63 up-regulation demonstrates a maximal but more continuous expression that peaks approximately 5 min later than phosphorylation of p38 MAPK. Specific inhibition of p38 MAPK reduced or almost completely abrogated up-regulation of CD63. Pre-incubation of the basophils with IL-3 produces a rapid p38 MAPK phosphorylation over basal levels, but this was weaker and shorter than for anti-IgE stimulation. Pre-incubation of the basophils with IL-3 did not potentiate anti-IgE-induced phosphorylation of p38 MAPK and did affect spontaneous or IgE-mediated CD63 up-regulation. Conclusions This study provides the proof that the flow cytometer allows an integrated analysis of basophilic intracellular signalling and immunophenotyping. Owing to its technical simplicity, the low number of cells required and rapid analysis, the technique seems promising for use in the clinic as a diagnostic tool or to monitor therapy. Capsule summary This study is the first to provide evidence for a combined analysis of basophilic intracellular signalling and immunophenotyping by flow cytometry. Owing to its technical simplicity, the low number of cells required and rapid analysis, the technique seems promising for use in the clinic as a diagnostic tool or to monitor therapy. [source] | |||||||||||||