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Vertebrate Cells (vertebrate + cell)
Selected AbstractsMyosin Va phosphorylated on Ser1650 is found in nuclear speckles and redistributes to nucleoli upon inhibition of transcriptionCYTOSKELETON, Issue 6 2008Maria Cristina S. Pranchevicius Abstract Nuclear actin and nuclear myosins have been implicated in the regulation of gene expression in vertebrate cells. Myosin V is a class of actin-based motor proteins involved in cytoplasmic vesicle transport and anchorage, spindle-pole alignment and mRNA translocation. In this study, myosin-Va, phosphorylated on a conserved serine in the tail domain (phospho-ser1650 MVa), was localized to subnuclear compartments. A monoclonal antibody, 9E6, raised against a peptide corresponding to phosphoserine1650 and flanking regions of the murine myosin Va sequence, was immunoreactive to myosin Va heavy chain in cellular and nuclear extracts of HeLa cells, PC12 cells and B16-F10 melanocytes. Immunofluorescence microscopy with this antibody revealed discrete irregular spots within the nucleoplasm that colocalized with SC35, a splicing factor that earmarks nuclear speckles. Phospho-ser1650 MVa was not detected in other nuclear compartments, such as condensed chromatin, Cajal bodies, gems and perinucleolar caps. Although nucleoli also were not labeled by 9E6 under normal conditions, inhibition of transcription in HeLa cells by actinomycin D caused the redistribution of phospho-ser1650 MVa to nucleoli, as well as separating a fraction of phospho-ser1650 MVa from SC35 into near-neighboring particles. These observations indicate a novel role for myosin Va in nuclear compartmentalization and offer a new lead towards the understanding of actomyosin-based gene regulation. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source] Structural and functional effects of hydrostatic pressure on centrosomes from vertebrate cellsCYTOSKELETON, Issue 4 2001A. Rousselet Abstract In an attempt to better understand the role of centrioles in vertebrate centrosomes, hydrostatic pressure was applied to isolated centrosomes as a means to disassemble centriole microtubules. Treatments of the centrosomes were monitored by analyzing their protein composition, ultrastructure, their ability to nucleate microtubules from pure tubulin, and their capability to induce parthenogenetic development of Xenopus eggs. Moderate hydrostatic pressure (95 MPa) already affected the organization of centriole microtubules in isolated centrosomes, and also impaired microtubule nucleation. At higher pressure, the protein composition of the peri-centriolar matrix (PCM) was also altered and the capacity to nucleate microtubules severely impaired. Incubation of the treated centrosomes in Xenopus egg extract could restore their capacity to nucleate microtubules after treatment at 95 MPa, but not after higher pressure treatment. However, the centriole structure was in no case restored. It is noteworthy that centrosomes treated with mild pressure did not allow parthenogenetic development after injection into Xenopus eggs, even if they had recovered their capacity to nucleate microtubules. This suggested that, in agreement with previous results, centrosomes in which centriole architecture is impaired, could not direct the biogenesis of new centrioles in Xenopus eggs. Centriole structure could also be affected by applying mild hydrostatic pressure directly to living cells. Comparison of the effect of hydrostatic pressure on cells at the G1/S border or on the corresponding cytoplasts suggests that pro-centrioles are very sensitive to pressure. However, cells can regrow a centriole after pressure-induced disassembly. In that case, centrosomes eventually recover an apparently normal duplication cycle although with some delay. Cell Motil. Cytoskeleton 48:262,276, 2001. © 2001 Wiley-Liss, Inc. [source] Development of a solvent-free, solid-phase in vitro bioassay using vertebrate cellsENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 5 2006Stephanie K. Bopp Abstract Miniaturized bioassays offer many advantages in exploring the toxic potential of chemicals, including small sample volumes and compatibility with high-throughput screening. One problem common to miniaturized systems, however, is the loss of test chemicals because of sorption. The idea of the current study was to use the sorption phenomenon in a positive way. It was found that contaminants sorbed to the growth surface in wells of tissue-culture plates or to the surface of selected sorbent bead materials are available to vertebrate cells growing in direct contact with the contaminant-coated surface. The use of beads provided more flexibility with regard to surface area, materials, and assay format. Biosilon, a bead cell-culture carrier made of polystyrene, was found to be most suitable. It supported cell adherence and allowed the detection of reproducible dose-response curves of an increase in cytochrome CYP1A enzyme activity by sorbed polycyclic aromatic hydrocarbons in the rainbow trout (Oncorhynchus mykiss) liver cell line, RTL-W1. The resulting bead assay provides a miniaturized, solvent-free exposure system. Potential future applications include the coupling to environmental sampling, in which the bead material is used as solid receiving phase before serving as a surface for vertebrate cells to attach and respond. [source] Porous silicon substrates for neurons culturing and bio-photonic sensingPHYSICA STATUS SOLIDI (A) APPLICATIONS AND MATERIALS SCIENCE, Issue 8 2005S. Ben-Tabou de-Leon Abstract In this work we report on culturing of Aplysia neurons and vertebrate cells to porous silicon substrates and on the first steps toward characterizing porous silicon as a biosensor of neural activity. Neurons cultured on porous silicon substrates survived for at least one week showing normal passive membrane properties and generation of action potentials. We have investigated several mechanisms that take advantage of the optical properties of porous silicon for transducing both electrical and chemical neuronal activities into photonic signals. For example, the photoluminescence response to voltage and the reflectivity response to chemical changes were investigated. (© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Making sense of cilia in disease: The human ciliopathies,AMERICAN JOURNAL OF MEDICAL GENETICS, Issue 4 2009Kate Baker Abstract Ubiquitous in nature, cilia and flagella comprise nearly identical structures with similar functions. The most obvious example of the latter is motility: driving movement of the organism or particle flow across the epithelial surface in fixed structures. In vertebrates, such motile cilia are evident in the respiratory epithelia, ependyma, and oviducts. For over a century, non-motile cilia have been observed on the surface of most vertebrate cells but until recently their function has eluded us. Gathering evidence now points to critical roles for the mono-cilium in sensing the extracellular environment, and perturbation of this function gives rise to a predictable panoply of clinical problems. We review the common clinical phenotypes associated with ciliopathies and interrogate Online Mendelian Inheritance in Man (OMIM) to compile a comprehensive list of putative disorders in which ciliary dysfunction may play a role. © 2009 Wiley-Liss, Inc. [source] Influence of cell cycle on ecdysteroid receptor in CHO-K1 cellsARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 3 2009Katarzyna Betanska Abstract CHO-K1 cells are routinely used for characterization of ecdysone receptor (EcR) function, because these vertebrate cells are devoid of endogenous ecdysone receptor protein. Moreover, the endogenous expression of RXR, the vertebrate orthologue of Ultraspiracle (Usp), the most important heterodimerization partner, is neglectable. In contrast to insect cells, there is also no influence of moulting hormone on CHO-K1 cells on cell proliferation either in the absence or presence of transiently expressed EcR. In contrast to Usp, which is exclusively found in nuclei, EcR is heterogeneously distributed between cytoplasm and nuclei in non-synchronized cells. Synchronization of CHO-K1 cells by nocodazole revealed that the cell cycle influences receptor concentration with lowest amounts in late S-phase and G2/M phase and intracellular distribution of the receptor protein showing a minimum of receptors present in nuclei during S-phase. EcR, but not Usp reduces cyclin D1 expression and cyclin D1 concentration is impaired by cyclin D1. Coimmunoprecipitation studies reveal physical interaction of EcR and cyclin D1. © 2009 Wiley Periodicals, Inc. [source] DNA-binding properties of Drosophila ecdysone receptor isoforms and their modification by the heterodimerization partner ultraspiracleARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 3 2009Simone Braun Abstract Transcriptional activity of ecdysone receptor (EcR) isoforms varies considerably and is modified further by the heterodimerization partner and hormone treatment. To investigate whether differences in DNA binding of receptor complexes are responsible for these variations in transcriptional activity, interaction of Drosophila EcR isoforms, and variants of Ultraspiracle (Usp), the orthologue of RXR, with the ecdysone response elements (EcRE) hsp 27, PAL-1, and DR-1, were determined by electrophoretic mobility shift assays. Receptor proteins were expressed in vertebrate cells (CHO-K1) in order to rule out an influence of endogenous receptor proteins. In the absence of a heterodimerization partner, weak DNA binding of EcR was detected even without hormone with EcR-A and -B1, but not EcR-B2. In the presence of hormone, all three isoforms show increased binding to the hsp 27 EcRE. The heterodimerization partner Usp increased DNA binding considerably. The hormone effect of heterodimers is more pronounced with both EcR-B isoforms compared to EcR-A. Two specific bands were obtained for EcR-A and B1 but only one band is visible with EcR-B2. Deletion of the C-domain of Usp still allows basal DNA binding of the heterodimer, but in contrast to full-length Usp, addition of hormone decreases the intensity of the retarded receptor band of all EcR isoforms and the EcREs hsp27 and DR-1 considerably, whereas interaction with the EcRE PAL-1 is only slightly affected. Synergistic effects on transcriptional activity are associated with the formation of different receptor DNA-complexes observed with 1×hsp27 and 3×hsp27. Comparison of DNA-binding properties of EcR isoforms and EcR/Usp heterodimers revealed that binding of receptor complexes to hsp 27 EcRE is dependent on the AB domain of EcR and the AB-, C-, and D-domains of the heterodimerization partner. Interaction with the hsp 27 EcRE correlates neither with ligand binding nor with transcriptional activity of the various receptor complexes. We, therefore, conclude that the different receptor functions are regulated separately, for example, by interaction with co-modulators or post-transcriptional modifications. © 2009 Wiley Periodicals, Inc. [source] Impact of heterodimerization on intracellular localization of the ecdysteroid receptor (EcR)ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 1 2008Claudia Nieva Abstract Initially, nuclear import of the ecdysteroid receptor (EcR) in vertebrate cells (CHO-K1 and COS-7) does not afford a heterodimerization partner. Later on, EcR is retained in the nucleus only in the presence of a heterodimerization partner. Ultraspiracle (Usp) is more efficient compared to its vertebrate orthologue RXR and leads to an exclusively nuclear localization of EcR even in the absence of ligand. The DNA binding domain of the heterodimerization partner is important for retainment of EcR in the nucleus as shown by Usp4 (UspR130C), which has lost its DNA binding capability. The C-terminal end of Usp (Usp,205-508) encompassing the C-terminal part of the D-domain and the E- and F-domains are essential for retainment of EcR in the nucleus. Nuclear localization is further influenced by cell-specific factors, since hormone and heterodimerization stabilizes the EcR protein in a cell-specific way. Arch. Insect Biochem. Physiol. 2008. © 2008 Wiley-Liss, Inc. [source] What can humans learn from flies about adenomatous polyposis coli?BIOESSAYS, Issue 9 2002Angela I.M. Barth Somatic or inherited mutations in the adenomatous polyposis coli (APC) gene are a frequent cause of colorectal cancer in humans. APC protein has an important tumor suppression function to reduce cellular levels of the signaling protein ,-catenin and, thereby, inhibit ,-catenin and T-cell-factor-mediated gene expression. In addition, APC protein binds to microtubules in vertebrate cells and localizes to actin-rich adherens junctions in epithelial cells of the fruit fly Drosophila (Fig. 1). Very little is known, however, about the function of these cytoskeletal associations. Recently, Hamada and Bienz have described a potential role for Drosophila E-APC in cellular adhesion,1 which offers new clues to APC function in embryonic development, and potentially colorectal adenoma formation and tumor progression in humans. BioEssays 24:771,774, 2002. © 2002 Wiley Periodicals, Inc. [source] | ||||||||||