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Sample records for cytoplasmic cell compartments

  1. Myosin-Powered Membrane Compartment Drives Cytoplasmic Streaming, Cell Expansion and Plant Development.

    Science.gov (United States)

    Peremyslov, Valera V; Cole, Rex A; Fowler, John E; Dolja, Valerian V

    2015-01-01

    Using genetic approaches, particle image velocimetry and an inert tracer of cytoplasmic streaming, we have made a mechanistic connection between the motor proteins (myosins XI), cargo transported by these motors (distinct endomembrane compartment defined by membrane-anchored MyoB receptors) and the process of cytoplasmic streaming in plant cells. It is shown that the MyoB compartment in Nicotiana benthamiana is highly dynamic moving with the mean velocity of ~3 μm/sec. In contrast, Golgi, mitochondria, peroxisomes, carrier vesicles and a cytosol flow tracer share distinct velocity profile with mean velocities of 0.6-1.5 μm/sec. Dominant negative inhibition of the myosins XI or MyoB receptors using overexpression of the N. benthamiana myosin cargo-binding domain or MyoB myosin-binding domain, respectively, resulted in velocity reduction for not only the MyoB compartment, but also each of the tested organelles, vesicles and cytoplasmic streaming. Furthermore, the extents of this reduction were similar for each of these compartments suggesting that MyoB compartment plays primary role in cytosol dynamics. Using gene knockout analysis in Arabidopsis thaliana, it is demonstrated that inactivation of MyoB1-4 results in reduced velocity of mitochondria implying slower cytoplasmic streaming. It is also shown that myosins XI and MyoB receptors genetically interact to contribute to cell expansion, plant growth, morphogenesis and proper onset of flowering. These results support a model according to which myosin-dependent, MyoB receptor-mediated transport of a specialized membrane compartment that is conserved in all land plants drives cytoplasmic streaming that carries organelles and vesicles and facilitates cell growth and plant development.

  2. A review of reagents for fluorescence microscopy of cellular compartments and structures, Part III: reagents for actin, tubulin, cellular membranes, and whole cell and cytoplasm.

    Science.gov (United States)

    Kilgore, Jason A; Dolman, Nick J; Davidson, Michael W

    2014-01-02

    Non-antibody commercial fluorescent reagents for imaging of cytoskeletal structures have been limited primarily to tubulin and actin, with the main factor in choice based mainly on whether cells are live or fixed and permeabilized. A wider range of options exist for cell membrane dyes, and the choice of reagent primarily depends on the preferred localization in the cell (i.e., all membranes or only the plasma membrane) and usage (i.e., whether the protocol involves fixation and permeabilization). For whole-cell or cytoplasmic imaging, the choice of reagent is determined mostly by the length of time that the cells need to be visualized (hours or days) and by fixation status. Presented here is a discussion on choosing commercially available reagents for these cellular structures, with an emphasis on use for microscopic imaging, with a featured reagent for each structure, a recommended protocol, troubleshooting guide, and example image. Copyright © 2014 John Wiley & Sons, Inc.

  3. Polarized trafficking: the palmitoylation cycle distributes cytoplasmic proteins to distinct neuronal compartments.

    Science.gov (United States)

    Tortosa, Elena; Hoogenraad, Casper C

    2018-02-01

    In neurons, polarized cargo distribution occurs mainly between the soma and axonal and dendritic compartments, and requires coordinated regulation of cytoskeletal remodeling and membrane trafficking. The Golgi complex plays a critical role during neuronal polarization and secretory trafficking has been shown to differentially transport proteins to both axons and dendrites. Besides the Golgi protein sorting, recent data revealed that palmitoylation cycles are an efficient mechanism to localize cytoplasmic, non-transmembrane proteins to particular neuronal compartments, such as the newly formed axon. Palmitoylation allows substrate proteins to bind to and ride with Golgi-derived secretory vesicles to all neuronal compartments. By allowing cytoplasmic proteins to 'hitchhike' on transport carriers in a non-polarized fashion, compartmentalized depalmitoylation may act as a selective retention mechanism. Copyright © 2018 Elsevier Ltd. All rights reserved.

  4. [The perichromatin compartment of the cell nucleus].

    Science.gov (United States)

    Bogoliubov, D S

    2014-01-01

    In this review, the data on the structure and composition of the perichromatin compartment, a special border area between the condensed chromatin and the interchromatin space of the cell nucleus, are discussed in the light of the concept of nuclear functions in complex nuclear architectonics. Morphological features, molecular composition and functions of main extrachromosomal structures of the perichromatin compartment, perichromatin fibrils (PFs) and perichromatin granules (PGs) including nuclear stress-bodies (nSBs) that are derivates of the PGs under heat shock, are presented. A special attention was paid to the features of the molecular compositions of PFs and PGs in different cell types and at different physiological conditions.

  5. Probing the biology of cell boundary conditions through confinement of Xenopus cell-free cytoplasmic extracts.

    Science.gov (United States)

    Bermudez, Jessica G; Chen, Hui; Einstein, Lily C; Good, Matthew C

    2017-01-01

    Cell-free cytoplasmic extracts prepared from Xenopus eggs and embryos have for decades provided a biochemical system with which to interrogate complex cell biological processes in vitro. Recently, the application of microfabrication and microfluidic strategies in biology has narrowed the gap between in vitro and in vivo studies by enabling formation of cell-size compartments containing functional cytoplasm. These approaches provide numerous advantages over traditional biochemical experiments performed in a test tube. Most notably, the cell-free cytoplasm is confined using a two- or three-dimensional boundary, which mimics the natural configuration of a cell. This strategy enables characterization of the spatial organization of a cell, and the role that boundaries play in regulating intracellular assembly and function. In this review, we describe the marriage of Xenopus cell-free cytoplasm and confinement technologies to generate synthetic cell-like systems, the recent biological insights they have enabled, and the promise they hold for future scientific discovery. © 2017 Wiley Periodicals, Inc.

  6. Cytoplasmic proteasomes are not indispensable for cell growth in Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Tsuchiya, Hikaru; Arai, Naoko; Tanaka, Keiji, E-mail: tanaka-kj@igakuken.or.jp; Saeki, Yasushi, E-mail: saeki-ys@igakuken.or.jp

    2013-07-05

    Highlights: •We succeeded to control the proteasome localization by the anchor-away technique. •Nuclear proteasome-depleted cells showed a lethal phenotype. •Cytoplasmic proteasomes are not indispensable for cell growth in dividing cells. -- Abstract: The 26S proteasome is an essential protease complex responsible for the degradation of ubiquitinated proteins in eukaryotic cells. In rapidly proliferating yeast cells, proteasomes are mainly localized in the nucleus, but the biological significance of the proteasome localization is still unclear. In this study, we investigated the relationship between the proteasome localization and the functions by the anchor-away technique, a ligand-dependent sequestration of a target protein into specific compartment(s). Anchoring of the proteasome to the plasma membrane or the ribosome resulted in conditional depletion of the nuclear proteasomes, whereas anchoring to histone resulted in the proteasome sequestration into the nucleus. We observed that the accumulation of ubiquitinated proteins in all the proteasome-targeted cells, suggesting that both the nuclear and cytoplasmic proteasomes have proteolytic functions and that the ubiquitinated proteins are produced and degraded in each compartment. Consistent with previous studies, the nuclear proteasome-depleted cells exhibited a lethal phenotype. In contrast, the nuclear sequestration of the proteasome resulted only in a mild growth defect, suggesting that the cytoplasmic proteasomes are not basically indispensable for cell growth in rapidly growing yeast cells.

  7. COMPARTMENTS

    DEFF Research Database (Denmark)

    Binder, Janos X; Pletscher-Frankild, Sune; Tsafou, Kalliopi

    2014-01-01

    of the localization of a protein, it is thus necessary to consult multiple databases and prediction tools. To address this, we present the COMPARTMENTS resource, which integrates all sources listed above as well as the results of automatic text mining. The resource is automatically kept up to date with source...

  8. Cytoplasmic Flow Enhances Organelle Dispersion in Eukaryotic Cells

    Science.gov (United States)

    Koslover, Elena; Mogre, Saurabh; Chan, Caleb; Theriot, Julie

    The cytoplasm of a living cell is an active environment through which intracellular components move and mix. We explore, using theoretical modeling coupled with microrheological measurements, the efficiency of particle dispersion via different modes of transport within this active environment. In particular, we focus on the role of cytoplasmic flow over different scales in contributing to organelle transport within two different cell types. In motile neutrophil cells, we show that bulk fluid flow associated with rapid cell deformation enhances particle transport to and from the cell periphery. In narrow fungal hyphae, localized flows due to hydrodynamic entrainment are shown to contribute to optimally efficient organelle dispersion. Our results highlight the importance of non-traditional modes of transport associated with flow of the cytoplasmic fluid in the distribution of organelles throughout eukaryotic cells.

  9. Hydrodynamic flow in the cytoplasm of plant cells.

    NARCIS (Netherlands)

    Esseling-Ozdoba, A.; Houtman, D.; Lammeren, A.A. van; Eiser, E.; Emons, A.M.C.

    2008-01-01

    Plant cells show myosin-driven organelle movement, called cytoplasmic streaming. Soluble molecules, such as metabolites do not move with motor proteins but by diffusion. However, is all of this streaming active motor-driven organelle transport? Our recent simulation study (Houtman et al., 2007)

  10. Hydrodynamic flow in the cytoplasm of plant cells

    NARCIS (Netherlands)

    Esseling-Ozdoba, A.; Houtman, D.; van Lammeren, A.A.M.; Eiser, E.; Emons, A.M.C.

    2008-01-01

    Plant cells show myosin-driven organelle movement, called cytoplasmic streaming. Soluble molecules, such as metabolites do not move with motor proteins but by diffusion. However, is all of this streaming active motor-driven organelle transport? Our recent simulation study (Houtman et al., 2007)

  11. Hydrodynamic flow in the cytoplasm of plant cells

    NARCIS (Netherlands)

    Esseling-Ozdoba, A.; Houtman, D.; Lammeren, van A.A.M.; Eiser, E.; Emons, A.M.C.

    2008-01-01

    Plant cells show myosin-driven organelle movement, called cytoplasmic streaming. Soluble molecules, such as metabolites do not move with motor proteins but by diffusion. However, is all of this streaming active motor-driven organelle transport? Our recent simulation study ( Houtman et al., 2007 )

  12. Algorithms for Cytoplasm Segmentation of Fluorescence Labelled Cells

    OpenAIRE

    Carolina Wählby; Joakim Lindblad; Mikael Vondrus; Ewert Bengtsson; Lennart Björkesten

    2002-01-01

    Automatic cell segmentation has various applications in cytometry, and while the nucleus is often very distinct and easy to identify, the cytoplasm provides a lot more challenge. A new combination of image analysis algorithms for segmentation of cells imaged by fluorescence microscopy is presented. The algorithm consists of an image pre?processing step, a general segmentation and merging step followed by a segmentation quality measurement. The quality measurement consists of a statistical ana...

  13. Amorphous areas in the cytoplasm of Dendrobium tepal cells

    Science.gov (United States)

    van Doorn, Wouter G.; Kirasak, Kanjana; Ketsa, Saichol

    2013-01-01

    In Dendrobium flowers some tepal mesophyll cells showed cytoplasmic areas devoid of large organelles. Such amorphous areas comprised up to about 40% of the cross-section of a cell. The areas were not bound by a membrane. The origin of these areas is not known. We show data suggesting that they can be formed from vesicle-like organelles. The data imply that these organelles and other material become degraded inside the cytoplasm. This can be regarded as a form of autophagy. The amorphous areas became surrounded by small vacuoles, vesicles or double membranes. These seemed to merge and thereby sequester the areas. Degradation of the amorphous areas therefore seemed to involve macroautophagy. PMID:23823702

  14. Algorithms for Cytoplasm Segmentation of Fluorescence Labelled Cells

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    Carolina Wählby

    2002-01-01

    Full Text Available Automatic cell segmentation has various applications in cytometry, and while the nucleus is often very distinct and easy to identify, the cytoplasm provides a lot more challenge. A new combination of image analysis algorithms for segmentation of cells imaged by fluorescence microscopy is presented. The algorithm consists of an image pre‐processing step, a general segmentation and merging step followed by a segmentation quality measurement. The quality measurement consists of a statistical analysis of a number of shape descriptive features. Objects that have features that differ to that of correctly segmented single cells can be further processed by a splitting step. By statistical analysis we therefore get a feedback system for separation of clustered cells. After the segmentation is completed, the quality of the final segmentation is evaluated. By training the algorithm on a representative set of training images, the algorithm is made fully automatic for subsequent images created under similar conditions. Automatic cytoplasm segmentation was tested on CHO‐cells stained with calcein. The fully automatic method showed between 89% and 97% correct segmentation as compared to manual segmentation.

  15. Isolation of cell nuclei using inert macromolecules to mimic the crowded cytoplasm.

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    Ronald Hancock

    Full Text Available Cell nuclei are commonly isolated and studied in media which include millimolar concentrations of cations, which conserve the nuclear volume by screening the negative charges on chromatin and maintaining its compaction. However, two factors question if these ionic conditions correctly reproduce the environment of nuclei in vivo: the small-scale motion and conformation of chromatin in vivo are not reproduced in isolated nuclei, and experiments and theory suggest that small ions in the cytoplasm are not free in the soluble phase but are predominantly bound to macromolecules. We studied the possible role in maintaining the structure and functions of nuclei in vivo of a further but frequently overlooked property of the cytoplasm, the crowding or osmotic effects caused by diffusible macromolecules whose concentration, measured in several studies, is in the range of 130 mg/ml. Nuclei which conserved their volume in the cell and their ultrastructure seen by electron microscopy were released from K562 cells in media containing the inert polymer 70 kDa Ficoll (50% w/v or 70 kDa dextran (35% w/v to replace the diffusible cytoplasmic molecules which were dispersed on cell lysis with digitonin, with 100 microM K-Hepes buffer as the only source of ions. Immunofluorescence labelling and experiments using cells expressing GFP-fusion proteins showed that internal compartments (nucleoli, PML and coiled bodies, foci of RNA polymerase II were conserved in these nuclei, and nascent RNA transcripts could be elongated. Our observations are consistent with the hypothesis that crowding by diffusible cytoplasmic macromolecules is a crucial but overlooked factor which supports the nucleus in vivo by equilibrating the opposing osmotic pressure cause by the high concentration of macromolecules in the nucleus, and suggest that crowded media provide more physiological conditions to study nuclear structure and functions. They may also help to resolve the long-standing paradox

  16. Actin and myosin regulate cytoplasm stiffness in plant cells: a study using optical tweezers

    NARCIS (Netherlands)

    Honing, van der H.S.; Ruijter, de N.C.A.; Emons, A.M.C.; Ketelaar, T.

    2010-01-01

    Here, we produced cytoplasmic protrusions with optical tweezers in mature BY-2 suspension cultured cells to study the parameters involved in the movement of actin filaments during changes in cytoplasmic organization and to determine whether stiffness is an actin-related property of plant cytoplasm.

  17. Multi-Scale Characean Experimental System: From Electrophysiology of Membrane Transporters to Cell-to-Cell Connectivity, Cytoplasmic Streaming and Auxin Metabolism

    Science.gov (United States)

    Beilby, Mary J.

    2016-01-01

    The morphology of characean algae could be mistaken for a higher plant: stem-like axes with leaf-like branchlets anchored in the soil by root-like rhizoids. However, all of these structures are made up of giant multinucleate cells separated by multicellular nodal complexes. The excised internodal cells survive long enough for the nodes to give rise to new thallus. The size of the internodes and their thick cytoplasmic layer minimize impalement injury and allow specific micro-electrode placement. The cell structure can be manipulated by centrifugation, perfusion of cell contents or creation of cytoplasmic droplets, allowing access to both vacuolar and cytoplasmic compartments and both sides of the cell membranes. Thousands of electrical measurements on intact or altered cells and cytoplasmic droplets laid down basis to modern plant electrophysiology. Furthermore, the giant internodal cells and whole thalli facilitate research into many other plant properties. As nutrients have to be transported from rhizoids to growing parts of the thallus and hormonal signals need to pass from cell to cell, Characeae possess very fast cytoplasmic streaming. The mechanism was resolved in the characean model. Plasmodesmata between the internodal cells and nodal complexes facilitate transport of ions, nutrients and photosynthates across the nodes. The internal structure was found to be similar to those of higher plants. Recent experiments suggest a strong circadian influence on metabolic pathways producing indole-3-acetic acid (IAA) and serotonin/melatonin. The review will discuss the impact of the characean models arising from fragments of cells, single cells, cell-to-cell transport or whole thalli on understanding of plant evolution and physiology. PMID:27504112

  18. Regulation of Cytoplasmic and Vacuolar Volumes by Plant Cells in Suspension Culture

    DEFF Research Database (Denmark)

    Owens, Trevor; Poole, Ronald J

    1979-01-01

    Quantitative microscopical measurements have been made of the proportion of cell volume occupied by cytoplasm in a cell suspension culture derived from cotyledons of bush bean (cv. Contender). On a 7-day culture cycle, the content of cytoplasm varies from 25% at the time of transfer to 45% at the...

  19. Clonal dominance and transplantation dynamics in hematopoietic stem cell compartments.

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    Peter Ashcroft

    2017-10-01

    Full Text Available Hematopoietic stem cells in mammals are known to reside mostly in the bone marrow, but also transitively passage in small numbers in the blood. Experimental findings have suggested that they exist in a dynamic equilibrium, continuously migrating between these two compartments. Here we construct an individual-based mathematical model of this process, which is parametrised using existing empirical findings from mice. This approach allows us to quantify the amount of migration between the bone marrow niches and the peripheral blood. We use this model to investigate clonal hematopoiesis, which is a significant risk factor for hematologic cancers. We also analyse the engraftment of donor stem cells into non-conditioned and conditioned hosts, quantifying the impact of different treatment scenarios. The simplicity of the model permits a thorough mathematical analysis, providing deeper insights into the dynamics of both the model and of the real-world system. We predict the time taken for mutant clones to expand within a host, as well as chimerism levels that can be expected following transplantation therapy, and the probability that a preconditioned host is reconstituted by donor cells.

  20. Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy reveal the cytoplasmic origination of loaded nuclear RISC in vivo in human cells.

    Science.gov (United States)

    Ohrt, Thomas; Mütze, Jörg; Staroske, Wolfgang; Weinmann, Lasse; Höck, Julia; Crell, Karin; Meister, Gunter; Schwille, Petra

    2008-11-01

    Studies of RNA interference (RNAi) provide evidence that in addition to the well-characterized cytoplasmic mechanisms, nuclear mechanisms also exist. The mechanism by which the nuclear RNA-induced silencing complex (RISC) is formed in mammalian cells, as well as the relationship between the RNA silencing pathways in nuclear and cytoplasmic compartments is still unknown. Here we show by applying fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS) in vivo that two distinct RISC exist: a large approximately 3 MDa complex in the cytoplasm and a 20-fold smaller complex of approximately 158 kDa in the nucleus. We further show that nuclear RISC, consisting only of Ago2 and a short RNA, is loaded in the cytoplasm and imported into the nucleus. The loaded RISC accumulates in the nucleus depending on the presence of a target, based on an miRNA-like interaction with impaired cleavage of the cognate RNA. Together, these results suggest a new RISC shuttling mechanism between nucleus and cytoplasm ensuring concomitant gene regulation by small RNAs in both compartments.

  1. Microparticle Shedding from Neural Progenitor Cells and Vascular Compartment Cells Is Increased in Ischemic Stroke.

    Science.gov (United States)

    Chiva-Blanch, Gemma; Suades, Rosa; Crespo, Javier; Peña, Esther; Padró, Teresa; Jiménez-Xarrié, Elena; Martí-Fàbregas, Joan; Badimon, Lina

    2016-01-01

    Ischemic stroke has shown to induce platelet and endothelial microparticle shedding, but whether stroke induces microparticle shedding from additional blood and vascular compartment cells is unclear. Neural precursor cells have been shown to replace dying neurons at sites of brain injury; however, if neural precursor cell activation is associated to microparticle shedding, and whether this activation is maintained at long term and associates to stroke type and severity remains unknown. We analyzed neural precursor cells and blood and vascular compartment cells microparticle shedding after an acute ischemic stroke. Forty-four patients were included in the study within the first 48h after the onset of stroke. The cerebral lesion size was evaluated at 3-7 days of the stroke. Circulating microparticles from neural precursor cells and blood and vascular compartment cells (platelets, endothelial cells, erythrocytes, leukocytes, lymphocytes, monocytes and smooth muscle cells) were analyzed by flow cytometry at the onset of stroke and at 7 and 90 days. Forty-four age-matched high cardiovascular risk subjects without documented vascular disease were used as controls. Compared to high cardiovascular risk controls, patients showed higher number of neural precursor cell- and all blood and vascular compartment cell-derived microparticles at the onset of stroke, and after 7 and 90 days. At 90 days, neural precursor cell-derived microparticles decreased and smooth muscle cell-derived microparticles increased compared to levels at the onset of stroke, but only in those patients with the highest stroke-induced cerebral lesions. Stroke increases blood and vascular compartment cell and neural precursor cell microparticle shedding, an effect that is chronically maintained up to 90 days after the ischemic event. These results show that stroke induces a generalized blood and vascular cell activation and the initiation of neuronal cell repair process after stroke. Larger cerebral lesions

  2. Microparticle Shedding from Neural Progenitor Cells and Vascular Compartment Cells Is Increased in Ischemic Stroke.

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    Gemma Chiva-Blanch

    Full Text Available Ischemic stroke has shown to induce platelet and endothelial microparticle shedding, but whether stroke induces microparticle shedding from additional blood and vascular compartment cells is unclear. Neural precursor cells have been shown to replace dying neurons at sites of brain injury; however, if neural precursor cell activation is associated to microparticle shedding, and whether this activation is maintained at long term and associates to stroke type and severity remains unknown. We analyzed neural precursor cells and blood and vascular compartment cells microparticle shedding after an acute ischemic stroke.Forty-four patients were included in the study within the first 48h after the onset of stroke. The cerebral lesion size was evaluated at 3-7 days of the stroke. Circulating microparticles from neural precursor cells and blood and vascular compartment cells (platelets, endothelial cells, erythrocytes, leukocytes, lymphocytes, monocytes and smooth muscle cells were analyzed by flow cytometry at the onset of stroke and at 7 and 90 days. Forty-four age-matched high cardiovascular risk subjects without documented vascular disease were used as controls.Compared to high cardiovascular risk controls, patients showed higher number of neural precursor cell- and all blood and vascular compartment cell-derived microparticles at the onset of stroke, and after 7 and 90 days. At 90 days, neural precursor cell-derived microparticles decreased and smooth muscle cell-derived microparticles increased compared to levels at the onset of stroke, but only in those patients with the highest stroke-induced cerebral lesions.Stroke increases blood and vascular compartment cell and neural precursor cell microparticle shedding, an effect that is chronically maintained up to 90 days after the ischemic event. These results show that stroke induces a generalized blood and vascular cell activation and the initiation of neuronal cell repair process after stroke. Larger

  3. Relative Roles of Gap Junction Channels and Cytoplasm in Cell-to-Cell Diffusion of Fluorescent Tracers

    Science.gov (United States)

    Safranyos, Richard G. A.; Caveney, Stanley; Miller, James G.; Petersen, Nils O.

    1987-04-01

    Intercellular (tissue) diffusion of molecules requires cytoplasmic diffusion and diffusion through gap junctional (or cell-to-cell) channels. The rates of tissue and cytoplasmic diffusion of fluorescent tracers, expressed as an effective diffusion coefficient, De, and a cytoplasmic diffusion coefficient, Dcyt, have been measured among the developing epidermal cells of a larval beetle, Tenebrio molitor L., to determine the contribution of the junctional channels to intercellular diffusion. Tracer diffusion was measured by injecting fluorescent tracers into cells and quantitating the rate of subsequent spread into adjacent cells. Cytoplasmic diffusion was determined by fluorescence photobleaching. These experiments show that gap junctional channels constitute approximately 70-80% of the total cell-to-cell resistance to the diffusion of organic tracers at high concentrations in this tissue. At low concentrations, however, the binding of tracer to cytoplasm slows down the cytoplasmic diffusion, which may limit intercellular diffusion.

  4. Water diffusion in cytoplasmic streaming in Elodea internodal cells under the effect of antimitotic agents.

    Science.gov (United States)

    Vorob'ev, Vladimir N; Anisimov, Alexander V; Dautova, Nailya R

    2008-07-01

    The translational displacement of the cytoplasmic water in Elodea stem cells resulting from protein motor activity was measured using the NMR method. A 24-h treatment with vincristine results in a reduction of the translational displacement of the cytoplasmic water. With a constant cytoplasmic streaming velocity, the dynamics of the translational displacement of the cytoplasmic water under the effect of taxol are characterized by a continuous increase at a concentration of 0.05 mM, and reaching a plateau at a concentration of 0.5 mM.

  5. Functional organization of cytoplasmic inclusion bodies in cells infected by respiratory syncytial virus.

    Science.gov (United States)

    Rincheval, Vincent; Lelek, Mickael; Gault, Elyanne; Bouillier, Camille; Sitterlin, Delphine; Blouquit-Laye, Sabine; Galloux, Marie; Zimmer, Christophe; Eleouet, Jean-François; Rameix-Welti, Marie-Anne

    2017-09-15

    Infection of cells by respiratory syncytial virus induces the formation of cytoplasmic inclusion bodies (IBs) where all the components of the viral RNA polymerase complex are concentrated. However, the exact organization and function of these IBs remain unclear. In this study, we use conventional and super-resolution imaging to dissect the internal structure of IBs. We observe that newly synthetized viral mRNA and the viral transcription anti-terminator M2-1 concentrate in IB sub-compartments, which we term "IB-associated granules" (IBAGs). In contrast, viral genomic RNA, the nucleoprotein, the L polymerase and its cofactor P are excluded from IBAGs. Live imaging reveals that IBAGs are highly dynamic structures. Our data show that IBs are the main site of viral RNA synthesis. They further suggest that shortly after synthesis in IBs, viral mRNAs and M2-1 transiently concentrate in IBAGs before reaching the cytosol and suggest a novel post-transcriptional function for M2-1.Respiratory syncytial virus (RSV) induces formation of inclusion bodies (IBs) sheltering viral RNA synthesis. Here, Rincheval et al. identify highly dynamic IB-associated granules (IBAGs) that accumulate newly synthetized viral mRNA and the viral M2-1 protein but exclude viral genomic RNA and RNA polymerase complexes.

  6. Experimental Analysis of Cell Function Using Cytoplasmic Streaming

    Science.gov (United States)

    Janssens, Peter; Waldhuber, Megan

    2012-01-01

    This laboratory exercise investigates the phenomenon of cytoplasmic streaming in the fresh water alga "Nitella". Students use the fungal toxin cytochalasin D, an inhibitor of actin polymerization, to investigate the mechanism of streaming. Students use simple statistical methods to analyze their data. Typical student data are provided. (Contains 3…

  7. The longest telomeres: a general signature of adult stem cell compartments

    Science.gov (United States)

    Flores, Ignacio; Canela, Andres; Vera, Elsa; Tejera, Agueda; Cotsarelis, George; Blasco, María A.

    2008-01-01

    Identification of adult stem cells and their location (niches) is of great relevance for regenerative medicine. However, stem cell niches are still poorly defined in most adult tissues. Here, we show that the longest telomeres are a general feature of adult stem cell compartments. Using confocal telomere quantitative fluorescence in situ hybridization (telomapping), we find gradients of telomere length within tissues, with the longest telomeres mapping to the known stem cell compartments. In mouse hair follicles, we show that cells with the longest telomeres map to the known stem cell compartments, colocalize with stem cell markers, and behave as stem cells upon treatment with mitogenic stimuli. Using K15-EGFP reporter mice, which mark hair follicle stem cells, we show that GFP-positive cells have the longest telomeres. The stem cell compartments in small intestine, testis, cornea, and brain of the mouse are also enriched in cells with the longest telomeres. This constitutes the description of a novel general property of adult stem cell compartments. Finally, we make the novel finding that telomeres shorten with age in different mouse stem cell compartments, which parallels a decline in stem cell functionality, suggesting that telomere loss may contribute to stem cell dysfunction with age. PMID:18283121

  8. Organization of the cytoplasmic reticulum in the central vacuole of parenchyma cells in Allium cepa L.

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    Tomasz J. Wodzicki

    2015-01-01

    Full Text Available An elaborate and complex cytoplasmic reticulum composed of fine filaments and lamellae ranging from 0.1 to 4 microns in size is revealed by viewing the central vacuole of onion bulb parenchyma cells with the scanning election microscope. The larger cytoplasmic strands, visible with the light microscope, are composed of numerous smaller filaments (some tubular which might explain the observed bidirectional movement of particles in these larger strands. The finely divided cytoplasmic network of filaments is continuous with the parietal cytoplasm inclosing the vacuolar sap. In these highly vacuolated cells the mass of the protoplast is in the form of an intravacuolar reticulum immersed in the cell sap. The probable significance of the vacuolar sap in relation to physiological processes of the cell is discussed.

  9. Cytoplasmic sequestration of cyclin D1 associated with cell cycle withdrawal of neuroblastoma cells

    International Nuclear Information System (INIS)

    Sumrejkanchanakij, Piyamas; Eto, Kazuhiro; Ikeda, Masa-Aki

    2006-01-01

    The regulation of D-type cyclin-dependent kinase activity is critical for neuronal differentiation and apoptosis. We recently showed that cyclin D1 is sequestered in the cytoplasm and that its nuclear localization induces apoptosis in postmitotic primary neurons. Here, we further investigated the role of the subcellular localization of cyclin D1 in cell cycle withdrawal during the differentiation of N1E-115 neuroblastoma cells. We show that cyclin D1 became predominantly cytoplasmic after differentiation. Targeting cyclin D1 expression to the nucleus induced phosphorylation of Rb and cdk2 kinase activity. Furthermore, cyclin D1 nuclear localization promoted differentiated N1E-115 cells to reenter the cell cycle, a process that was inhibited by p16 INK4a , a specific inhibitor of D-type cyclin activity. These results indicate that cytoplasmic sequestration of cyclin D1 plays a role in neuronal cell cycle withdrawal, and suggests that the abrogation of machinery involved in monitoring aberrant nuclear cyclin D1 activity contributes to neuronal tumorigenesis

  10. Cytoplasmic Acidification and Secondary Metabolite Production in Different Plant Cell Suspensions (A Comparative Study).

    Science.gov (United States)

    Hagendoorn, MJM.; Wagner, A. M.; Segers, G.; Van Der Plas, LHW.; Oostdam, A.; Van Walraven, H. S.

    1994-10-01

    In this study, a correlation is described between low cytoplasmic pH, measured with the fluorescent probes 2[prime],7[prime]-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (acetoxymethyl ester) and bis- [3-propyl-5-oxoisoxazol-4-yl]pentamethine oxonol, and the production of secondary metabolites for several plant cell-suspension systems. Anthraquinone production in Morinda citrifolia suspensions is negligible in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D), whereas with naphthalene acetic acid (NAA) a significant accumulation is realized. NAA-grown cells showed a lower cytoplasmic pH than did 2,4-D-grown cells. Addition of 2,4-D or parachlorophenoxy acetic acid to NAA-grown cells resulted in an inhibition of anthraquinone production and an increase of the cytoplasmic pH, whereas addition of parachlorophenyl acetic acid had no effect on either parameter. Lignin production in Petunia hybrida cells could be induced by subculturing them in a medium without iron. These cells showed a lower cytoplasmic pH than control cells. Addition of Fe3+ led to a decreased lignin content and an increased cytoplasmic pH. Two cell lines of Linum flavum showed a different level of coniferin and lignin concentration in their cells. Cells that accumulated coniferin and lignin had a lower cytoplasmic pH than cells that did not accumulate these secondary metabolites. Apparently, in different species and after different kinds of treatment there is a correlation between acidification of the cytoplasm and the production of different secondary metabolites. The possible role of this acidification in secondary metabolite production is discussed.

  11. The Composition and Organization of Cytoplasm in Prebiotic Cells

    Directory of Open Access Journals (Sweden)

    Jack T. Trevors

    2011-03-01

    Full Text Available This article discusses the hypothesized composition and organization of cytoplasm in prebiotic cells from a theoretical perspective and also based upon what is currently known about bacterial cytoplasm. It is unknown if the first prebiotic, microscopic scale, cytoplasm was initially contained within a primitive, continuous, semipermeable membrane, or was an uncontained gel substance, that later became enclosed by a continuous membrane. Another possibility is that the first cytoplasm in prebiotic cells and a primitive membrane organized at the same time, permitting a rapid transition to the first cell(s capable of growth and division, thus assisting with the emergence of life on Earth less than a billion years after the formation of the Earth. It is hypothesized that the organization and composition of cytoplasm progressed initially from an unstructured, microscopic hydrogel to a more complex cytoplasm, that may have been in the volume magnitude of about 0.1–0.2 µm3 (possibly less if a nanocell prior to the first cell division.

  12. α6-Integrin alternative splicing: distinct cytoplasmic variants in stem cell fate specification and niche interaction.

    Science.gov (United States)

    Zhou, Zijing; Qu, Jing; He, Li; Peng, Hong; Chen, Ping; Zhou, Yong

    2018-05-02

    α6-Integrin subunit (also known as CD49f) is a stemness signature that has been found on the plasma membrane of more than 30 stem cell populations. A growing body of studies have focused on the critical role of α6-containing integrins (α6β1 and α6β4) in the regulation of stem cell properties, lineage-specific differentiation, and niche interaction. α6-Integrin subunit can be alternatively spliced at the post-transcriptional level, giving rise to divergent isoforms which differ in the cytoplasmic and/or extracellular domains. The cytoplasmic domain of integrins is an important functional part of integrin-mediated signals. Structural changes in the cytoplasmic domain of α6 provide an efficient means for the regulation of stem cell responses to biochemical stimuli and/or biophysical cues in the stem cell niche, thus impacting stem cell fate determination. In this review, we summarize the current knowledge on the structural variants of the α6-integrin subunit and spatiotemporal expression of α6 cytoplasmic variants in embryonic and adult stem/progenitor cells. We highlight the roles of α6 cytoplasmic variants in stem cell fate decision and niche interaction, and discuss the potential mechanisms involved. Understanding of the distinct functions of α6 splicing variants in stem cell biology may inform the rational design of novel stem cell-based therapies for a range of human diseases.

  13. Matrix metalloproteinase-9 expression in the nuclear compartment of neurons and glial cells in aging and stroke.

    Science.gov (United States)

    Pirici, Daniel; Pirici, Ionica; Mogoanta, Laurentiu; Margaritescu, Otilia; Tudorica, Valerica; Margaritescu, Claudiu; Ion, Daniela A; Simionescu, Cristiana; Coconu, Marieta

    2012-10-01

    Matrix metalloproteinases (MMPs) are well-recognized denominators for extracellular matrix remodeling in the pathology of both ischemic and hemorrhagic strokes. Recent data on non-nervous system tissue showed intracellular and even intranuclear localizations for different MMPs, and together with this, a plethora of new functions have been proposed for these intracellular active enzymes, but are mostly related to apoptosis induction and malign transformation. In neurons and glial cells, on human tissue, animal models and cell cultures, different active MMPs have been also proven to be located in the intra-cytoplasmic or intra-nuclear compartments, with no clear-cut function. In the present study we show for the first time on human tissue the nuclear expression of MMP-9, mainly in neurons and to a lesser extent in astrocytes. We have studied ischemic and hemorrhagic stroke patients, as well as aged control patients. Age and ischemic suffering seemed to be the best predictors for an elevated MMP-9 nuclear expression, and there was no evidence of a clear-cut extracellular proteolytic activity for this compartment, as revealed by intact vascular basement membranes and assessment of vascular densities. More, the majority of the cells expressing MMP-9 in the nuclear compartment also co-expressed activated-caspase 3, indicating a possible link between nuclear MMP-9 localization and apoptosis in neuronal and glial cells following an ischemic or hemorrhagic event. These results, besides showing for the first time the nuclear localization of MMP-9 on a large series of human stroke and aged brain tissues, raise new questions regarding the unknown spectrum of the functions MMPs in human CNS pathology. © 2011 Japanese Society of Neuropathology.

  14. Delivery of folates to the cytoplasm of MA104 cells is mediated by a surface membrane receptor that recycles

    International Nuclear Information System (INIS)

    Kamen, B.A.; Wang, M.T.; Streckfuss, A.J.; Peryea, X.; Anderson, R.G.

    1988-01-01

    MA104 cells, as well as several other rapidly dividing tissue culture cells, have a folate-binding protein associated with their cell surface. The protein has the properties of a membrane receptor: (a) 5-methyl[ 3 H]tetrahydrofolic acid binds with high affinity (Kd approximately equal to 3 nM); (b) the protein is an integral membrane protein; (c) it appears to deliver physiological concentrations of 5-methyl[ 3 H]tetrahydrofolic acid to the inside of the cell; (d) binding activity is regulated by the concentration of folate within the cell. To better understand the mechanism of action of this receptor, we have studied the pathway of folate internalization. We present evidence that during internalization: (a) folate binds to the membrane receptor; (b) the ligand-receptor complex moves into the cell; (c) the ligand is released from the receptor in an acidic intracellular compartment and moves into the cytoplasm; and (d) the unoccupied receptor returns to the cell surface

  15. A stromal cell free culture system generates mouse pro-T cells that can reconstitute T-cell compartments in vivo.

    Science.gov (United States)

    Gehre, Nadine; Nusser, Anja; von Muenchow, Lilly; Tussiwand, Roxane; Engdahl, Corinne; Capoferri, Giuseppina; Bosco, Nabil; Ceredig, Rhodri; Rolink, Antonius G

    2015-03-01

    T-cell lymphopenia following BM transplantation or diseases such as AIDS result in immunodeficiency. Novel approaches to ameliorate this situation are urgently required. Herein, we describe a novel stromal cell free culture system in which Lineage(-) Sca1(+)c-kit(+) BM hematopoietic progenitors very efficiently differentiate into pro-T cells. This culture system consists of plate-bound Delta-like 4 Notch ligand and the cytokines SCF and IL-7. The pro-T cells developing in these cultures express CD25, CD117, and partially CD44; express cytoplasmic CD3ε; and have their TCRβ locus partially D-J rearranged. They could be expanded for over 3 months and used to reconstitute the T-cell compartments of sublethally irradiated T-cell-deficient CD3ε(-/-) mice or lethally irradiated WT mice. Pro-T cells generated in this system could partially correct the T-cell lymphopenia of pre-Tα(-/-) mice. However, reconstituted CD3ε(-/-) mice suffered from a wasting disease that was prevented by co-injection of purified CD4(+) CD25(high) WT Treg cells. In a T-cell-sufficient or T-lymphopenic setting, the development of disease was not observed. Thus, this in vitro culture system represents a powerful tool to generate large numbers of pro-T cells for transplantation and possibly with clinical applications. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Cell cycle-dependent microtubule-based dynamic transport of cytoplasmic dynein in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Takuya Kobayashi

    Full Text Available BACKGROUND: Cytoplasmic dynein complex is a large multi-subunit microtubule (MT-associated molecular motor involved in various cellular functions including organelle positioning, vesicle transport and cell division. However, regulatory mechanism of the cell-cycle dependent distribution of dynein has not fully been understood. METHODOLOGY/PRINCIPAL FINDINGS: Here we report live-cell imaging of cytoplasmic dynein in HeLa cells, by expressing multifunctional green fluorescent protein (mfGFP-tagged 74-kDa intermediate chain (IC74. IC74-mfGFP was successfully incorporated into functional dynein complex. In interphase, dynein moved bi-directionally along with MTs, which might carry cargos such as transport vesicles. A substantial fraction of dynein moved toward cell periphery together with EB1, a member of MT plus end-tracking proteins (+TIPs, suggesting +TIPs-mediated transport of dynein. In late-interphase and prophase, dynein was localized at the centrosomes and the radial MT array. In prometaphase and metaphase, dynein was localized at spindle MTs where it frequently moved from spindle poles toward chromosomes or cell cortex. +TIPs may be involved in the transport of spindle dyneins. Possible kinetochore and cortical dyneins were also observed. CONCLUSIONS AND SIGNIFICANCE: These findings suggest that cytoplasmic dynein is transported to the site of action in preparation for the following cellular events, primarily by the MT-based transport. The MT-based transport may have greater advantage than simple diffusion of soluble dynein in rapid and efficient transport of the limited concentration of the protein.

  17. Cytoplasmic PELP1 and ERRgamma protect human mammary epithelial cells from Tam-induced cell death.

    Science.gov (United States)

    Girard, Brian J; Regan Anderson, Tarah M; Welch, Siya Lem; Nicely, Julie; Seewaldt, Victoria L; Ostrander, Julie H

    2015-01-01

    Tamoxifen (Tam) is the only FDA-approved chemoprevention agent for pre-menopausal women at high risk for developing breast cancer. While Tam reduces a woman's risk of developing estrogen receptor positive (ER+) breast cancer, the molecular mechanisms associated with risk reduction are poorly understood. Prior studies have shown that cytoplasmic proline, glutamic acid and leucine rich protein 1 (PELP1) promotes Tam resistance in breast cancer cell lines. Herein, we tested for PELP1 localization in breast epithelial cells from women at high risk for developing breast cancer and found that PELP1 was localized to the cytoplasm in 36% of samples. In vitro, immortalized HMECs expressing a nuclear localization signal (NLS) mutant of PELP1 (PELP1-cyto) were resistant to Tam-induced death. Furthermore, PELP1-cyto signaling through estrogen-related receptor gamma (ERRγ) promoted cell survival in the presence of Tam. Overexpression of ERRγ in immortalized HMECs protected cells from Tam-induced death, while knockdown of ERRγ sensitized PELP1-cyto expressing HMECs to Tam. Moreover, Tam-induced HMEC cell death was independent of apoptosis and involved accumulation of the autophagy marker LC3-II. Expression of PELP1-cyto and ERRγ reduced Tam-induced LC3-II accumulation, and knockdown of ERRγ increased LC3-II levels in response to Tam. Additionally, PELP1-cyto expression led to the upregulation of MMP-3 and MAOB, known PELP1 and ERRγ target genes, respectively. Our data indicate that cytoplasmic PELP1 induces signaling pathways that converge on ERRγ to promote cell survival in the presence of Tam. These data suggest that PELP1 localization and/or ERRγ activation could be developed as tissue biomarkers for Tam responsiveness.

  18. Transcytosis of Aminopeptidase N in caco-2 cells is mediated by a Non-cytoplasmic Signal

    DEFF Research Database (Denmark)

    Vogel, L K; Norén, Ove; Sjöström, H

    1995-01-01

    In Caco-2 cells, aminopeptidase N is transported to the apical membrane from the trans Golgi network by both the direct and the indirect pathway (Matter, K., Brauchbar, M., Bucher, K., and Hauri, H.-P. (1990) Cell 60, 429-437). The aim of this study was to determine the importance...... of the transmembrane or cytoplasmic domain of aminopeptidase N for transport of aminopeptidase N by the indirect pathway by analysis of mutated forms of aminopeptidase N recombinantly expressed in Caco-2 cells. A tail-less and two secretory forms of aminopeptidase N, all deprived of the cytoplasmic tail, were...

  19. Cytoplasmic RAP1 mediates cisplatin resistance of non-small cell lung cancer.

    Science.gov (United States)

    Xiao, Lu; Lan, Xiaoying; Shi, Xianping; Zhao, Kai; Wang, Dongrui; Wang, Xuejun; Li, Faqian; Huang, Hongbiao; Liu, Jinbao

    2017-05-18

    Cytotoxic chemotherapy agents (e.g., cisplatin) are the first-line drugs to treat non-small cell lung cancer (NSCLC) but NSCLC develops resistance to the agent, limiting therapeutic efficacy. Despite many approaches to identifying the underlying mechanism for cisplatin resistance, there remains a lack of effective targets in the population that resist cisplatin treatment. In this study, we sought to investigate the role of cytoplasmic RAP1, a previously identified positive regulator of NF-κB signaling, in the development of cisplatin resistance in NSCLC cells. We found that the expression of cytoplasmic RAP1 was significantly higher in high-grade NSCLC tissues than in low-grade NSCLC; compared with a normal pulmonary epithelial cell line, the A549 NSCLC cells exhibited more cytoplasmic RAP1 expression as well as increased NF-κB activity; cisplatin treatment resulted in a further increase of cytoplasmic RAP1 in A549 cells; overexpression of RAP1 desensitized the A549 cells to cisplatin, and conversely, RAP1 depletion in the NSCLC cells reduced their proliferation and increased their sensitivity to cisplatin, indicating that RAP1 is required for cell growth and has a key mediating role in the development of cisplatin resistance in NSCLC cells. The RAP1-mediated cisplatin resistance was associated with the activation of NF-κB signaling and the upregulation of the antiapoptosis factor BCL-2. Intriguingly, in the small portion of RAP1-depleted cells that survived cisplatin treatment, no induction of NF-κB activity and BCL-2 expression was observed. Furthermore, in established cisplatin-resistant A549 cells, RAP1 depletion caused BCL2 depletion, caspase activation and dramatic lethality to the cells. Hence, our results demonstrate that the cytoplasmic RAP1-NF-κB-BCL2 axis represents a key pathway to cisplatin resistance in NSCLC cells, identifying RAP1 as a marker and a potential therapeutic target for cisplatin resistance of NSCLC.

  20. The GOD of Hematopoietic Stem Cells: A Clonal Diversity Model of the Stem Cell Compartment

    OpenAIRE

    Muller-Sieburg, C.E.; Sieburg, H.B.

    2006-01-01

    Hematopoietic stem cells (HSC) show heterogeneous behavior even when isolated as phenotypically homogeneous populations. The cellular and molecular mechanisms that control the generation of diversity (GOD) in the HSC compartment are not well understood, but have been the focus of much debate. There is increasing evidence that the most important HSC functions, self-renewal and differentiation, are epigenetically preprogrammed and therefore predictable. Indeed, recent data show that the adult H...

  1. Cytoplasmic Kaiso is associated with poor prognosis in non-small cell lung cancer

    International Nuclear Information System (INIS)

    Dai, Shun-Dong; Wang, Yan; Miao, Yuan; Zhao, Yue; Zhang, Yong; Jiang, Gui-Yang; Zhang, Peng-Xin; Yang, Zhi-Qiang; Wang, En-Hua

    2009-01-01

    Kaiso has been identified as a new member of the POZ-zinc finger family of transcription factors that are implicated in development and cancer. Although controversy still exists, Kaiso is supposed to be involved in human cancer. However, there is limited information regarding the clinical significance of cytoplasmic/nuclear Kaiso in human lung cancer. In this study, immunohistochemical studies were performed on 20 cases of normal lung tissues and 294 cases of non-small cell lung cancer (NSCLC), including 50 cases of paired lymph node metastases and 88 cases with complete follow-up records. Three lung cancer cell lines showing primarily nuclear localization of Kaiso were selected to examine whether roles of Kaiso in cytoplasm and in nucleus are identical. Nuclear Kaiso was down-regulated by shRNA technology or addition a specific Kaiso antibody in these cell lines. The proliferative and invasive abilities were evaluated by MTT and Matrigel invasive assay, transcription of Kaiso's target gene matrilysin was detected by RT-PCR. Kaiso was primarily expressed in the cytoplasm of lung cancer tissues. Overall positive cytoplasmic expression rate was 63.61% (187/294). The positive cytoplasmic expression of Kaiso was higher in advanced TNM stages (III+IV) of NSCLC, compared to lower stages (I+II) (p = 0.019). A correlation between cytoplasmic Kaiso expression and lymph node metastasis was found (p = 0.003). In 50 paired cases, cytoplasmic expression of Kaiso was 78.0% (41/50) in primary sites and 90.0% (45/50) in lymph node metastases (p = 0.001). The lung cancer-related 5-year survival rate was significantly lower in patients who were cytoplasmic Kaiso-positive (22.22%), compared to those with cytoplasmic Kaiso-negative tumors (64.00%) (p = 0.005). Nuclear Kaiso staining was seen in occasional cases with only a 5.10% (15/294) positive rate and was not associated with any clinicopathological features of NSCLC. Furthermore, after the down-regulation of the nuclear

  2. Making it big : how characean algae use cytoplasmic streaming to enhance transport in giant cells

    NARCIS (Netherlands)

    Meent, Jan Willem van de

    2010-01-01

    Organisms show a remarkable variation in sizes, yet cell sizes are surprisingly similar across species, typically ranging from 10 μm to 100 μm. A striking exception are the giant cells of the algal weed Chara, which can exceed 10 cm in length and 1 mm in diameter. A circulation known as cytoplasmic

  3. Localization and function of KLF4 in cytoplasm of vascular smooth muscle cell

    International Nuclear Information System (INIS)

    Liu, Yan; Zheng, Bin; Zhang, Xin-hua; Nie, Chan-juan; Li, Yong-hui; Wen, Jin-kun

    2013-01-01

    Highlights: •PDGF-BB prompts the translocation of KLF4 to the cytoplasm. •PDGF-BB promotes interaction between KLF4 and actin in the cytoplasm. •Phosphorylation and SUMOylation of KLF4 participates in regulation of cytoskeletal organization. •KLF4 regulates cytoskeleton by promoting the expression of contraction-associated genes. -- Abstract: The Krüppel-like factor 4 is a DNA-binding transcriptional regulator that regulates a diverse array of cellular processes, including development, differentiation, proliferation, and apoptosis. The previous studies about KLF4 functions mainly focused on its role as a transcription factor, its functions in the cytoplasm are still unknown. In this study, we found that PDGF-BB could prompt the translocation of KLF4 to the cytoplasm through CRM1-mediated nuclear export pathway in vascular smooth muscle cells (VSMCs) and increased the interaction of KLF4 with actin in the cytoplasm. Further study showed that both KLF4 phosphorylation and SUMOylation induced by PDGF-BB participates in regulation of cytoskeletal organization by stabilizing the actin cytoskeleton in VSMCs. In conclusion, these results identify that KLF4 participates in the cytoskeletal organization by stabilizing cytoskeleton in the cytoplasm of VSMCs

  4. Localization and function of KLF4 in cytoplasm of vascular smooth muscle cell

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Yan [Department of Biochemistry and Molecular Biology, The Key Laboratory of Neurobiology and Vascular Biology (China); The Third Hospital of Hebei Medical University, Shijazhuang (China); Zheng, Bin; Zhang, Xin-hua; Nie, Chan-juan; Li, Yong-hui [Department of Biochemistry and Molecular Biology, The Key Laboratory of Neurobiology and Vascular Biology (China); Wen, Jin-kun, E-mail: wjk@hebmu.edu.cn [Department of Biochemistry and Molecular Biology, The Key Laboratory of Neurobiology and Vascular Biology (China)

    2013-06-28

    Highlights: •PDGF-BB prompts the translocation of KLF4 to the cytoplasm. •PDGF-BB promotes interaction between KLF4 and actin in the cytoplasm. •Phosphorylation and SUMOylation of KLF4 participates in regulation of cytoskeletal organization. •KLF4 regulates cytoskeleton by promoting the expression of contraction-associated genes. -- Abstract: The Krüppel-like factor 4 is a DNA-binding transcriptional regulator that regulates a diverse array of cellular processes, including development, differentiation, proliferation, and apoptosis. The previous studies about KLF4 functions mainly focused on its role as a transcription factor, its functions in the cytoplasm are still unknown. In this study, we found that PDGF-BB could prompt the translocation of KLF4 to the cytoplasm through CRM1-mediated nuclear export pathway in vascular smooth muscle cells (VSMCs) and increased the interaction of KLF4 with actin in the cytoplasm. Further study showed that both KLF4 phosphorylation and SUMOylation induced by PDGF-BB participates in regulation of cytoskeletal organization by stabilizing the actin cytoskeleton in VSMCs. In conclusion, these results identify that KLF4 participates in the cytoskeletal organization by stabilizing cytoskeleton in the cytoplasm of VSMCs.

  5. Optochemical Control of Protein Localization and Activity within Cell-like Compartments.

    Science.gov (United States)

    Caldwell, Reese M; Bermudez, Jessica G; Thai, David; Aonbangkhen, Chanat; Schuster, Benjamin S; Courtney, Taylor; Deiters, Alexander; Hammer, Daniel A; Chenoweth, David M; Good, Matthew C

    2018-05-08

    We report inducible dimerization strategies for controlling protein positioning, enzymatic activity, and organelle assembly inside synthetic cell-like compartments upon photostimulation. Using a photocaged TMP-Haloligand compound, we demonstrate small molecule and light-induced dimerization of DHFR and Haloenzyme to localize proteins to a compartment boundary and reconstitute tripartite sfGFP assembly. Using photocaged rapamycin and fragments of split TEV protease fused to FRB and FKBP, we establish optical triggering of protease activity inside cell-size compartments. We apply light-inducible protease activation to initiate assembly of membraneless organelles, demonstrating the applicability of these tools for characterizing cell biological processes in vitro. This modular toolkit, which affords spatial and temporal control of protein function in a minimal cell-like system, represents a critical step toward the reconstitution of a tunable synthetic cell, built from the bottom up.

  6. Human B cells fail to secrete type I interferons upon cytoplasmic DNA exposure.

    Science.gov (United States)

    Gram, Anna M; Sun, Chenglong; Landman, Sanne L; Oosenbrug, Timo; Koppejan, Hester J; Kwakkenbos, Mark J; Hoeben, Rob C; Paludan, Søren R; Ressing, Maaike E

    2017-11-01

    Most cells are believed to be capable of producing type I interferons (IFN I) as part of an innate immune response against, for instance, viral infections. In macrophages, IFN I is potently induced upon cytoplasmic exposure to foreign nucleic acids. Infection of these cells with herpesviruses leads to triggering of the DNA sensors interferon-inducible protein 16 (IFI16) and cyclic GMP-AMP (cGAMP) synthase (cGAS). Thereby, the stimulator of interferon genes (STING) and the downstream molecules TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) are sequentially activated culminating in IFN I secretion. Human gamma-herpesviruses, such as Epstein-Barr virus (EBV), exploit B cells as a reservoir for persistent infection. In this study, we investigated whether human B cells, similar to macrophages, engage the cytoplasmic DNA sensing pathway to induce an innate immune response. We found that the B cells fail to secrete IFN I upon cytoplasmic DNA exposure, although they express the DNA sensors cGAS and IFI16 and the signaling components TBK1 and IRF3. In primary human B lymphocytes and EBV-negative B cell lines, this deficiency is explained by a lack of detectable levels of the central adaptor protein STING. In contrast, EBV-transformed B cell lines did express STING, yet both these lines as well as STING-reconstituted EBV-negative B cells did not produce IFN I upon dsDNA or cGAMP stimulation. Our combined data show that the cytoplasmic DNA sensing pathway is dysfunctional in human B cells. This exemplifies that certain cell types cannot induce IFN I in response to cytoplasmic DNA exposure providing a potential niche for viral persistence. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  7. Dynamics of vegetative cytoplasm during generative cell formation and pollen maturation in Arabidopsis thaliana

    Science.gov (United States)

    Kuang, A.; Musgrave, M. E.

    1996-01-01

    Ultrastructural changes of pollen cytoplasm during generative cell formation and pollen maturation in Arabidopsis thaliana were studied. The pollen cytoplasm develops a complicated ultrastructure and changes dramatically during these stages. Lipid droplets increase after generative cell formation and their organization and distribution change with the developmental stage. Starch grains in amyloplasts increase in number and size during generative and sperm cell formation and decrease at pollen maturity. The shape and membrane system of mitochondria change only slightly. Dictyosomes become very prominent, and numerous associated vesicles are observed during and after sperm cell formation. Endoplasmic reticulum appears extensively as stacks during sperm cell formation. Free and polyribosomes are abundant in the cytoplasm at all developmental stages although they appear denser at certain stages and in some areas. In mature pollen, all organelles are randomly distributed throughout the vegetative cytoplasm and numerous small particles appear. Organization and distribution of storage substances and appearance of these small particles during generative and sperm cell formation and pollen maturation are discussed.

  8. Coordinate expansion of murine hematopoietic and mesenchymal stem cell compartments by SHIPi.

    Science.gov (United States)

    Brooks, R; Iyer, S; Akada, H; Neelam, S; Russo, C M; Chisholm, J D; Kerr, W G

    2015-03-01

    Promoting the expansion of adult stem cell populations offers the potential to ameliorate radiation or chemotherapy-induced bone marrow failure and allows for expedited recovery for patients undergoing these therapies. Previous genetic studies suggested a pivotal role for SH2 domain-containing inositol-5-phosphatase 1 (SHIP1) in limiting the size of the hematopoietic stem cell (HSC) compartment. The aim of this study was to determine whether our recent development of small molecule SHIP1 inhibitors offers the potential for pharmacological expansion of the HSC compartment in vivo. We show here that treatment of mice with aminosteroid inhibitors of SHIP1 (SHIPi) more than doubles the size of the adult mesenchymal stem cell (MSC) compartment while simultaneously expanding the HSC pool sixfold. Consistent with its ability to target SHIP1 function in vivo, SHIPi also significantly increases plasma granulocyte colony-stimulating factor (G-CSF) levels, a growth factor that supports proliferation of HSC. Here, we show that SHIPi-induced G-CSF production mediates HSC and MSC expansion, as in vivo neutralization of G-CSF abrogates the SHIPi-induced expansion of both the HSC and MSC compartments. Due to its expansionary effect on adult stem cell compartments, SHIPi represents a potential novel strategy to improve declining stem cell function in both therapy induced and genetically derived bone marrow failure syndromes. © 2014 AlphaMed Press.

  9. Cell nuclei and cytoplasm joint segmentation using the sliding band filter.

    Science.gov (United States)

    Quelhas, Pedro; Marcuzzo, Monica; Mendonça, Ana Maria; Campilho, Aurélio

    2010-08-01

    Microscopy cell image analysis is a fundamental tool for biological research. In particular, multivariate fluorescence microscopy is used to observe different aspects of cells in cultures. It is still common practice to perform analysis tasks by visual inspection of individual cells which is time consuming, exhausting and prone to induce subjective bias. This makes automatic cell image analysis essential for large scale, objective studies of cell cultures. Traditionally the task of automatic cell analysis is approached through the use of image segmentation methods for extraction of cells' locations and shapes. Image segmentation, although fundamental, is neither an easy task in computer vision nor is it robust to image quality changes. This makes image segmentation for cell detection semi-automated requiring frequent tuning of parameters. We introduce a new approach for cell detection and shape estimation in multivariate images based on the sliding band filter (SBF). This filter's design makes it adequate to detect overall convex shapes and as such it performs well for cell detection. Furthermore, the parameters involved are intuitive as they are directly related to the expected cell size. Using the SBF filter we detect cells' nucleus and cytoplasm location and shapes. Based on the assumption that each cell has the same approximate shape center in both nuclei and cytoplasm fluorescence channels, we guide cytoplasm shape estimation by the nuclear detections improving performance and reducing errors. Then we validate cell detection by gathering evidence from nuclei and cytoplasm channels. Additionally, we include overlap correction and shape regularization steps which further improve the estimated cell shapes. The approach is evaluated using two datasets with different types of data: a 20 images benchmark set of simulated cell culture images, containing 1000 simulated cells; a 16 images Drosophila melanogaster Kc167 dataset containing 1255 cells, stained for DNA and

  10. Probing cytoplasmic organization and the actin cytoskeleton of plant cells with optical tweezers

    NARCIS (Netherlands)

    Ketelaar, T.; Honing, van der H.S.; Emons, A.M.C.

    2010-01-01

    In interphase plant cells, the actin cytoskeleton is essential for intracellular transport and organization. To fully understand how the actin cytoskeleton functions as the structural basis for cytoplasmic organization, both molecular and physical aspects of the actin organization have to be

  11. Perturbation of nucleo-cytoplasmic transport affects size of nucleus and nucleolus in human cells.

    Science.gov (United States)

    Ganguly, Abira; Bhattacharjee, Chumki; Bhave, Madhura; Kailaje, Vaishali; Jain, Bhawik K; Sengupta, Isha; Rangarajan, Annapoorni; Bhattacharyya, Dibyendu

    2016-03-01

    Size regulation of human cell nucleus and nucleolus are poorly understood subjects. 3D reconstruction of live image shows that the karyoplasmic ratio (KR) increases by 30-80% in transformed cell lines compared to their immortalized counterpart. The attenuation of nucleo-cytoplasmic transport causes the KR value to increase by 30-50% in immortalized cell lines. Nucleolus volumes are significantly increased in transformed cell lines and the attenuation of nucleo-cytoplasmic transport causes a significant increase in the nucleolus volume of immortalized cell lines. A cytosol and nuclear fraction swapping experiment emphasizes the potential role of unknown cytosolic factors in nuclear and nucleolar size regulation. © 2016 Federation of European Biochemical Societies.

  12. Cytoplasm localization of aminopeptidase M1 and its functional activity in root hair cells and BY-2 cells.

    Science.gov (United States)

    Lee, Ok Ran; Cho, Hyung-Taeg

    2012-12-01

    Aminopeptidase M1 (APM1) was the first M1 metallopeptidase family member identified in Arabidopsis, isolated by its affinity for the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). A loss-of-function mutation showed various developmental defects in cell division and auxin transport. APM1 was shown to be localized in endomembrane structures, the cytoplasm, and the plasma membrane. These previous results suggested that APM1 has diverse functional roles in different cell and tissue types. Here we report that APM1 localized to the cytoplasm, and its over-expression in the root hair cell caused longer root hair phenotypes. Treatment of aminopeptidase inhibitors caused internalization of auxin efflux PIN-FORMED proteins in root hair cells and suppressed short root hair phenotype of PIN3 overexpression line (PIN3ox). APM1 also localized to the cytoplasm in tobacco BY-2 cells, its over-expression had little effect on auxin transport in these cells.

  13. Diffusive Promotion by Velocity Gradient of Cytoplasmic Streaming (CPS in Nitella Internodal Cells.

    Directory of Open Access Journals (Sweden)

    Kenji Kikuchi

    Full Text Available Cytoplasmic streaming (CPS is well known to assist the movement of nutrients, organelles and genetic material by transporting all of the cytoplasmic contents of a cell. CPS is generated by motility organelles that are driven by motor proteins near a membrane surface, where the CPS has been found to have a flat velocity profile in the flow field according to the sliding theory. There is a consistent mixing of contents inside the cell by CPS if the velocity gradient profile is flattened, which is not assisted by advection diffusion but is only supported by Brownian diffusion. Although the precise flow structure of the cytoplasm has an important role for cellular metabolism, the hydrodynamic mechanism of its convection has not been clarified. We conducted an experiment to visualise the flow of cytoplasm in Nitella cells by injecting tracer fluorescent nanoparticles and using a flow visualisation system in order to understand how the flow profile affects their metabolic system. We determined that the velocity field in the cytosol has an obvious velocity gradient, not a flattened gradient, which suggests that the gradient assists cytosolic mixing by Taylor-Aris dispersion more than by Brownian diffusion.

  14. Effect of heterogeneity on the characterization of cell membrane compartments: I. Uniform size and permeability.

    Science.gov (United States)

    Hall, Damien

    2010-03-15

    Observations of the motion of individual molecules in the membrane of a number of different cell types have led to the suggestion that the outer membrane of many eukaryotic cells may be effectively partitioned into microdomains. A major cause of this suggested partitioning is believed to be due to the direct/indirect association of the cytosolic face of the cell membrane with the cortical cytoskeleton. Such intimate association is thought to introduce effective hydrodynamic barriers into the membrane that are capable of frustrating molecular Brownian motion over distance scales greater than the average size of the compartment. To date, the standard analytical method for deducing compartment characteristics has relied on observing the random walk behavior of a labeled lipid or protein at various temporal frequencies and different total lengths of time. Simple theoretical arguments suggest that the presence of restrictive barriers imparts a characteristic turnover to a plot of mean squared displacement versus sampling period that can be interpreted to yield the average dimensions of the compartment expressed as the respective side lengths of a rectangle. In the following series of articles, we used computer simulation methods to investigate how well the conventional analytical strategy coped with heterogeneity in size, shape, and barrier permeability of the cell membrane compartments. We also explored questions relating to the necessary extent of sampling required (with regard to both the recorded time of a single trajectory and the number of trajectories included in the measurement bin) for faithful representation of the actual distribution of compartment sizes found using the SPT technique. In the current investigation, we turned our attention to the analytical characterization of diffusion through cell membrane compartments having both a uniform size and permeability. For this ideal case, we found that (i) an optimum sampling time interval existed for the analysis

  15. Antigen storage compartments in mature dendritic cells facilitate prolonged cytotoxic T lymphocyte cross-priming capacity.

    Science.gov (United States)

    van Montfoort, Nadine; Camps, Marcel G; Khan, Selina; Filippov, Dmitri V; Weterings, Jimmy J; Griffith, Janice M; Geuze, Hans J; van Hall, Thorbald; Verbeek, J Sjef; Melief, Cornelis J; Ossendorp, Ferry

    2009-04-21

    Dendritic cells (DCs) are crucial for priming of naive CD8(+) T lymphocytes to exogenous antigens, so-called "cross-priming." We report that exogenous protein antigen can be conserved for several days in mature DCs, coinciding with strong cytotoxic T lymphocyte cross-priming potency in vivo. After MHC class I peptide elution, protein antigen-derived peptide presentation is efficiently restored, indicating the presence of an intracellular antigen depot. We characterized this depot as a lysosome-like organelle, distinct from MHC class II compartments and recently described early endosomal compartments that allow acute antigen presentation in MHC class I. The storage compartments we report here facilitate continuous supply of MHC class I ligands. This mechanism ensures sustained cross-presentation by DCs, despite the short-lived expression of MHC class I-peptide complexes at the cell surface.

  16. Replacement of the cytoplasmic domain alters sorting of a viral glycoprotein in polarized cells.

    OpenAIRE

    Puddington, L; Woodgett, C; Rose, J K

    1987-01-01

    The envelope glycoprotein (G protein) of vesicular stomatitis virus (VSV) is transported to the basolateral plasma membrane of polarized epithelial cells, whereas the hemagglutinin glycoprotein (HA protein) of influenza virus is transported to the apical plasma membrane. To determine if the cytoplasmic domain of VSV G protein might be important in directing G protein to the basolateral membrane, we derived polarized Madin-Darby canine kidney cell lines expressing G protein or G protein with i...

  17. Engaging the lysosomal compartment to combat B cell malignancies

    DEFF Research Database (Denmark)

    Gronbaek, K.; Jaattela, M.

    2009-01-01

    The combination of rituximab, a type I anti-CD20 mAb, with conventional chemotherapy has significantly improved the outcome of patients with B cell malignancies. Regardless of this success, many patients still relapse with therapy-resistant disease, highlighting the need for the development of m...

  18. The epidermis comprises autonomous compartments maintained by distinct stem cell populations

    DEFF Research Database (Denmark)

    Page, Mahalia E; Lombard, Patrick; Ng, Felicia

    2013-01-01

    populations. In contrast, upon wounding, stem cell progeny from multiple compartments acquire lineage plasticity and make permanent contributions to regenerating tissue. We further show that oncogene activation in Lrig1(+ve) cells drives hyperplasia but requires auxiliary stimuli for tumor formation....... In summary, our data demonstrate that epidermal stem cells are lineage restricted during homeostasis and suggest that compartmentalization may constitute a conserved mechanism underlying epithelial tissue maintenance....

  19. Persistent Inflammation Alters the Function of the Endogenous Brain Stem Cell Compartment

    OpenAIRE

    Pluchino, Stefano; Muzio, Luca; Alfaro-Cervello, Clara; Salani, Giuliana; Porcheri, Cristina; Brambilla, Elena; Cavasinni, Francesca; Bergamaschi, Andrea; Garcia-Verdugo, Jose Manuel; Comi, Giancarlo; Martino, Gianvito; Imitola, Jaime; Deleidi, Michela; Khoury, Samia Joseph

    2008-01-01

    Endogenous neural stem/precursor cells (NPCs) are considered a functional reservoir for promoting tissue homeostasis and repair after injury, therefore regenerative strategies that mobilize these cells have recently been proposed. Despite evidence of increased neurogenesis upon acute inflammatory insults (e.g. ischaemic stroke), the plasticity of the endogenous brain stem cell compartment in chronic CNS inflammatory disorders remains poorly characterized. Here we show that persistent brain in...

  20. Quantifying Multistate Cytoplasmic Molecular Diffusion in Bacterial Cells via Inverse Transform of Confined Displacement Distribution.

    Science.gov (United States)

    Chen, Tai-Yen; Jung, Won; Santiago, Ace George; Yang, Feng; Krzemiński, Łukasz; Chen, Peng

    2015-11-12

    Single-molecule tracking (SMT) of fluorescently tagged cytoplasmic proteins can provide valuable information on the underlying biological processes in living cells via subsequent analysis of the displacement distributions; however, the confinement effect originated from the small size of a bacterial cell skews the protein's displacement distribution and complicates the quantification of the intrinsic diffusive behaviors. Using the inverse transformation method, we convert the skewed displacement distribution (for both 2D and 3D imaging conditions) back to that in free space for systems containing one or multiple (non)interconverting Brownian diffusion states, from which we can reliably extract the number of diffusion states as well as their intrinsic diffusion coefficients and respective fractional populations. We further demonstrate a successful application to experimental SMT data of a transcription factor in living E. coli cells. This work allows a direct quantitative connection between cytoplasmic SMT data with diffusion theory for analyzing molecular diffusive behavior in live bacteria.

  1. The subapical compartment and its role in intracellular trafficking and cell polarity

    NARCIS (Netherlands)

    Van Ijzendoorn, Sven C. D.; Maier, Olaf; Van Der Wouden, Johanna M.; Hoekstra, Dick

    In polarized epithelial cells and hepatocytes, apical and basolateral plasma membrane surfaces are maintained, each displaying a distinct molecular composition. In recent years, it has become apparent that a subapical compartment, referred to as SAC, plays a prominent if not crucial role in the

  2. Sensitivity to radiation and cycle-active drugs as a function of stem cell compartment repletion

    International Nuclear Information System (INIS)

    Degowin, R.L.; Gibson, D.P.

    1976-01-01

    We have studied the sensitivity of normal mouse hemopoietic tissue to radiation and cycle-active drugs in relation to stem cell compartment repletion. Recovery of erythropoiesis in endogenous spleen colonies, blood reticulocytes, and 30-day survivals were determined in mice after an initial large dose of partial-body irradiation. We found that the normal stem cell compartment is more sensitive to cycle-independent modes of therapy, like radiation and cyclophosphamide, than it is to cycle-active agents like cytosine arabinoside and methotrexate. The depleted stem cell compartment exhibits marked sensitivity to cycle-independent agents but less to cycle-active agents, which, however, suppress its recovery more than they do the normal. The overshoot phase of recovery is relatively resistant to either cycle-independent or cycle-active agents. A reticulocytosis following a reticulocytopenia signals the overshoot phase of stem cell compartment recovery and relatively increased resistance. These findings may prove useful in designing chemotherapy regimens and in anticipating marrow recovery in planning for supportive care in patients with neoplastic disease

  3. Isotachophoresis for fractionation and recovery of cytoplasmic RNA and nucleus from single cells.

    Science.gov (United States)

    Kuriyama, Kentaro; Shintaku, Hirofumi; Santiago, Juan G

    2015-07-01

    There is a substantial need for simultaneous analyses of RNA and DNA from individual single cells. Such analysis provides unique evidence of cell-to-cell differences and the correlation between gene expression and genomic mutation in highly heterogeneous cell populations. We present a novel microfluidic system that leverages isotachophoresis to fractionate and isolate cytoplasmic RNA and genomic DNA (gDNA) from single cells. The system uniquely enables independent, sequence-specific analyses of these critical markers. Our system uses a microfluidic chip with a simple geometry and four end-channel electrodes, and completes the entire process in RNA output reservoirs, each containing high quality and purity aliquots with no measurable cross-contamination of cytoplasmic RNA versus gDNA. We demonstrate our system with simultaneous, sequence-specific quantitation using off-chip RT-qPCR and qPCR for simultaneous cytoplasmic RNA and gDNA analyses, respectively. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Receptor trafficking via the perinuclear recycling compartment accompanied by cell division is necessary for permanent neurotensin cell sensitization and leads to chronic mitogen-activated protein kinase activation.

    Science.gov (United States)

    Toy-Miou-Leong, Mireille; Cortes, Catherine Llorens; Beaudet, Alain; Rostène, William; Forgez, Patricia

    2004-03-26

    Most G protein-coupled receptors are internalized after interaction with their respective ligand, a process that subsequently contributes to cell desensitization, receptor endocytosis, trafficking, and finally cell resensitization. Although cellular mechanisms leading to cell desensitization have been widely studied, those responsible for cell resensitization are still poorly understood. We examined here the traffic of the high affinity neurotensin receptor (NT1 receptor) following prolonged exposure to high agonist concentration. Fluorescence and confocal microscopy of Chinese hamster ovary, human neuroblastoma (CHP 212), and murine neuroblastoma (N1E-115) cells expressing green fluorescent protein-tagged NT1 receptor revealed that under prolonged treatment with saturating concentrations of neurotensin (NT) agonist, NT1 receptor and NT transiently accumulated in the perinuclear recycling compartment (PNRC). During this cellular event, cell surface receptors remained markedly depleted as detected by both confocal microscopy and (125)I-NT binding assays. In dividing cells, we observed that following prolonged NT agonist stimulation, NT1 receptors were removed from the PNRC, accumulated in dispersed vesicles inside the cytoplasm, and subsequently reappeared at the cell surface. This NT binding recovery allowed for constant cell sensitization and led to a chronic activation of mitogen-activated protein kinases p42 and p44. Under these conditions, the constant activation of NT1 receptor generates an oncogenic regulation. These observations support the potent role for neuropeptides, such as NT, in cancer progression.

  5. Cytoplasmic pH dynamics in maize pulvinal cells induced by gravity vector changes

    Science.gov (United States)

    Johannes, E.; Collings, D. A.; Rink, J. C.; Allen, N. S.; Brown, C. S. (Principal Investigator)

    2001-01-01

    In maize (Zea mays) and other grasses, changes in orientation of stems are perceived by pulvinal tissue, which responds to the stimulus by differential growth resulting in upward bending of the stem. The amyloplast-containing bundle sheath cells are the sites of gravity perception, although the initial steps of gravity perception and transmission remain unclear. In columella cells of Arabidopsis roots, we previously found that cytoplasmic pH (pH(c)) is a mediator in early gravitropic signaling (A.C. Scott, N.S. Allen [1999] Plant Physiol 121: 1291-1298). The question arises whether pH(c) has a more general role in signaling gravity vector changes. Using confocal ratiometric imaging and the fluorescent pH indicator carboxy seminaphtorhodafluor acetoxymethyl ester acetate, we measured pH(c) in the cells composing the maize pulvinus. When stem slices were gravistimulated and imaged on a horizontally mounted confocal microscope, pH(c) changes were only apparent within the bundle sheath cells, and not in the parenchyma cells. After turning, cytoplasmic acidification was observed at the sides of the cells, whereas the cytoplasm at the base of the cells where plastids slowly accumulated became more basic. These changes were most apparent in cells exhibiting net amyloplast sedimentation. Parenchyma cells and isolated bundle sheath cells did not show any gravity-induced pH(c) changes although all cell types responded to external stimuli in the predicted way: Propionic acid and auxin treatments induced acidification, whereas raising the external pH caused alkalinization. The results suggest that pH(c) has an important role in the early signaling pathways of maize stem gravitropism.

  6. Functional and morphological recovery of the T-cell compartment in lethally irradiated and reconstituted mice

    International Nuclear Information System (INIS)

    Kraal, G.; Hilst, B. van der; Boden, D.

    1979-01-01

    The recovery of the T-cell compartment in mice after lethal irradiation and reconstitution was studied using functional and morphological parameters. T-helper cell activity, determined by the direct SRBC-plaque-forming cell (PFC) response, recovered in a similar fashion as T-memory function which was studied by adoptive transfer of carrier-primed cells. Both functions returned to control levels in 2.5 to 3 months. Using immunoperoxidase staining of frozen sections with anti-T cell serum, the morphological recovery of the T-cell dependent areas in the white pulp of the spleen could be studied and compared with the functional recovery. (author)

  7. The cell-mediated immunity of Drosophila melanogaster: hemocyte lineages, immune compartments, microanatomy and regulation.

    Science.gov (United States)

    Honti, Viktor; Csordás, Gábor; Kurucz, Éva; Márkus, Róbert; Andó, István

    2014-01-01

    In the animal kingdom, innate immunity is the first line of defense against invading pathogens. The dangers of microbial and parasitic attacks are countered by similar mechanisms, involving the prototypes of the cell-mediated immune responses, the phagocytosis and encapsulation. Work on Drosophila has played an important role in promoting an understanding of the basic mechanisms of phylogenetically conserved modules of innate immunity. The aim of this review is to survey the developments in the identification and functional definition of immune cell types and the immunological compartments of Drosophila melanogaster. We focus on the molecular and developmental aspects of the blood cell types and compartments, as well as the dynamics of blood cell development and the immune response. Further advances in the characterization of the innate immune mechanisms in Drosophila will provide basic clues to the understanding of the importance of the evolutionary conserved mechanisms of innate immune defenses in the animal kingdom. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Cytoplasmic pH and the regulation of the dictyostelium cell cycle

    NARCIS (Netherlands)

    Aerts, R.J.; Durston, A.J.; Moolenaar, W.H.

    1985-01-01

    Cytoplasmic pH (pHl) was monitored during the cell cycle of synchronous populations of Dictyostelium discoideum by means of a pH “null point” method. There is a cycle of pHl that closely corresponds to the DNA replication cycle, with a minimum of pH 7.20 in interphase and a peak of pH 7.45 during S

  9. Susceptibility to virus-cell fusion at the plasma membrane is reduced through expression of HIV gp41 cytoplasmic domains

    International Nuclear Information System (INIS)

    Malinowsky, Katharina; Luksza, Julia; Dittmar, Matthias T.

    2008-01-01

    The cytoplasmic tail of the HIV transmembrane protein plays an important role in viral infection. In this study we analyzed the role of retroviral cytoplasmic tails in modulating the cytoskeleton and interfering with virus-cell fusion. HeLaP4 cells expressing different HIV cytoplasmic tail constructs showed reduced acetylated tubulin levels whereas the cytoplasmic tail of MLV did not alter microtubule stability indicating a unique function for the lentiviral cytoplasmic tail. The effect on tubulin is mediated through the membrane proximal region of the HIV cytoplasmic tail and was independent of membrane localization. Site-directed mutagenesis identified three motifs in the HIV-2 cytoplasmic tail required to effect the reduction in acetylated tubulin. Both the YxxΦ domain and amino acids 21 to 45 of the HIV-2 cytoplasmic tail need to be present to change the level of acetylated tubulin in transfected cells. T-cells stably expressing one HIV-2 cytoplasmic tail derived construct showed also a reduction in acetylated tubulin thus confirming the importance of this effect not only for HeLaP4 and 293T cells. Challenge experiments using transiently transfected HeLaP4 cells and T cells stably expressing an HIV cytoplasmic tail construct revealed both reduced virus-cell fusion and replication of HIV-1 NL4.3 compared to control cells. In the virus-cell fusion assay only virions pseudotyped with either HIV or MLV envelopes showed reduced fusion efficiency, whereas VSV-G pseudotyped virions where not affected by the expression of HIV derived cytoplasmic tail constructs, indicating that fusion at the plasma but not endosomal membrane is affected. Overexpression of human histone-deacetylase 6 (HDAC6) and constitutively active RhoA resulted in a reduction of acetylated tubulin and reduced virus-cell fusion as significant as that observed following expression of HIV cytoplasmic tail constructs. Inhibition of HDAC6 showed a strong increase in acetylated tubulin and increase of

  10. Cytoplasmic inheritance of parent-offspring cell structure in the clonal diatom Cyclotella meneghiniana.

    Science.gov (United States)

    Shirokawa, Yuka; Shimada, Masakazu

    2016-11-16

    In cytoplasmic inheritance, structural states of a parent cell could be transmitted to offspring cells via two mechanisms. The first is referred to as the hangover of parent structure, where the structure itself remains and faithfully transmits within offspring cells; the second is structural inheritance, wherein the parent structure functions as a template for development of new offspring structure. We estimated to what extent the parent structure affects the development of offspring structure by structural inheritance, using a clone of the diatom Cyclotella meneghiniana The cell has two siliceous valves (a cell wall part at both cell poles): one is inherited from the parent and the other is newly formed. We estimated cytoplasmic heritability by comparing valve traits (central fultoportulae (CTFP), striae, central area, and cell diameter) of parent and new offspring valves, using single-cell isolation and valve labelling. Parent-offspring valve trait regressions showed that all traits, except CTFP, were significantly correlated. We formulated a quantitative genetic model considering the diatom inheritance system and revealed short-term rapid evolution compared with other inheritance systems. Diatom structural inheritance will have evolved to enable clonal populations to rapidly acquire and maintain suitable structures for temporal changes in environments and life-cycle stages. © 2016 The Author(s).

  11. Green tea polyphenol epigallocatechin-3-gallate differentially modulates oxidative stress in PC12 cell compartments

    International Nuclear Information System (INIS)

    Raza, Haider; John, Annie

    2005-01-01

    Tea polyphenols have been reported to be potent antioxidants and beneficial in oxidative stress related diseases. Prooxidant effects of tea polyphenols have also been reported in cell culture systems. In the present study, we have studied oxidative stress in the subcellular compartments of PC12 cells after treatment with different concentrations of the green tea polyphenol, epigallocatechin-3-gallate (EGCG). We have demonstrated that EGCG has differentially affected the production of reactive oxygen species (ROS), glutathione (GSH) metabolism and cytochrome P450 2E1 activity in the different subcellular compartments in PC12 cells. Our results have shown that although the cell survival was not inhibited by EGCG, there was, however, an increased DNA breakdown and activation of apoptotic markers, caspase 3 and poly- (ADP-ribose) polymerase (PARP) at higher concentrations of EGCG treatment. Our results suggest that the differential effects of EGCG might be related to the alterations in oxidative stress, GSH pools and CYP2E1 activity in different cellular compartments. These results may have implications in determining the chemopreventive therapeutic use of tea polyphenols in vivo

  12. Developmental potential of human oocytes reconstructed by transferring somatic cell nuclei into polyspermic zygote cytoplasm

    International Nuclear Information System (INIS)

    Fan, Yong; Chen, Xinjie; Luo, Yumei; Chen, Xiaolin; Li, Shaoying; Huang, Yulin; Sun, Xiaofang

    2009-01-01

    The generation of patient-specific nuclear transfer embryonic stem cells holds huge promise in modern regenerative medicine and cell-based drug discovery. Since human in vivo matured oocytes are not readily available, human therapeutic cloning is developing slowly. Here, we investigated for the first time whether human polyspermic zygotes could support preimplantation development of cloned embryos. Our results showed that polyspermic zygotes could be used as recipients for human somatic cell nuclear transfer (SCNT). The preimplantation developmental potential of SCNT embryos from polyspermic zygotes was limited to the 8-cell stage. Since ES cell lines can be derived from single blastomeres, these results may have important significance for human ES cells derived by SCNT. In addition, confocal images demonstrated that all of the SCNT embryos that failed to cleave showed abnormal microtubule organization. The results of the present study suggest that polyspermic human zygotes could be used as a potential source of recipient cytoplasm for SCNT.

  13. Developmental potential of human oocytes reconstructed by transferring somatic cell nuclei into polyspermic zygote cytoplasm

    Energy Technology Data Exchange (ETDEWEB)

    Fan, Yong; Chen, Xinjie; Luo, Yumei; Chen, Xiaolin; Li, Shaoying; Huang, Yulin [Institute of Gynecology and Obstetrics, The Third Affiliated Hospital of Guangzhou Medical College, Duobao Road 63, Guangzhou, Guangdong (China); Sun, Xiaofang, E-mail: xiaofangsun@hotmail.com [Institute of Gynecology and Obstetrics, The Third Affiliated Hospital of Guangzhou Medical College, Duobao Road 63, Guangzhou, Guangdong (China)

    2009-04-24

    The generation of patient-specific nuclear transfer embryonic stem cells holds huge promise in modern regenerative medicine and cell-based drug discovery. Since human in vivo matured oocytes are not readily available, human therapeutic cloning is developing slowly. Here, we investigated for the first time whether human polyspermic zygotes could support preimplantation development of cloned embryos. Our results showed that polyspermic zygotes could be used as recipients for human somatic cell nuclear transfer (SCNT). The preimplantation developmental potential of SCNT embryos from polyspermic zygotes was limited to the 8-cell stage. Since ES cell lines can be derived from single blastomeres, these results may have important significance for human ES cells derived by SCNT. In addition, confocal images demonstrated that all of the SCNT embryos that failed to cleave showed abnormal microtubule organization. The results of the present study suggest that polyspermic human zygotes could be used as a potential source of recipient cytoplasm for SCNT.

  14. Methuosis: nonapoptotic cell death associated with vacuolization of macropinosome and endosome compartments.

    Science.gov (United States)

    Maltese, William A; Overmeyer, Jean H

    2014-06-01

    Apoptosis is the most widely recognized form of physiological programmed cell death. During the past three decades, various nonapoptotic forms of cell death have gained increasing attention, largely because of their potential importance in pathological processes, toxicology, and cancer therapy. A recent addition to the panoply of cell death phenotypes is methuosis. The neologism is derived from the Greek methuo (to drink to intoxication) because the hallmark of this form of cell death is displacement of the cytoplasm by large fluid-filled vacuoles derived from macropinosomes. The demise of the cell resembles many forms of necrosis, insofar as there is a loss of metabolic capacity and plasma membrane integrity, without the cell shrinkage and nuclear fragmentation associated with apoptosis. Methuosis was initially defined in glioblastoma cells after ectopic expression of activated Ras, but recent reports have described small molecules that can induce the features of methuosis in a broad spectrum of cancer cells, including those that are resistant to conventional apoptosis-inducing drugs. This review summarizes the available information about the distinguishing morphological characteristics and underlying mechanisms of methuosis. We compare and contrast methuosis with other cytopathological conditions in which accumulation of clear cytoplasmic vacuoles is a prominent feature. Finally, we highlight key questions that need to be answered to determine whether methuosis truly represents a unique form of regulated cell death. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  15. Cytoplasmic streaming in plant cells emerges naturally by microfilament self-organization.

    Science.gov (United States)

    Woodhouse, Francis G; Goldstein, Raymond E

    2013-08-27

    Many cells exhibit large-scale active circulation of their entire fluid contents, a process termed cytoplasmic streaming. This phenomenon is particularly prevalent in plant cells, often presenting strikingly regimented flow patterns. The driving mechanism in such cells is known: myosin-coated organelles entrain cytoplasm as they process along actin filament bundles fixed at the periphery. Still unknown, however, is the developmental process that constructs the well-ordered actin configurations required for coherent cell-scale flow. Previous experimental works on streaming regeneration in cells of Characean algae, whose longitudinal flow is perhaps the most regimented of all, hint at an autonomous process of microfilament self-organization driving the formation of streaming patterns during morphogenesis. Working from first principles, we propose a robust model of streaming emergence that combines motor dynamics with both microscopic and macroscopic hydrodynamics to explain how several independent processes, each ineffectual on its own, can reinforce to ultimately develop the patterns of streaming observed in the Characeae and other streaming species.

  16. High ASMA+ Fibroblasts and Low Cytoplasmic HMGB1+ Breast Cancer Cells Predict Poor Prognosis.

    Science.gov (United States)

    Amornsupak, Kamolporn; Jamjuntra, Pranisa; Warnnissorn, Malee; O-Charoenrat, Pornchai; Sa-Nguanraksa, Doonyapat; Thuwajit, Peti; Eccles, Suzanne A; Thuwajit, Chanitra

    2017-10-01

    The influence of cancer-associated fibroblasts (CAFs) and high mobility group box 1 (HMGB1) has been recognized in several cancers, although their roles in breast cancer are unclear. The present study aimed to determine the levels and prognostic significance of α-smooth muscle actin-positive (ASMA + ) CAFs, plus HMGB1 and receptor for advanced glycation end products (RAGE) in cancer cells. A total of 127 breast samples, including 96 malignant and 31 benign, were examined for ASMA, HMGB1, and RAGE by immunohistochemistry. The χ 2 test and Fisher's exact test were used to test the association of each protein with clinicopathologic parameters. The Kaplan-Meier method or log-rank test and Cox regression were used for survival analysis. ASMA + fibroblast infiltration was significantly increased in the tumor stroma compared with that in benign breast tissue. The levels of cytoplasmic HMGB1 and RAGE were significantly greater in the breast cancer tissue than in the benign breast tissues. High ASMA expression correlated significantly with large tumor size, clinical stage III-IV, and angiolymphatic and perinodal invasion. In contrast, increased cytoplasmic HMGB1 correlated significantly with small tumor size, pT stage, early clinical stage, luminal subtype (but not triple-negative subtype), and estrogen receptor and progesterone receptor expression. The levels of ASMA (hazard ratio, 14.162; P = .010) and tumor cytoplasmic HMGB1 (hazard ratio, 0.221; P = .005) could serve as independent prognostic markers for metastatic relapse in breast cancer patients. The ASMA-high/HMGB1-low profile provided the most reliable prediction of metastatic relapse. We present for the first time, to the best of our knowledge, the potential clinical implications of the combined assessment of ASMA + fibroblasts and cytoplasmic HMGB1 in breast cancer. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  17. Cellular compartments cause multistability and allow cells to process more information

    DEFF Research Database (Denmark)

    Harrington, Heather A; Feliu, Elisenda; Wiuf, Carsten

    2013-01-01

    recent developments from dynamical systems and chemical reaction network theory to identify and characterize the key-role of the spatial organization of eukaryotic cells in cellular information processing. In particular, the existence of distinct compartments plays a pivotal role in whether a system...... is capable of multistationarity (multiple response states), and is thus directly linked to the amount of information that the signaling molecules can represent in the nucleus. Multistationarity provides a mechanism for switching between different response states in cell signaling systems and enables multiple...

  18. Loss of γ-cytoplasmic actin triggers myofibroblast transition of human epithelial cells.

    Science.gov (United States)

    Lechuga, Susana; Baranwal, Somesh; Li, Chao; Naydenov, Nayden G; Kuemmerle, John F; Dugina, Vera; Chaponnier, Christine; Ivanov, Andrei I

    2014-10-15

    Transdifferentiation of epithelial cells into mesenchymal cells and myofibroblasts plays an important role in tumor progression and tissue fibrosis. Such epithelial plasticity is accompanied by dramatic reorganizations of the actin cytoskeleton, although mechanisms underlying cytoskeletal effects on epithelial transdifferentiation remain poorly understood. In the present study, we observed that selective siRNA-mediated knockdown of γ-cytoplasmic actin (γ-CYA), but not β-cytoplasmic actin, induced epithelial-to-myofibroblast transition (EMyT) of different epithelial cells. The EMyT manifested by increased expression of α-smooth muscle actin and other contractile proteins, along with inhibition of genes responsible for cell proliferation. Induction of EMyT in γ-CYA-depleted cells depended on activation of serum response factor and its cofactors, myocardial-related transcriptional factors A and B. Loss of γ-CYA stimulated formin-mediated actin polymerization and activation of Rho GTPase, which appear to be essential for EMyT induction. Our findings demonstrate a previously unanticipated, unique role of γ-CYA in regulating epithelial phenotype and suppression of EMyT that may be essential for cell differentiation and tissue fibrosis. © 2014 Lechuga, Baranwal, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  19. Measuring cell viscoelastic properties using a force-spectrometer: influence of protein-cytoplasm interactions.

    Science.gov (United States)

    Canetta, Elisabetta; Duperray, Alain; Leyrat, Anne; Verdier, Claude

    2005-01-01

    Cell adhesive and rheological properties play a very important role in cell transmigration through the endothelial barrier, in particular in the case of inflammation (leukocytes) or cancer metastasis (cancer cells). In order to characterize cell viscoelastic properties, we have designed a force spectrometer (AFM) which can stretch cells thereby allowing measurement of their rheological properties. This custom-made force spectrometer allows two different visualizations, one lateral and one from below. It allows investigation of the effects of rheology involved during cell stretching. To test the ability of our system to characterize such viscoelastic properties, ICAM-1 transfected CHO cells were analyzed. Two forms of ICAM-1 were tested; wild type ICAM-1, which can interact with the cytoskeleton, and a mutant form which lacks the cytoplasmic domain, and is unable to associate with the cytoskeleton. Stretching experiments carried out on these cells show the formation of long filaments. Using a previous model of filament elongation, we could determine the viscoelastic properties of a single cell. As expected, different viscoelastic components were found between the wild type and the mutant, which reveal that the presence of interactions between ICAM-1 and the cytoskeleton increases the stiffness of the cell.

  20. Design and application of optical nanosensors for pH imaging in cell compartments

    DEFF Research Database (Denmark)

    Benjaminsen, Rikke Vicki; Almdal, Kristoffer

    the last two decades. However, even though these sensor systems have proven themselves as superior to conventional methods, there are still questions about the use of these sensors that need to be addressed, especially regarding sensor design and calibration. We have developed a new triple-labelled p......Measurements of pH in acidic cellular compartments of mammalian cells is important for our understanding of cell metabolism, and organelle acidification is an essential event in living cells especially in the endosomal-lysosomal pathway where pH is critical for cellular sorting of internalized...... material. Intracellular pH can be measured by the use of fluorescence ratio imaging microscopy (FRIM), however, available methods for pH measurements in living cells are not optimal. Nanoparticle based optical sensor technology for quantification of metabolites in living cells has been developed over...

  1. Extracellular signal-regulated kinase 2 (ERK-2) mediated phosphorylation regulates nucleo-cytoplasmic shuttling and cell growth control of Ras-associated tumor suppressor protein, RASSF2

    International Nuclear Information System (INIS)

    Kumari, Gita; Mahalingam, S.

    2009-01-01

    Ras GTPase controls the normal cell growth through binding with an array of effector molecules, such as Raf and PI3-kinase in a GTP-dependent manner. RASSF2, a member of the Ras association domain family, is known to be involved in the suppression of cell growth and is frequently down-regulated in various tumor tissues by promoter hypermethylation. In the present study, we demonstrate that RASSF2 shuttles between nucleus and cytoplasm by a signal-mediated process and its export from the nucleus is sensitive to leptomycin B. Amino acids between 240 to 260 in the C-terminus of RASSF2 harbor a functional nuclear export signal (NES), which is necessary and sufficient for efficient export of RASSF2 from the nucleus. Substitution of conserved Ile254, Val257 and Leu259 within the minimal NES impaired RASSF2 export from the nucleus. In addition, wild type but not the nuclear export defective RASSF2 mutant interacts with export receptor, CRM-1 and exported from the nucleus. Surprisingly, we observed nucleolar localization for the nuclear export defective mutant suggesting the possibility that RASSF2 may localize in different cellular compartments transiently in a cell cycle dependent manner and the observed nuclear localization for wild type protein may be due to faster export kinetics from the nucleolus. Furthermore, our data suggest that RASSF2 is specifically phosphorylated by MAPK/ERK-2 and the inhibitors of MAPK pathway impair the phosphorylation and subsequently block the export of RASSF2 from the nucleus. These data clearly suggest that ERK-2 mediated phosphorylation plays an important role in regulating the nucleo-cytoplasmic shuttling of RASSF2. Interestingly, nuclear import defective mutant of RASSF2 failed to induce cell cycle arrest at G1/S phase and apoptosis suggesting that RASSF2 regulates cell growth in a nuclear localization dependent manner. Collectively, these data provided evidence for the first time that MAPK/ERK-2 mediated phosphorylation regulates

  2. Extracellular signal-regulated kinase 2 (ERK-2) mediated phosphorylation regulates nucleo-cytoplasmic shuttling and cell growth control of Ras-associated tumor suppressor protein, RASSF2

    Energy Technology Data Exchange (ETDEWEB)

    Kumari, Gita [Laboratory of Molecular Virology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad 500076 (India); Mahalingam, S., E-mail: mahalingam@iitm.ac.in [Laboratory of Molecular Virology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad 500076 (India); Department of Biotechnology, Laboratory of Molecular Virology and Cell Biology, Indian Institute of Technology-Madras, Chennai 600 036 (India)

    2009-10-01

    Ras GTPase controls the normal cell growth through binding with an array of effector molecules, such as Raf and PI3-kinase in a GTP-dependent manner. RASSF2, a member of the Ras association domain family, is known to be involved in the suppression of cell growth and is frequently down-regulated in various tumor tissues by promoter hypermethylation. In the present study, we demonstrate that RASSF2 shuttles between nucleus and cytoplasm by a signal-mediated process and its export from the nucleus is sensitive to leptomycin B. Amino acids between 240 to 260 in the C-terminus of RASSF2 harbor a functional nuclear export signal (NES), which is necessary and sufficient for efficient export of RASSF2 from the nucleus. Substitution of conserved Ile254, Val257 and Leu259 within the minimal NES impaired RASSF2 export from the nucleus. In addition, wild type but not the nuclear export defective RASSF2 mutant interacts with export receptor, CRM-1 and exported from the nucleus. Surprisingly, we observed nucleolar localization for the nuclear export defective mutant suggesting the possibility that RASSF2 may localize in different cellular compartments transiently in a cell cycle dependent manner and the observed nuclear localization for wild type protein may be due to faster export kinetics from the nucleolus. Furthermore, our data suggest that RASSF2 is specifically phosphorylated by MAPK/ERK-2 and the inhibitors of MAPK pathway impair the phosphorylation and subsequently block the export of RASSF2 from the nucleus. These data clearly suggest that ERK-2 mediated phosphorylation plays an important role in regulating the nucleo-cytoplasmic shuttling of RASSF2. Interestingly, nuclear import defective mutant of RASSF2 failed to induce cell cycle arrest at G1/S phase and apoptosis suggesting that RASSF2 regulates cell growth in a nuclear localization dependent manner. Collectively, these data provided evidence for the first time that MAPK/ERK-2 mediated phosphorylation regulates

  3. The effect of ionizing irradiation on motion of cytoplasm in cells of Elodea canadensis

    International Nuclear Information System (INIS)

    Tordyiya, N.V.; Grodzyins'kij, D.M.; Danil'chenko, O.O.

    1999-01-01

    The effect of acute irradiation on the velocity of cytoplasm is investigated. It is shown that, for small doses, there is a strong nonlinearity between the velocity of cytoplasm and dose. The nonlinear behavior disappears with increasing a dose

  4. Iron repletion relocalizes hephaestin to a proximal basolateral compartment in polarized MDCK and Caco2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Seung-Min [Department of Biological Sciences, University of Columbia, NY (United States); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Attieh, Zouhair K. [Department of Laboratory Science and Technology, American University of Science and Technology, Ashrafieh (Lebanon); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Son, Hee Sook [Department of Food Science and Human Nutrition, College of Human Ecology, Chonbuk National University (Korea, Republic of); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Chen, Huijun [Medical School, Nanjing University, Nanjing 210008, Jiangsu Province (China); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Bacouri-Haidar, Mhenia [Department of Biology, Faculty of Sciences (I), Lebanese University, Hadath (Lebanon); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Vulpe, Chris D., E-mail: vulpe@berkeley.edu [Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States)

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer Hephaestin localizes in the perinuclear space in non-polarized cells. Black-Right-Pointing-Pointer Hephaestin localizes in the perinuclear space in iron deficient and polarized cells. Black-Right-Pointing-Pointer Hephaestin with apical iron moves near to basolateral membrane of polarized cells. Black-Right-Pointing-Pointer Peri-basolateral location of hephaestin is accessible to the extracellular space. Black-Right-Pointing-Pointer Hephaestin is involved in iron mobilization from the intestine to circulation. -- Abstract: While intestinal cellular iron entry in vertebrates employs multiple routes including heme and non-heme routes, iron egress from these cells is exclusively channeled through the only known transporter, ferroportin. Reduced intestinal iron export in sex-linked anemia mice implicates hephaestin, a ferroxidase, in this process. Polarized cells are exposed to two distinct environments. Enterocytes contact the gut lumen via the apical surface of the cell, and through the basolateral surface, to the body. Previous studies indicate both local and systemic control of iron uptake. We hypothesized that differences in iron availability at the apical and/or basolateral surface may modulate iron uptake via cellular localization of hephaestin. We therefore characterized the localization of hephaestin in two models of polarized epithelial cell lines, MDCK and Caco2, with varying iron availability at the apical and basolateral surfaces. Our results indicate that hephaestin is expressed in a supra-nuclear compartment in non-polarized cells regardless of the iron status of the cells and in iron deficient and polarized cells. In polarized cells, we found that both apical (as FeSO{sub 4}) and basolateral iron (as the ratio of apo-transferrin to holo-transferrin) affect mobilization of hephaestin from the supra-nuclear compartment. We find that the presence of apical iron is essential for relocalization of hephaestin to a

  5. DNA precursor compartmentation in mammalian cells: distribution and rates of equilibration between nucleus and cytoplasm

    International Nuclear Information System (INIS)

    Leeds, J.M.

    1986-01-01

    A rapid nuclear isolation technique was adapted in order to examine the question of DNA precursor compartmentation in mammalian cells. By using this method a reproducible proportion of the cellular nucleotides remained associated with the isolated nuclei. Examination, at several different cell densities, of exponentially growing HeLa cells showed that the nuclei contained a constant but distinct proportion of each dNTP. The nuclear dATP and dTTP concentrations were equal at all densities examined even though the dTTP pool was 150% of the dATP whole-cell pool. The nuclear portion of the whole-cell pools was roughly equal to the volume occupied by the nucleus. The nuclear-cytoplasmic dNTP pool distribution did not change throughout the cell cycle of synchronized Chinese hamster ovary (CHO) cells. The rates at which either radiolabeled cytidine or deoxycytidine equilibrated with the nuclear and whole-cell dCTP pools of G1 and S phase CHO cells were compared. Experiments comparing the labeling kinetics of 3 H-thymidine in G1, S phase, and exponentially growing cells revealed that the S phase dTTP pool equilibrated with exogenously added thymidine faster than the G1 phase pool. The rate of equilibration in exponentially growing cells appeared to be a combination of that seen in G1 and S phases. A linear rate of 3 H-thymidine incorporation into DNA occurred at the same rate in S phase and exponentially growing cells

  6. Mapping of ESE-1 subdomains required to initiate mammary epithelial cell transformation via a cytoplasmic mechanism

    Directory of Open Access Journals (Sweden)

    Tentler John J

    2011-08-01

    Full Text Available Abstract Background The ETS family transcription factor ESE-1 is often overexpressed in human breast cancer. ESE-1 initiates transformation of MCF-12A cells via a non-transcriptional, cytoplasmic process that is mediated by a unique 40-amino acid serine and aspartic acid rich (SAR subdomain, whereas, ESE-1's nuclear transcriptional property is required to maintain the transformed phenotype of MCF7, ZR-75-1 and T47D breast cancer cells. Results To map the minimal functional nuclear localization (NLS and nuclear export (NES signals, we fused in-frame putative NLS and NES motifs between GFP and the SAR domain. Using these GFP constructs as reporters of subcellular localization, we mapped a single NLS to six basic amino acids (242HGKRRR247 in the AT-hook and two CRM1-dependent NES motifs, one to the pointed domain (NES1: 102LCNCALEELRL112 and another to the DNA binding domain (DBD, (NES2: 275LWEFIRDILI284. Moreover, analysis of a putative NLS located in the DBD (316GQKKKNSN323 by a similar GFP-SAR reporter or by internal deletion of the DBD, revealed this sequence to lack NLS activity. To assess the role of NES2 in regulating ESE-1 subcellular localization and subsequent transformation potency, we site-specifically mutagenized NES2, within full-length GFP-ESE-1 and GFP-NES2-SAR reporter constructs. These studies show that site-specific mutation of NES2 completely abrogates ESE-1 transforming activity. Furthermore, we show that exclusive cytoplasmic targeting of the SAR domain is sufficient to initiate transformation, and we report that an intact SAR domain is required, since block mutagenesis reveals that an intact SAR domain is necessary to maintain its full transforming potency. Finally, using a monoclonal antibody targeting the SAR domain, we demonstrate that the SAR domain contains a region accessible for protein - protein interactions. Conclusions These data highlight that ESE-1 contains NLS and NES signals that play a critical role in

  7. Coulomb interactions between cytoplasmic electric fields and phosphorylated messenger proteins optimize information flow in cells.

    Directory of Open Access Journals (Sweden)

    Robert A Gatenby

    2010-08-01

    Full Text Available Normal cell function requires timely and accurate transmission of information from receptors on the cell membrane (CM to the nucleus. Movement of messenger proteins in the cytoplasm is thought to be dependent on random walk. However, Brownian motion will disperse messenger proteins throughout the cytosol resulting in slow and highly variable transit times. We propose that a critical component of information transfer is an intracellular electric field generated by distribution of charge on the nuclear membrane (NM. While the latter has been demonstrated experimentally for decades, the role of the consequent electric field has been assumed to be minimal due to a Debye length of about 1 nanometer that results from screening by intracellular Cl- and K+. We propose inclusion of these inorganic ions in the Debye-Huckel equation is incorrect because nuclear pores allow transit through the membrane at a rate far faster than the time to thermodynamic equilibrium. In our model, only the charged, mobile messenger proteins contribute to the Debye length.Using this revised model and published data, we estimate the NM possesses a Debye-Huckel length of a few microns and find this is consistent with recent measurement using intracellular nano-voltmeters. We demonstrate the field will accelerate isolated messenger proteins toward the nucleus through Coulomb interactions with negative charges added by phosphorylation. We calculate transit times as short as 0.01 sec. When large numbers of phosphorylated messenger proteins are generated by increasing concentrations of extracellular ligands, we demonstrate they generate a self-screening environment that regionally attenuates the cytoplasmic field, slowing movement but permitting greater cross talk among pathways. Preliminary experimental results with phosphorylated RAF are consistent with model predictions.This work demonstrates that previously unrecognized Coulomb interactions between phosphorylated messenger

  8. A Multi-Compartment Hybrid Computational Model Predicts Key Roles for Dendritic Cells in Tuberculosis Infection

    Directory of Open Access Journals (Sweden)

    Simeone Marino

    2016-10-01

    Full Text Available Tuberculosis (TB is a world-wide health problem with approximately 2 billion people infected with Mycobacterium tuberculosis (Mtb, the causative bacterium of TB. The pathologic hallmark of Mtb infection in humans and Non-Human Primates (NHPs is the formation of spherical structures, primarily in lungs, called granulomas. Infection occurs after inhalation of bacteria into lungs, where resident antigen-presenting cells (APCs, take up bacteria and initiate the immune response to Mtb infection. APCs traffic from the site of infection (lung to lung-draining lymph nodes (LNs where they prime T cells to recognize Mtb. These T cells, circulating back through blood, migrate back to lungs to perform their immune effector functions. We have previously developed a hybrid agent-based model (ABM, labeled GranSim describing in silico immune cell, bacterial (Mtb and molecular behaviors during tuberculosis infection and recently linked that model to operate across three physiological compartments: lung (infection site where granulomas form, lung draining lymph node (LN, site of generation of adaptive immunity and blood (a measurable compartment. Granuloma formation and function is captured by a spatio-temporal model (i.e., ABM, while LN and blood compartments represent temporal dynamics of the whole body in response to infection and are captured with ordinary differential equations (ODEs. In order to have a more mechanistic representation of APC trafficking from the lung to the lymph node, and to better capture antigen presentation in a draining LN, this current study incorporates the role of dendritic cells (DCs in a computational fashion into GranSim. Results: The model was calibrated using experimental data from the lungs and blood of NHPs. The addition of DCs allowed us to investigate in greater detail mechanisms of recruitment, trafficking and antigen presentation and their role in tuberculosis infection. Conclusion: The main conclusion of this study is

  9. Cell density-dependent nuclear/cytoplasmic localization of NORPEG (RAI14) protein

    International Nuclear Information System (INIS)

    Kutty, R. Krishnan; Chen, Shanyi; Samuel, William; Vijayasarathy, Camasamudram; Duncan, Todd; Tsai, Jen-Yue; Fariss, Robert N.; Carper, Deborah; Jaworski, Cynthia; Wiggert, Barbara

    2006-01-01

    NORPEG (RAI14), a developmentally regulated gene induced by retinoic acid, encodes a 980 amino acid (aa) residue protein containing six ankyrin repeats and a long coiled-coil domain [Kutty et al., J. Biol. Chem. 276 (2001), pp. 2831-2840]. We have expressed aa residues 1-287 of NORPEG and used the recombinant protein to produce an anti-NORPEG polyclonal antibody. Confocal immunofluorescence analysis showed that the subcellular localization of NORPEG in retinal pigment epithelial (ARPE-19) cells varies with cell density, with predominantly nuclear localization in nonconfluent cells, but a cytoplasmic localization, reminiscent of cytoskeleton, in confluent cultures. Interestingly, an evolutionarily conserved putative monopartite nuclear localization signal (P 27 KKRKAP 276 ) was identified by analyzing the sequences of NORPEG and its orthologs. GFP-NORPEG (2-287 aa), a fusion protein containing this signal, was indeed localized to nuclei when expressed in ARPE-19 or COS-7 cells. Deletion and mutation analysis indicated that the identified nuclear localization sequence is indispensable for nuclear targeting

  10. Pollen mitochondria in cytoplasmically male sterile tobacco zygotic and embryonic cells

    International Nuclear Information System (INIS)

    Symillides, Y.

    1985-09-01

    An attempt is being made to establish cytoplasmic organelles transmission during the process of fertilization, by using tobacco grain pollen labelled with leucine 14 C and tritiated thymidine. Through autoradiography the fate of pollen germination and its entry into the embryo sac has been studied. A few days after fertilization, labelled cytoplasmic organelles - mainly mitochondria - were detected in the embryo sac. However, labelling was not observed in cytoplasmic organelles by using tritiated thymidine. For more conclusive results labelled DNA incorporated in cytoplasmic organelles have to be traced during the embryo and endosperm development

  11. Human herpesvirus-8 infection leads to expansion of the preimmune/natural effector B cell compartment.

    Directory of Open Access Journals (Sweden)

    Silvia Della Bella

    Full Text Available BACKGROUND: Human herpesvirus-8 (HHV-8 is the etiological agent of Kaposi's sarcoma (KS and of some lymphoproliferative disorders of B cells. Most malignancies develop after long-lasting viral dormancy, and a preventing role for both humoral and cellular immune control is suggested by the high frequency of these pathologies in immunosuppressed patients. B cells, macrophages and dendritic cells of peripheral lymphoid organs and blood represent the major reservoir of HHV-8. Due to the dual role of B cells in HHV-8 infection, both as virus reservoir and as agents of humoral immune control, we analyzed the subset distribution and the functional state of peripheral blood B cells in HHV-8-infected individuals with and without cKS. METHODOLOGY/PRINCIPAL FINDINGS: Circulating B cells and their subsets were analyzed by 6-color flow cytometry in the following groups: 1- patients HHV-8 positive with classic KS (cKS (n = 47; 2- subjects HHV-8 positive and cKS negative (HSP (n = 10; 3- healthy controls, HHV-8 negative and cKS negative (HC (n = 43. The number of B cells belonging to the preimmune/natural effector compartment, including transitional, pre-naïve, naïve and MZ-like subsets, was significantly higher among HHV-8 positive subjects, with or without cKS, while was comparable to healthy controls in the antigen-experienced T-cell dependent compartment. The increased number of preimmune/natural effector B cells was associated with increased resistance to spontaneous apoptosis, while it did not correlate with HHV-8 viral load. CONCLUSIONS/SIGNIFICANCE: Our results indicate that long-lasting HHV-8 infection promotes an imbalance in peripheral B cell subsets, perturbing the equilibrium between earlier and later steps of maturation and activation processes. This observation may broaden our understanding of the complex interplay between viral and immune factors leading HHV-8-infected individuals to develop HHV-8-associated malignancies.

  12. The nectin-1α transmembrane domain, but not the cytoplasmic tail, influences cell fusion induced by HSV-1 glycoproteins

    International Nuclear Information System (INIS)

    Subramanian, Ravi P.; Dunn, Jennifer E.; Geraghty, Robert J.

    2005-01-01

    Nectin-1 is a receptor for herpes simplex virus (HSV), a member of the immunoglobulin superfamily, and a cellular adhesion molecule. To study domains of nectin-1α involved in cell fusion, we measured the ability of nectin-1α/nectin-2α chimeras, nectin-1α/CD4 chimeras, and transmembrane domain and cytoplasmic tail mutants of nectin-1α to promote cell fusion induced by HSV-1 glycoproteins. Our results demonstrate that only chimeras and mutants containing the entire V-like domain and a link to the plasma membrane conferred cell-fusion activity. The transmembrane domain and cytoplasmic tail of nectin-1 were not required for any viral receptor or cell adhesion function tested. Cellular cytoplasmic factors that bind to the nectin-1α cytoplasmic tail, therefore, did not influence virus entry or cell fusion. Interestingly, the efficiency of cell fusion was reduced when membrane-spanning domains of nectin-1α and gD were replaced by glycosylphosphatidylinositol tethers, indicating that transmembrane domains may play a modulatory role in the gD/nectin-1α interaction in fusion

  13. Stat5 Exerts Distinct, Vital Functions in the Cytoplasm and Nucleus of Bcr-Abl+ K562 and Jak2(V617F)+ HEL Leukemia Cells

    International Nuclear Information System (INIS)

    Weber, Axel; Borghouts, Corina; Brendel, Christian; Moriggl, Richard; Delis, Natalia; Brill, Boris; Vafaizadeh, Vida; Groner, Bernd

    2015-01-01

    Signal transducers and activators of transcription (Stats) play central roles in the conversion of extracellular signals, e.g., cytokines, hormones and growth factors, into tissue and cell type specific gene expression patterns. In normal cells, their signaling potential is strictly limited in extent and duration. The persistent activation of Stat3 or Stat5 is found in many human tumor cells and contributes to their growth and survival. Stat5 activation plays a pivotal role in nearly all hematological malignancies and occurs downstream of oncogenic kinases, e.g., Bcr-Abl in chronic myeloid leukemias (CML) and Jak2(V617F) in other myeloproliferative diseases (MPD). We defined the mechanisms through which Stat5 affects growth and survival of K562 cells, representative of Bcr-Abl positive CML, and HEL cells, representative for Jak2(V617F) positive acute erythroid leukemia. In our experiments we suppressed the protein expression levels of Stat5a and Stat5b through shRNA mediated downregulation and demonstrated the dependence of cell survival on the presence of Stat5. Alternatively, we interfered with the functional capacities of the Stat5 protein through the interaction with a Stat5 specific peptide ligand. This ligand is a Stat5 specific peptide aptamer construct which comprises a 12mer peptide integrated into a modified thioredoxin scaffold, S5-DBD-PA. The peptide sequence specifically recognizes the DNA binding domain (DBD) of Stat5. Complex formation of S5-DBD-PA with Stat5 causes a strong reduction of P-Stat5 in the nuclear fraction of Bcr-Abl-transformed K562 cells and a suppression of Stat5 target genes. Distinct Stat5 mediated survival mechanisms were detected in K562 and Jak2(V617F)-transformed HEL cells. Stat5 is activated in the nuclear and cytosolic compartments of K562 cells and the S5-DBD-PA inhibitor most likely affects the viability of Bcr-Abl + K562 cells through the inhibition of canonical Stat5 induced target gene transcription. In HEL cells, Stat5

  14. NMR quantification of diffusional exchange in cell suspensions with relaxation rate differences between intra and extracellular compartments.

    Science.gov (United States)

    Eriksson, Stefanie; Elbing, Karin; Söderman, Olle; Lindkvist-Petersson, Karin; Topgaard, Daniel; Lasič, Samo

    2017-01-01

    Water transport across cell membranes can be measured non-invasively with diffusion NMR. We present a method to quantify the intracellular lifetime of water in cell suspensions with short transverse relaxation times, T2, and also circumvent the confounding effect of different T2 values in the intra- and extracellular compartments. Filter exchange spectroscopy (FEXSY) is specifically sensitive to exchange between compartments with different apparent diffusivities. Our investigation shows that FEXSY could yield significantly biased results if differences in T2 are not accounted for. To mitigate this problem, we propose combining FEXSY with diffusion-relaxation correlation experiment, which can quantify differences in T2 values in compartments with different diffusivities. Our analysis uses a joint constrained fitting of the two datasets and considers the effects of diffusion, relaxation and exchange in both experiments. The method is demonstrated on yeast cells with and without human aquaporins.

  15. Somatic cell cloning in Buffalo (Bubalus bubalis): effects of interspecies cytoplasmic recipients and activation procedures.

    Science.gov (United States)

    Kitiyanant, Y; Saikhun, J; Chaisalee, B; White, K L; Pavasuthipaisit, K

    2001-01-01

    Successful nuclear transfer (NT) of somatic cell nuclei from various mammalian species to enucleated bovine oocytes provides a universal cytoplast for NT in endangered or extinct species. Buffalo fetal fibroblasts were isolated from a day 40 fetus and were synchronized in presumptive G(0) by serum deprivation. Buffalo and bovine oocytes from abattoir ovaries were matured in vitro and enucleated at 22 h. In the first experiment, we compared the ability of buffalo and bovine oocyte cytoplasm to support in vitro development of NT embryos produced by buffalo fetal fibroblasts as donor nuclei. There were no significant differences (p > 0.05) between the NT embryos derived from buffalo and bovine oocytes, in fusion (74% versus 71%) and cleavage (77% versus 75%) rates, respectively. No significant differences were also observed in blastocyst development (39% versus 33%) and the mean cell numbers of day 7 cloned blastocysts (88.5 +/- 25.7 versus 51.7 +/- 5.4). In the second experiment, we evaluated the effects of activation with calcium ionophore A23187 on development of NT embryos after electrical fusion. A significantly higher (p cloned buffalo blastocysts similar to those transferred into buffalo oocytes. Calcium ionophore used in conjunction with 6-DMAP effectively induces NT embryo development.

  16. Cross-species genomics matches driver mutations and cell compartments to model ependymoma

    Science.gov (United States)

    Johnson, Robert A.; Wright, Karen D.; Poppleton, Helen; Mohankumar, Kumarasamypet M.; Finkelstein, David; Pounds, Stanley B.; Rand, Vikki; Leary, Sarah E.S.; White, Elsie; Eden, Christopher; Hogg, Twala; Northcott, Paul; Mack, Stephen; Neale, Geoffrey; Wang, Yong-Dong; Coyle, Beth; Atkinson, Jennifer; DeWire, Mariko; Kranenburg, Tanya A.; Gillespie, Yancey; Allen, Jeffrey C.; Merchant, Thomas; Boop, Fredrick A.; Sanford, Robert. A.; Gajjar, Amar; Ellison, David W.; Taylor, Michael D.; Grundy, Richard G.; Gilbertson, Richard J.

    2010-01-01

    Understanding the biology that underlies histologically similar but molecularly distinct subgroups of cancer has proven difficult since their defining genetic alterations are often numerous, and the cellular origins of most cancers remain unknown1–3. We sought to decipher this heterogeneity by integrating matched genetic alterations and candidate cells of origin to generate accurate disease models. First, we identified subgroups of human ependymoma, a form of neural tumor that arises throughout the central nervous system (CNS). Subgroup specific alterations included amplifications and homozygous deletions of genes not yet implicated in ependymoma. To select cellular compartments most likely to give rise to subgroups of ependymoma, we matched the transcriptomes of human tumors to those of mouse neural stem cells (NSCs), isolated from different regions of the CNS at different developmental stages, with an intact or deleted Ink4a/Arf locus. The transcriptome of human cerebral ependymomas with amplified EPHB2 and deleted INK4A/ARF matched only that of embryonic cerebral Ink4a/Arf−/− NSCs. Remarkably, activation of Ephb2 signaling in these, but not other NSCs, generated the first mouse model of ependymoma, which is highly penetrant and accurately models the histology and transcriptome of one subgroup of human cerebral tumor. Further comparative analysis of matched mouse and human tumors revealed selective deregulation in the expression and copy number of genes that control synaptogenesis, pinpointing disruption of this pathway as a critical event in the production of this ependymoma subgroup. Our data demonstrate the power of cross-species genomics to meticulously match subgroup specific driver mutations with cellular compartments to model and interrogate cancer subgroups. PMID:20639864

  17. Urinary CD4+ Effector Memory T Cells Reflect Renal Disease Activity in Antineutrophil Cytoplasmic Antibody-Associated Vasculitis

    NARCIS (Netherlands)

    Abdulahad, Wayel H.; Kallenberg, Cees G. M.; Limburg, Pieter C.; Stegeman, Coen A.

    Objective. Numbers of circulating CD4+ effector memory T cells are proportionally increased in patients with proteinase 3 antineutrophil cytoplasmic antibody-associated vasculitis (AAV) whose disease is in remission and are decreased during active disease, which presumably reflects their migration

  18. Nanoparticle uptake and their co-localization with cell compartments - a confocal Raman microscopy study at single cell level

    International Nuclear Information System (INIS)

    Estrela-Lopis, I; Donath, E; Romero, G; Rojas, E; Moya, S E

    2011-01-01

    Confocal Raman Microscopy, a non-invasive, non-destructive and label-free technique, was employed to study the uptake and localization of nanoparticles (NPs) in the Hepatocarcinoma human cell line HepG2 at the level of single cells. Cells were exposed to carbon nanotubes (CNTs) the surface of which was engineered with polyelectrolytes and lipid layers, aluminium oxide and cerium dioxide nanoparticles. Raman spectra deconvolution was applied to obtain the spatial distributions of NPs together with lipids/proteins in cells. The colocalization of the NPs with different intracellular environments, lipid bodies, protein and DNA, was inferred. Lipid coated CNTs associated preferentially with lipid rich regions, whereas polyelectrolyte coated CNTs were excluded from lipid rich regions. Al 2 O 3 NPs were found in the cytoplasm. CeO 2 NPs were readily taken up and have been observed all over the cell. Raman z-scans proved the intracellular distribution of the respective NPs.

  19. Nanoparticle uptake and their co-localization with cell compartments - a confocal Raman microscopy study at single cell level

    Science.gov (United States)

    Estrela-Lopis, I.; Romero, G.; Rojas, E.; Moya, S. E.; Donath, E.

    2011-07-01

    Confocal Raman Microscopy, a non-invasive, non-destructive and label-free technique, was employed to study the uptake and localization of nanoparticles (NPs) in the Hepatocarcinoma human cell line HepG2 at the level of single cells. Cells were exposed to carbon nanotubes (CNTs) the surface of which was engineered with polyelectrolytes and lipid layers, aluminium oxide and cerium dioxide nanoparticles. Raman spectra deconvolution was applied to obtain the spatial distributions of NPs together with lipids/proteins in cells. The colocalization of the NPs with different intracellular environments, lipid bodies, protein and DNA, was inferred. Lipid coated CNTs associated preferentially with lipid rich regions, whereas polyelectrolyte coated CNTs were excluded from lipid rich regions. Al2O3 NPs were found in the cytoplasm. CeO2 NPs were readily taken up and have been observed all over the cell. Raman z-scans proved the intracellular distribution of the respective NPs.

  20. A One-compartment direct glucose alkaline fuel cell with methyl viologen as electron mediator

    International Nuclear Information System (INIS)

    Liu, Xianhua; Hao, Miaoqing; Feng, Mengnan; Zhang, Lin; Zhao, Yong; Du, Xiwen; Wang, Guangyi

    2013-01-01

    Highlights: ► A glucose–air alkaline fuel cell without using noble metal catalysts has been developed. ► The rudimentary fuel cell generates a maximum power density of 0.62 mW m −2 . ► The high performance is attributed to the use of MV and nickel foam. ► Main oxidation products are small organic acids indicating deep oxidation of glucose. - Abstract: Glucose is abundant, renewable, non-toxic and convenient as a fuel for fuel cells, but current technologies are unavailable for us to directly oxidize it to obtain energy. Fuel cells using enzymes and micro-organisms as catalysts are limited by their extremely low power output and rather short durability. Fuel cells using precious metal catalyst are expensive for large-scale use. In this work, a one-compartment direct glucose alkaline fuel cell has been developed that use methyl viologen (MV) as electron mediator and nickel foam as the anode. The rudimentary fuel cell generates a maximum power density of 0.62 mW cm −2 , while the maximum current density is 5.03 mA cm −2 . Electro-catalytic activities of MV and the nickel foam in alkaline conditions were studied by cyclic voltammetry. It is indicated that the high performance of the fuel cell is attributed to the combined use of MV and nickel foam. 13 C-NMR and HPLC were used to analyze oxidation products of glucose. The result shows that the principal oxidation products are short-chain organic acids indicating deep oxidation of glucose is achieved

  1. The effect of conditional EFNB1 deletion in the T cell compartment on T cell development and function

    Directory of Open Access Journals (Sweden)

    Jin Wei

    2011-12-01

    Full Text Available Abstract Background Eph kinases are the largest family of cell surface receptor tyrosine kinases. The ligands of Ephs, ephrins (EFNs, are also cell surface molecules. Ephs interact with EFNs transmitting signals in both directions, i.e., from Ephs to EFNs and from EFNs to Ephs. EFNB1 is known to be able to co-stimulate T cells in vitro and to modulate thymocyte development in a model of foetal thymus organ culture. To further understand the role of EFNB1 in T cell immunity, we generated T-cell-specific EFNB1 gene knockout mice to assess T cell development and function in these mice. Results The mice were of normal size and cellularity in the thymus and spleen and had normal T cell subpopulations in these organs. The bone marrow progenitors from KO mice and WT control mice repopulated host spleen T cell pool to similar extents. The activation and proliferation of KO T cells was comparable to that of control mice. Naïve KO CD4 cells showed an ability to differentiate into Th1, Th2, Th17 and Treg cells similar to control CD4 cells. Conclusions Our results suggest that the function of EFNB1 in the T cell compartment could be compensated by other members of the EFN family, and that such redundancy safeguards the pivotal roles of EFNB1 in T cell development and function.

  2. Physical change in cytoplasmic messenger ribonucleoproteins in cells treated with inhibitors of mRNA transcription

    International Nuclear Information System (INIS)

    Dreyfuss, G.; Adam, S.A.; Choi, Y.D.

    1984-01-01

    Exposure of intact cells to UV light brings about cross-linking of polyadenylated mRNA to a set of cytoplasmic proteins which are in direct contact with the mRNA in vivo. Substantial amounts of an additional protein of molecular weight 38,000 become cross-linked to the mRNA when cells are treated with inhibitors of mRNA synthesis (actinomycin D, camptothecin, and 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole) or after infection with vesicular stomatitis virus. Cordycepin, which inhibits polyadenylation but not mRNA synthesis, has no such effect. Inhibitors of protein synthesis and of rRNA synthesis are also without effect on 38K cross-linking to mRNA. The onset of the effect of inhibitors of mRNA synthesis on the UV cross-linkable interaction between mRNA and 38K is rapid and reaches a maximal level in less than 60 min, and it is completely and rapidly reversible. In cells treated with actinomycin D, the amount of 38K which becomes cross-linked to mRNA is proportional to the extent of inhibition of mRNA synthesis. The association of 38K with mRNA during transcriptional arrest does not require protein synthesis because simultaneous treatment with the protein synthesis inhibitor emetine does not interfere with it. The effectors which promote the interaction of 38K with mRNA do not affect the proteins which are in contact with polyadenylated heterogeneous nuclear RNA and do not markedly affect protein synthesis in the cell. The 38K protein can be isolated with the polyribosomal polyadenylated fraction from which it was purified, and monoclonal antibodies against it were prepared

  3. Crane system with remote actuation mechanism for use in argon compartment in ACPF hot cell

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jong Kwang, E-mail: leejk@kaeri.re.kr; Park, Byung-Suk; Yu, Seung-Nam; Kim, Kiho; Cho, Il-je

    2016-10-15

    Highlights: • Novel crane system with a remote actuation mechanism for feasible maintenance under limited space conditions is proposed. • Linear drive systems are implemented for accurate positioning. • Modular design concepts for easy maintenance are introduced. • The motion controller and the off-the-shelf camera controller are integrated to provide more efficient operation. - Abstract: The Advanced spent fuel Conditioning Process Facility (ACPF) at the Korea Atomic Energy Research Institute (KAERI) has recently been successfully renovated. One of the highlights of this renovation project was the installation of a small argon compartment within the atmospheric hot cell of the facility. Even though a crane system was considered necessary for the remote handling of the processing equipment inside the argon compartment, no suitable commercial cranes were available. This was because a limited amount of space had been reserved for the installation of the crane. Moreover, a master-slave manipulator (MSM), the only available means of maintenance of the crane, was unable to reach it in the limited workspace. To address the difficulties in the design of this crane, in this study, a remote actuation mechanism is devised where the mechanical and electrical parts of the crane system are separated, positioned far away from each other, and connected through power transmission shafts. This approach has two main advantages. First, the electrical parts can be placed inside the workspace of the MSM, hence allowing for remote maintenance. Second, the space occupied by the electrical parts and their cables, which are separate from the crane in the proposed design, can be considered and exploited in designing the mechanical parts of the crane. This enables the construction of a short, special crane in order to maximize the workspace. Furthermore, the mechanical parts for the MSM located outside the workspace are designed to possess a high safety margin to ensure durability

  4. Autophagy is involved in the reduction of myelinating Schwann cell cytoplasm during myelin maturation of the peripheral nerve.

    Directory of Open Access Journals (Sweden)

    So Young Jang

    Full Text Available Peripheral nerve myelination involves dynamic changes in Schwann cell morphology and membrane structure. Recent studies have demonstrated that autophagy regulates organelle biogenesis and plasma membrane dynamics. In the present study, we investigated the role of autophagy in the development and differentiation of myelinating Schwann cells during sciatic nerve myelination. Electron microscopy and biochemical assays have shown that Schwann cells remove excess cytoplasmic organelles during myelination through macroautophagy. Inhibition of autophagy via Schwann cell-specific removal of ATG7, an essential molecule for macroautophagy, using a conditional knockout strategy, resulted in abnormally enlarged abaxonal cytoplasm in myelinating Schwann cells that contained a large number of ribosomes and an atypically expanded endoplasmic reticulum. Small fiber hypermyelination and minor anomalous peripheral nerve functions are observed in this mutant. Rapamycin-induced suppression of mTOR activity during the early postnatal period enhanced not only autophagy but also developmental reduction of myelinating Schwann cells cytoplasm in vivo. Together, our findings suggest that autophagy is a regulatory mechanism of Schwann cells structural plasticity during myelination.

  5. Comparative Characterization of Cells from the Various Compartments of the Human Umbilical Cord Shows that the Wharton's Jelly Compartment Provides the Best Source of Clinically Utilizable Mesenchymal Stem Cells.

    Directory of Open Access Journals (Sweden)

    Arjunan Subramanian

    Full Text Available The human umbilical cord (UC is an attractive source of mesenchymal stem cells (MSCs with unique advantages over other MSC sources. They have been isolated from different compartments of the UC but there has been no rigorous comparison to identify the compartment with the best clinical utility. We compared the histology, fresh and cultured cell numbers, morphology, proliferation, viability, stemness characteristics and differentiation potential of cells from the amnion (AM, subamnion (SA, perivascular (PV, Wharton's jelly (WJ and mixed cord (MC of five UCs. The WJ occupied the largest area in the UC from which 4.61 ± 0.57 x 106 /cm fresh cells could be isolated without culture compared to AM, SA, PV and MC that required culture. The WJ and PV had significantly lesser CD40+ non-stem cell contaminants (26-27% compared to SA, AM and MC (51-70%. Cells from all compartments were proliferative, expressed the typical MSC-CD, HLA, and ESC markers, telomerase, had normal karyotypes and differentiated into adipocyte, chondrocyte and osteocyte lineages. The cells from WJ showed significantly greater CD24+ and CD108+ numbers and fluorescence intensities that discriminate between MSCs and non-stem cell mesenchymal cells, were negative for the fibroblast-specific and activating-proteins (FSP, FAP and showed greater osteogenic and chondrogenic differentiation potential compared to AM, SA, PV and MC. Cells from the WJ offer the best clinical utility as (i they have less non-stem cell contaminants (ii can be generated in large numbers with minimal culture avoiding changes in phenotype, (iii their derivation is quick and easy to standardize, (iv they are rich in stemness characteristics and (v have high differentiation potential. Our results show that when isolating MSCs from the UC, the WJ should be the preferred compartment, and a standardized method of derivation must be used so as to make meaningful comparisons of data between research groups.

  6. Cystatin E/M Suppresses Tumor Cell Growth through Cytoplasmic Retention of NF-κB

    Science.gov (United States)

    Soh, Hendrick; Venkatesan, Natarajan; Veena, Mysore S.; Ravichandran, Sandhiya; Zinabadi, Alborz; Basak, Saroj K.; Parvatiyar, Kislay; Srivastava, Meera; Liang, Li-Jung; Gjertson, David W.; Torres, Jorge Z.; Moatamed, Neda A.

    2016-01-01

    We and others have shown that the cystatin E/M gene is inactivated in primary human tumors, pointing to its role as a tumor suppressor gene. However, the molecular mechanism of tumor suppression is not yet understood. Using plasmid-directed cystatin E/M gene overexpression, a lentivirus-mediated tetracycline-inducible vector system, and human papillomavirus 16 (HPV 16) E6 and E7 gene-immortalized normal human epidermal keratinocytes, we demonstrated intracellular and non-cell-autonomous apoptotic growth inhibition of tumor cell lines and that growth inhibition is associated with cytoplasmic retention of NF-κB. We further demonstrated decreased phosphorylation of IκB kinase (IKKβ) and IκBα in the presence of tumor necrosis factor alpha (TNF-α), confirming the role of cystatin E/M in the regulation of the NF-κB signaling pathway. Growth suppression of nude mouse xenograft tumors carrying a tetracycline-inducible vector system was observed with the addition of doxycycline in drinking water, confirming that the cystatin E/M gene is a tumor suppressor gene. Finally, immunohistochemical analyses of cervical carcinoma in situ and primary tumors have shown a statistically significant inverse relationship between the expression of cystatin E/M and cathepsin L and a direct relationship between the loss of cystatin E/M expression and nuclear expression of NF-κB. We therefore propose that the cystatin E/M suppressor gene plays an important role in the regulation of NF-κB. PMID:27090639

  7. The Networks of Genes Encoding Palmitoylated Proteins in Axonal and Synaptic Compartments Are Affected in PPT1 Overexpressing Neuronal-Like Cells

    Directory of Open Access Journals (Sweden)

    Francesco Pezzini

    2017-08-01

    Full Text Available CLN1 disease (OMIM #256730 is an early childhood ceroid-lipofuscinosis associated with mutated CLN1, whose product Palmitoyl-Protein Thioesterase 1 (PPT1 is a lysosomal enzyme involved in the removal of palmitate residues from S-acylated proteins. In neurons, PPT1 expression is also linked to synaptic compartments. The aim of this study was to unravel molecular signatures connected to CLN1. We utilized SH-SY5Y neuroblastoma cells overexpressing wild type CLN1 (SH-p.wtCLN1 and five selected CLN1 patients’ mutations. The cellular distribution of wtPPT1 was consistent with regular processing of endogenous protein, partially detected inside Lysosomal Associated Membrane Protein 2 (LAMP2 positive vesicles, while the mutants displayed more diffuse cytoplasmic pattern. Transcriptomic profiling revealed 802 differentially expressed genes (DEGs in SH-p.wtCLN1 (as compared to empty-vector transfected cells, whereas the number of DEGs detected in the two mutants (p.L222P and p.M57Nfs*45 was significantly lower. Bioinformatic scrutiny linked DEGs with neurite formation and neuronal transmission. Specifically, neuritogenesis and proliferation of neuronal processes were predicted to be hampered in the wtCLN1 overexpressing cell line, and these findings were corroborated by morphological investigations. Palmitoylation survey identified 113 palmitoylated protein-encoding genes in SH-p.wtCLN1, including 25 ones simultaneously assigned to axonal growth and synaptic compartments. A remarkable decrease in the expression of palmitoylated proteins, functionally related to axonal elongation (GAP43, CRMP1 and NEFM and of the synaptic marker SNAP25, specifically in SH-p.wtCLN1 cells was confirmed by immunoblotting. Subsequent, bioinformatic network survey of DEGs assigned to the synaptic annotations linked 81 DEGs, including 23 ones encoding for palmitoylated proteins. Results obtained in this experimental setting outlined two affected functional modules (connected to

  8. Levels of small molecules and enzymes in the mother cell compartment and the forespore of sporulating Bacillus megaterium.

    Science.gov (United States)

    Singh, R P; Setlow, B; Setlow, P

    1977-06-01

    We have determined the amounts of a number of small molecules and enzymes in the mother cell compartment and the developing forespore during sporulation of Bacillus megaterium. Significant amounts of adenosine 5'-triphosphate and reduced nicotinamide adenine dinucleotide were present in the forespore compartment before accumulation of dipicolinic acid (DPA), but these compounds disappeared as DPA was accumulated. 3-Phosphoglyceric acid (3-PGA) accumulated only within the developing forespore, beginning 1 to 2 h before DPA accumulation. Throughout its development the forespore contained constant levels of enzymes of both 3-PGA synthesis (phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase) and 3-PGA utilization (phosphoglycerate mutase, enolase, and pyruvate kinase) at levels similar to those in the mother cell and the dormant spore. Despite the presence of enzymes for 3-PGA utilization, this compound was stable within isolated forespores. Two acid-soluble proteins (A and B proteins) also accumulated only in the forespore, beginning 1 to 2 h before DPA accumulation. At this time the specific protease involved in degradation of the A and B proteins during germination also appeared, but only in the forespore compartment. Nevertheless, the A and B proteins were stable within isolated forespores. Arginine and glutamic acid accumulated within the forespore in parallel with DPA accumulation. The forespore also contained the enzyme arginase at a level similar to that in the mother cell and a level of glutamic acid decarboxylase 2- to 25-fold higher than that in the mother cell, depending on when in sporulation the forespores were isolated. The specific activities of several other enzymes (protease active on hemoglobin, ornithine transcarbamylase, malate dehydrogenase, aconitase, and isocitrate dehydrogenase) in forespores were about 10% or less of the values in the mother cell. Aminopeptidase was present at similar levels in both compartments; threonine

  9. Induction of cytoplasmic rods and rings structures by inhibition of the CTP and GTP synthetic pathway in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Wendy C Carcamo

    Full Text Available Cytoplasmic filamentous rods and rings (RR structures were identified using human autoantibodies as probes. In the present study, the formation of these conserved structures in mammalian cells and functions linked to these structures were examined.Distinct cytoplasmic rods (∼3-10 µm in length and rings (∼2-5 µm in diameter in HEp-2 cells were initially observed in immunofluorescence using human autoantibodies. Co-localization studies revealed that, although RR had filament-like features, they were not enriched in actin, tubulin, or vimentin, and not associated with centrosomes or other known cytoplasmic structures. Further independent studies revealed that two key enzymes in the nucleotide synthetic pathway cytidine triphosphate synthase 1 (CTPS1 and inosine monophosphate dehydrogenase 2 (IMPDH2 were highly enriched in RR. CTPS1 enzyme inhibitors 6-diazo-5-oxo-L-norleucine and Acivicin as well as the IMPDH2 inhibitor Ribavirin exhibited dose-dependent induction of RR in >95% of cells in all cancer cell lines tested as well as mouse primary cells. RR formation by lower concentration of Ribavirin was enhanced in IMPDH2-knockdown HeLa cells whereas it was inhibited in GFP-IMPDH2 overexpressed HeLa cells. Interestingly, RR were detected readily in untreated mouse embryonic stem cells (>95%; upon retinoic acid differentiation, RR disassembled in these cells but reformed when treated with Acivicin.RR formation represented response to disturbances in the CTP or GTP synthetic pathways in cancer cell lines and mouse primary cells and RR are the convergence physical structures in these pathways. The availability of specific markers for these conserved structures and the ability to induce formation in vitro will allow further investigations in structure and function of RR in many biological systems in health and diseases.

  10. Influence of the fuel and dosage on the performance of double-compartment microbial fuel cells.

    Science.gov (United States)

    Asensio, Y; Fernandez-Marchante, C M; Lobato, J; Cañizares, P; Rodrigo, M A

    2016-08-01

    This manuscript focuses on the evaluation of the use of different types and dosages of fuels in the performance of double-compartment microbial fuel cell equipped with carbon felt electrodes and cationic membrane. Five types of fuels (ethanol, glycerol, acetate, propionate and fructose) have been tested for the same organic load (5,000 mg L(-1) measured as COD) and for one of them (acetate), the range of dosages between 500 and 20,000 mg L(-1) of COD was also studied. Results demonstrate that production of electricity depends strongly on the fuel used. Carboxylic acids are much more efficient than alcohols or fructose for the same organic load and within the range 500-5,000 mg L(-1) of acetate the production of electricity increases linearly with the amount of acetate fed but over these concentrations a change in the population composition may explain a worse performance. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. PTEN and PI-3 kinase inhibitors control LPS signaling and the lymphoproliferative response in the CD19+ B cell compartment

    International Nuclear Information System (INIS)

    Singh, Alok R.; Peirce, Susan K.; Joshi, Shweta; Durden, Donald L.

    2014-01-01

    Pattern recognition receptors (PRRs), e.g. toll receptors (TLRs) that bind ligands within the microbiome have been implicated in the pathogenesis of cancer. LPS is a ligand for two TLR family members, TLR4 and RP105 which mediate LPS signaling in B cell proliferation and migration. Although LPS/TLR/RP105 signaling is well-studied; our understanding of the underlying molecular mechanisms controlling these PRR signaling pathways remains incomplete. Previous studies have demonstrated a role for PTEN/PI-3K signaling in B cell selection and survival, however a role for PTEN/PI-3K in TLR4/RP105/LPS signaling in the B cell compartment has not been reported. Herein, we crossed a CD19cre and PTEN fl/fl mouse to generate a conditional PTEN knockout mouse in the CD19+ B cell compartment. These mice were further crossed with an IL-14α transgenic mouse to study the combined effect of PTEN deletion, PI-3K inhibition and expression of IL-14α (a cytokine originally identified as a B cell growth factor) in CD19+ B cell lymphoproliferation and response to LPS stimulation. Targeted deletion of PTEN and directed expression of IL-14α in the CD19+ B cell compartment (IL-14+PTEN-/-) lead to marked splenomegaly and altered spleen morphology at baseline due to expansion of marginal zone B cells, a phenotype that was exaggerated by treatment with the B cell mitogen and TLR4/RP105 ligand, LPS. Moreover, LPS stimulation of CD19+ cells isolated from these mice display increased proliferation, augmented AKT and NFκB activation as well as increased expression of c-myc and cyclinD1. Interestingly, treatment of LPS treated IL-14+PTEN-/- mice with a pan PI-3K inhibitor, SF1126, reduced splenomegaly, cell proliferation, c-myc and cyclin D1 expression in the CD19+ B cell compartment and normalized the splenic histopathologic architecture. These findings provide the direct evidence that PTEN and PI-3K inhibitors control TLR4/RP105/LPS signaling in the CD19+ B cell compartment and that pan PI-3

  12. PTEN and PI-3 kinase inhibitors control LPS signaling and the lymphoproliferative response in the CD19+ B cell compartment

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Alok R. [UCSD Department of Pediatrics, Moores UCSD Cancer Center, University of California School of Medicine, San Diego, CA 92093 (United States); Peirce, Susan K. [Department of Pediatrics, Emory University School of Medicine, Atlanta, GA (United States); Joshi, Shweta [UCSD Department of Pediatrics, Moores UCSD Cancer Center, University of California School of Medicine, San Diego, CA 92093 (United States); Durden, Donald L., E-mail: ddurden@ucsd.edu [UCSD Department of Pediatrics, Moores UCSD Cancer Center, University of California School of Medicine, San Diego, CA 92093 (United States); Division of Pediatric Hematology-Oncology, UCSD Rady Children' s Hospital, La Jolla, CA (United States)

    2014-09-10

    Pattern recognition receptors (PRRs), e.g. toll receptors (TLRs) that bind ligands within the microbiome have been implicated in the pathogenesis of cancer. LPS is a ligand for two TLR family members, TLR4 and RP105 which mediate LPS signaling in B cell proliferation and migration. Although LPS/TLR/RP105 signaling is well-studied; our understanding of the underlying molecular mechanisms controlling these PRR signaling pathways remains incomplete. Previous studies have demonstrated a role for PTEN/PI-3K signaling in B cell selection and survival, however a role for PTEN/PI-3K in TLR4/RP105/LPS signaling in the B cell compartment has not been reported. Herein, we crossed a CD19cre and PTEN{sup fl/fl} mouse to generate a conditional PTEN knockout mouse in the CD19+ B cell compartment. These mice were further crossed with an IL-14α transgenic mouse to study the combined effect of PTEN deletion, PI-3K inhibition and expression of IL-14α (a cytokine originally identified as a B cell growth factor) in CD19+ B cell lymphoproliferation and response to LPS stimulation. Targeted deletion of PTEN and directed expression of IL-14α in the CD19+ B cell compartment (IL-14+PTEN-/-) lead to marked splenomegaly and altered spleen morphology at baseline due to expansion of marginal zone B cells, a phenotype that was exaggerated by treatment with the B cell mitogen and TLR4/RP105 ligand, LPS. Moreover, LPS stimulation of CD19+ cells isolated from these mice display increased proliferation, augmented AKT and NFκB activation as well as increased expression of c-myc and cyclinD1. Interestingly, treatment of LPS treated IL-14+PTEN-/- mice with a pan PI-3K inhibitor, SF1126, reduced splenomegaly, cell proliferation, c-myc and cyclin D1 expression in the CD19+ B cell compartment and normalized the splenic histopathologic architecture. These findings provide the direct evidence that PTEN and PI-3K inhibitors control TLR4/RP105/LPS signaling in the CD19+ B cell compartment and that pan PI

  13. Patellofemoral compartment

    International Nuclear Information System (INIS)

    Brown, T.; Quinn, S.F.; Erickson, S.J.; Cox, I.

    1990-01-01

    This paper evaluates the normal and abnormal patellofemoral compartment with axial MR imaging. Anatomic cryotome sections of the patellofemoral compartment were correlated with the corresponding MR images for identification of ligamentous structures and cartilaginous surfaces. Two hundred fifty-four patients who underwent both arthroscopy and axial MR imaging of the patellofemoral compartment underwent axial MR examinations, which included gradient-echo (TR 23, TE 14, flip angle 30 degrees), T1- weighted (TR 400, TE 20), and proton and T2-weighted (2,500/20/80) sequences. The results from the cryotome-MR correlation show that axial MR images of the patellofemoral compartment accurately depict the major ligamentous and cartilaginous components. The MR arthroscopic correlation showed that all pulse sequences were unreliable in depicting the more superficial changes of chondromalacia and the evaluation on synovial plica

  14. Compartment syndromes

    Science.gov (United States)

    Mubarak, S. J.; Pedowitz, R. A.; Hargens, A. R.

    1989-01-01

    The compartment syndrome is defined as a condition in which high pressure within a closed fascial space (muscle compartment) reduces capillary blood perfusion below the level necessary for tissue viability'. This condition occurs in acute and chronic (exertional) forms, and may be secondary to a variety of causes. The end-result of an extended period of elevated intramuscular pressure may be the development of irreversible tissue injury and Volkmann's contracture. The goal of treatment of the compartment syndrome is the reduction of intracompartmental pressure thus facilitating reperfusion of ischaemic tissue and this goal may be achieved by decompressive fasciotomy. Controversy exists regarding the critical pressure-time thresholds for surgical decompression and the optimal diagnostic methods of measuring intracompartmental pressures. This paper will update and review some current knowledge regarding the pathophysiology, aetiology, diagnosis, and treatment of the acute compartment syndrome.

  15. Protein synthesis and the recovery of both survival and cytoplasmic ''petite'' mutation in ultraviolet-treated yeast cells

    International Nuclear Information System (INIS)

    Heude, M.; Chanet, R.

    1975-01-01

    The contribution of mitochondrial proteins in the repair of UV-induced lethal and cytoplasmic genetic damages was studied in dark liquid-held exponential and stationary phase yeast cells. This was performed by using the specific inhibitors, erythromycin and chloramphenicol. It was shown that mitochondrial proteins are involved in the recovery and survival of UV-treated exponential phase cells, but not in the recovery of stationary phase cells. Mitochondrial proteins are partly implicated in the mechanisms leading to the restoration of the e + genotype in UV-irradiated dark liquid-held exponential phase cells. Here again, in stationary phase cells, mitochondrial enzymes do not seem to participate in the negative liquid-holding process for the e - induction, as shown by inhibiting mitochondrial protein synthesis of both mitochondrial and nuclear protein synthesis. When cells are grown in glycerol, the response after dark liquid-holding of the UV-treated cells in the different growth stages are similar to that found for glucose-grown cells. In other words, the fate of cytoplasmic genetic damage in particular is not correlated with the repressed or derepressed state of the mitochondria

  16. Porphyromonas gingivalis Outer Membrane Vesicles Enter Human Epithelial Cells via an Endocytic Pathway and Are Sorted to Lysosomal Compartments

    Science.gov (United States)

    Furuta, Nobumichi; Tsuda, Kayoko; Omori, Hiroko; Yoshimori, Tamotsu; Yoshimura, Fuminobu; Amano, Atsuo

    2009-01-01

    Porphyromonas gingivalis, a periodontal pathogen, secretes outer membrane vesicles (MVs) that contain major virulence factors, including major fimbriae and proteases termed gingipains, although it is not confirmed whether MVs enter host cells. In this study, we analyzed the mechanisms involved in the interactions of P. gingivalis MVs with human epithelial cells. Our results showed that MVs swiftly adhered to HeLa and immortalized human gingival epithelial cells in a fimbria-dependent manner and then entered via a lipid raft-dependent endocytic pathway. The intracellular MVs were subsequently routed to early endosome antigen 1-associated compartments and then were sorted to lysosomal compartments within 90 min, suggesting that intracellular MVs were ultimately degraded by the cellular digestive machinery. However, P. gingivalis MVs remained there for over 24 h and significantly induced acidified compartment formation after being taken up by the cellular digestive machinery. In addition, MV entry was shown to be mediated by a novel pathway for transmission of bacterial products into host cells, a Rac1-regulated pinocytic pathway that is independent of caveolin, dynamin, and clathrin. Our findings indicate that P. gingivalis MVs efficiently enter host cells via an endocytic pathway and survive within the endocyte organelles for an extended period, which provides better understanding of the role of MVs in the etiology of periodontitis. PMID:19651865

  17. Cell-free protein synthesis in micro compartments: building a minimal cell from biobricks.

    Science.gov (United States)

    Jia, Haiyang; Heymann, Michael; Bernhard, Frank; Schwille, Petra; Kai, Lei

    2017-10-25

    The construction of a minimal cell that exhibits the essential characteristics of life is a great challenge in the field of synthetic biology. Assembling a minimal cell requires multidisciplinary expertise from physics, chemistry and biology. Scientists from different backgrounds tend to define the essence of 'life' differently and have thus proposed different artificial cell models possessing one or several essential features of living cells. Using the tools and methods of molecular biology, the bottom-up engineering of a minimal cell appears in reach. However, several challenges still remain. In particular, the integration of individual sub-systems that is required to achieve a self-reproducing cell model presents a complex optimization challenge. For example, multiple self-organisation and self-assembly processes have to be carefully tuned. We review advances and developments of new methods and techniques, for cell-free protein synthesis as well as micro-fabrication, for their potential to resolve challenges and to accelerate the development of minimal cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Lacking deoxygenation-linked interaction between cytoplasmic domain of band 3 and HbF from fetal red blood cells

    DEFF Research Database (Denmark)

    Weber, Roy E.

    2007-01-01

    Aim: Several of the red blood cell's metabolic and membrane functions display dependence on haemoglobin oxygenation. In adult human red cells, the increased glycolytic rate at low O2 tension results from binding of deoxygenated HbA at negatively charged, N-terminal, cytoplasmic domain of the memb......Aim: Several of the red blood cell's metabolic and membrane functions display dependence on haemoglobin oxygenation. In adult human red cells, the increased glycolytic rate at low O2 tension results from binding of deoxygenated HbA at negatively charged, N-terminal, cytoplasmic domain...... of the membrane protein band 3, which liberates glycolytic enzymes from this site. This study aims to investigate the role of fetal HbF (that has lower anion-binding capacity than HbA) in fetal red cells (that are subjected to low O2 tensions), and to elucidate possible linkage (e.g. via the major red cell...... membrane organising centre, band 3) between the individual oxygenation-linked reactions encountered in red cells. Methods: The interaction between band 3 and Hb is analysed in terms of the effects, measured under different conditions, of a 10-mer peptide that corresponds to the N-terminus of human band 3...

  19. Proliferative compensation of residual radiation damage in the compartment of hematopoietic early progenitor cells of the mouse

    International Nuclear Information System (INIS)

    Huebner, G.E.; Wangenheim, K.H. von; Feinendegen, L.E.

    1984-01-01

    The rate of cell entry from the compartment of hematopoietic early progenitor cells into differentiation was determined in sublethally irradiated mice. By use of the criterion of repopulating ability, transplantation of 5-( 125 I) iodo-2'-deoxyuridine labeled bone marrow cells into fatally irradiated syngeneic recipients allows to measure the relative number of early progenitor cells lodging in the spleen and the turnover of these cells in the donors. Following 450 rad the relative number of transplantable early progenitor cells in S-phase recovers to normal within 2 weeks and stabilizes after 5 weeks. At this time, the labeled progenitors turn over with a half-time of 1.4-2.2 days; the respective times for unirradiated mice are 1.5-1.8 days. This, quantitative and qualitative residual radiation damage that is known to exist in the compartment of CFU-S, is disguised within 2-5 weeks after irradiation by proliferative compensation in the entirety of early hemopoietic precursor cells which are here defined by their capacity of selfrenewal and delivery of differentiated cells and of seeding to spleens of lethally irradiated recipients. (orig.)

  20. The human Vδ2+ T-cell compartment comprises distinct innate-like Vγ9+ and adaptive Vγ9- subsets.

    Science.gov (United States)

    Davey, Martin S; Willcox, Carrie R; Hunter, Stuart; Kasatskaya, Sofya A; Remmerswaal, Ester B M; Salim, Mahboob; Mohammed, Fiyaz; Bemelman, Frederike J; Chudakov, Dmitriy M; Oo, Ye H; Willcox, Benjamin E

    2018-05-02

    Vδ2 + T cells form the predominant human γδ T-cell population in peripheral blood and mediate T-cell receptor (TCR)-dependent anti-microbial and anti-tumour immunity. Here we show that the Vδ2 + compartment comprises both innate-like and adaptive subsets. Vγ9 + Vδ2 + T cells display semi-invariant TCR repertoires, featuring public Vγ9 TCR sequences equivalent in cord and adult blood. By contrast, we also identify a separate, Vγ9 - Vδ2 + T-cell subset that typically has a CD27 hi CCR7 + CD28 + IL-7Rα + naive-like phenotype and a diverse TCR repertoire, however in response to viral infection, undergoes clonal expansion and differentiation to a CD27 lo CD45RA + CX 3 CR1 + granzymeA/B + effector phenotype. Consistent with a function in solid tissue immunosurveillance, we detect human intrahepatic Vγ9 - Vδ2 + T cells featuring dominant clonal expansions and an effector phenotype. These findings redefine human γδ T-cell subsets by delineating the Vδ2 + T-cell compartment into innate-like (Vγ9 + ) and adaptive (Vγ9 - ) subsets, which have distinct functions in microbial immunosurveillance.

  1. Tubulation of Class II MHC Compartments Is Microtubule Dependent and Involves Multiple Endolysosomal Membrane Proteins in Primary Dendritic Cells1

    Science.gov (United States)

    Vyas, Jatin M.; Kim, You-Me; Artavanis-Tsakonas, Katerina; Love, J. Christopher; Van der Veen, Annemarthe G.; Ploegh, Hidde L.

    2009-01-01

    Immature dendritic cells (DCs) capture exogenous Ags in the periphery for eventual processing in endolysosomes. Upon maturation by TLR agonists, DCs deliver peptide-loaded class II MHC molecules from these compartments to the cell surface via long tubular structures (endolysosomal tubules). The nature and rules that govern the movement of these DC compartments are unknown. In this study, we demonstrate that the tubules contain multiple proteins including the class II MHC molecules and LAMP1, a lysosomal resident protein, as well as CD63 and CD82, members of the tetraspanin family. Endolysosomal tubules can be stained with acidotropic dyes, indicating that they are extensions of lysosomes. However, the proper trafficking of class II MHC molecules themselves is not necessary for endolysosomal tubule formation. DCs lacking MyD88 can also form endolysosomal tubules, demonstrating that MyD88-dependent TLR activation is not necessary for the formation of this compartment. Endolysosomal tubules in DCs exhibit dynamic and saltatory movement, including bidirectional travel. Measured velocities are consistent with motor-based movement along microtubules. Indeed, nocodazole causes the collapse of endolysosomal tubules. In addition to its association with microtubules, endolysosomal tubules follow the plus ends of microtubules as visualized in primary DCs expressing end binding protein 1 (EB1)-enhanced GFP. PMID:17513769

  2. Tubulation of class II MHC compartments is microtubule dependent and involves multiple endolysosomal membrane proteins in primary dendritic cells.

    Science.gov (United States)

    Vyas, Jatin M; Kim, You-Me; Artavanis-Tsakonas, Katerina; Love, J Christopher; Van der Veen, Annemarthe G; Ploegh, Hidde L

    2007-06-01

    Immature dendritic cells (DCs) capture exogenous Ags in the periphery for eventual processing in endolysosomes. Upon maturation by TLR agonists, DCs deliver peptide-loaded class II MHC molecules from these compartments to the cell surface via long tubular structures (endolysosomal tubules). The nature and rules that govern the movement of these DC compartments are unknown. In this study, we demonstrate that the tubules contain multiple proteins including the class II MHC molecules and LAMP1, a lysosomal resident protein, as well as CD63 and CD82, members of the tetraspanin family. Endolysosomal tubules can be stained with acidotropic dyes, indicating that they are extensions of lysosomes. However, the proper trafficking of class II MHC molecules themselves is not necessary for endolysosomal tubule formation. DCs lacking MyD88 can also form endolysosomal tubules, demonstrating that MyD88-dependent TLR activation is not necessary for the formation of this compartment. Endolysosomal tubules in DCs exhibit dynamic and saltatory movement, including bidirectional travel. Measured velocities are consistent with motor-based movement along microtubules. Indeed, nocodazole causes the collapse of endolysosomal tubules. In addition to its association with microtubules, endolysosomal tubules follow the plus ends of microtubules as visualized in primary DCs expressing end binding protein 1 (EB1)-enhanced GFP.

  3. Cytoplasmic location of α1A voltage-gated calcium channel C-terminal fragment (Cav2.1-CTF aggregate is sufficient to cause cell death.

    Directory of Open Access Journals (Sweden)

    Makoto Takahashi

    Full Text Available The human α1A voltage-dependent calcium channel (Cav2.1 is a pore-forming essential subunit embedded in the plasma membrane. Its cytoplasmic carboxyl(C-tail contains a small poly-glutamine (Q tract, whose length is normally 4∼19 Q, but when expanded up to 20∼33Q, the tract causes an autosomal-dominant neurodegenerative disorder, spinocerebellar ataxia type 6 (SCA6. A recent study has shown that a 75-kDa C-terminal fragment (CTF containing the polyQ tract remains soluble in normal brains, but becomes insoluble mainly in the cytoplasm with additional localization to the nuclei of human SCA6 Purkinje cells. However, the mechanism by which the CTF aggregation leads to neurodegeneration is completely elusive, particularly whether the CTF exerts more toxicity in the nucleus or in the cytoplasm. We tagged recombinant (rCTF with either nuclear-localization or nuclear-export signal, created doxycyclin-inducible rat pheochromocytoma (PC12 cell lines, and found that the CTF is more toxic in the cytoplasm than in the nucleus, the observations being more obvious with Q28 (disease range than with Q13 (normal-length. Surprisingly, the CTF aggregates co-localized both with cAMP response element-binding protein (CREB and phosphorylated-CREB (p-CREB in the cytoplasm, and Western blot analysis showed that the quantity of CREB and p-CREB were both decreased in the nucleus when the rCTF formed aggregates in the cytoplasm. In human brains, polyQ aggregates also co-localized with CREB in the cytoplasm of SCA6 Purkinje cells, but not in other conditions. Collectively, the cytoplasmic Cav2.1-CTF aggregates are sufficient to cause cell death, and one of the pathogenic mechanisms may be abnormal CREB trafficking in the cytoplasm and reduced CREB and p-CREB levels in the nuclei.

  4. Cytoplasmic Location of α1A Voltage-Gated Calcium Channel C-Terminal Fragment (Cav2.1-CTF) Aggregate Is Sufficient to Cause Cell Death

    Science.gov (United States)

    Takahashi, Makoto; Obayashi, Masato; Ishiguro, Taro; Sato, Nozomu; Niimi, Yusuke; Ozaki, Kokoro; Mogushi, Kaoru; Mahmut, Yasen; Tanaka, Hiroshi; Tsuruta, Fuminori; Dolmetsch, Ricardo; Yamada, Mitsunori; Takahashi, Hitoshi; Kato, Takeo; Mori, Osamu; Eishi, Yoshinobu; Mizusawa, Hidehiro; Ishikawa, Kinya

    2013-01-01

    The human α1A voltage-dependent calcium channel (Cav2.1) is a pore-forming essential subunit embedded in the plasma membrane. Its cytoplasmic carboxyl(C)-tail contains a small poly-glutamine (Q) tract, whose length is normally 4∼19 Q, but when expanded up to 20∼33Q, the tract causes an autosomal-dominant neurodegenerative disorder, spinocerebellar ataxia type 6 (SCA6). A recent study has shown that a 75-kDa C-terminal fragment (CTF) containing the polyQ tract remains soluble in normal brains, but becomes insoluble mainly in the cytoplasm with additional localization to the nuclei of human SCA6 Purkinje cells. However, the mechanism by which the CTF aggregation leads to neurodegeneration is completely elusive, particularly whether the CTF exerts more toxicity in the nucleus or in the cytoplasm. We tagged recombinant (r)CTF with either nuclear-localization or nuclear-export signal, created doxycyclin-inducible rat pheochromocytoma (PC12) cell lines, and found that the CTF is more toxic in the cytoplasm than in the nucleus, the observations being more obvious with Q28 (disease range) than with Q13 (normal-length). Surprisingly, the CTF aggregates co-localized both with cAMP response element-binding protein (CREB) and phosphorylated-CREB (p-CREB) in the cytoplasm, and Western blot analysis showed that the quantity of CREB and p-CREB were both decreased in the nucleus when the rCTF formed aggregates in the cytoplasm. In human brains, polyQ aggregates also co-localized with CREB in the cytoplasm of SCA6 Purkinje cells, but not in other conditions. Collectively, the cytoplasmic Cav2.1-CTF aggregates are sufficient to cause cell death, and one of the pathogenic mechanisms may be abnormal CREB trafficking in the cytoplasm and reduced CREB and p-CREB levels in the nuclei. PMID:23505410

  5. The B7-1 cytoplasmic tail enhances intracellular transport and mammalian cell surface display of chimeric proteins in the absence of a linear ER export motif.

    Directory of Open Access Journals (Sweden)

    Yi-Chieh Lin

    Full Text Available Membrane-tethered proteins (mammalian surface display are increasingly being used for novel therapeutic and biotechnology applications. Maximizing surface expression of chimeric proteins on mammalian cells is important for these applications. We show that the cytoplasmic domain from the B7-1 antigen, a commonly used element for mammalian surface display, can enhance the intracellular transport and surface display of chimeric proteins in a Sar1 and Rab1 dependent fashion. However, mutational, alanine scanning and deletion analysis demonstrate the absence of linear ER export motifs in the B7 cytoplasmic domain. Rather, efficient intracellular transport correlated with the presence of predicted secondary structure in the cytoplasmic tail. Examination of the cytoplasmic domains of 984 human and 782 mouse type I transmembrane proteins revealed that many previously identified ER export motifs are rarely found in the cytoplasmic tail of type I transmembrane proteins. Our results suggest that efficient intracellular transport of B7 chimeric proteins is associated with the structure rather than to the presence of a linear ER export motif in the cytoplasmic tail, and indicate that short (less than ~ 10-20 amino acids and unstructured cytoplasmic tails should be avoided to express high levels of chimeric proteins on mammalian cells.

  6. Phosphorous recovery from sewage sludge ash suspended in water in a two-compartment electrodialytic cell

    DEFF Research Database (Denmark)

    Ottosen, Lisbeth M.; Jensen, Pernille Erland; Kirkelund, Gunvor Marie

    2016-01-01

    was suspended in water in the anolyte, which was separated from the catholyte by a cation exchange membrane. Electrolysis at the anode acidified the SSA suspension, and hereby P, Cu, Pb, Cd and Zn were extracted. The heavy metal ions electromigrated into the catholyte and were thus separated from the filtrate......Phosphorus (P) is indispensable for all forms of life on Earth and as P is a finite resource, it is highly important to increase recovery of P from secondary resources. This investigation is focused on P recovery from sewage sludge ash (SSA) by a two-compartment electrodialytic separation (EDS......) technique. Two SSAs are included in the investigation and they contained slightly less P than phosphate rock used in commercial fertilizer production and more heavy metals. The two-compartment electrodialytic technique enabled simultaneous recovery of P and separation of heavy metals. During EDS the SSA...

  7. Interferometric measurements of dry mass content in nuclei and cytoplasm in the life cycle of antheridial filaments cells of Chara vulgaris L. in their successive developmental stages

    Directory of Open Access Journals (Sweden)

    Hanna Kuran

    2015-01-01

    Full Text Available Interferometric measurements of the nucleus and cytoplasm dry mass during interphase in the successive stages of development of antheridial filaments of Chara vulgaris demonstrated that the dry mass and surface area of cell nuclei double in size in each of the successive generations of the filaments, whereas neither the surface nor the dry mass of the cytoplasm increase in such proportion in the same period. In the successive stages of development of the antheridial filaments the dry mass and surface area of the nuclei and cytoplasm gradually diminish.

  8. CD4+ virtual memory: Antigen-inexperienced T cells reside in the naïve, regulatory, and memory T cell compartments at similar frequencies, implications for autoimmunity.

    Science.gov (United States)

    Marusina, Alina I; Ono, Yoko; Merleev, Alexander A; Shimoda, Michiko; Ogawa, Hiromi; Wang, Elizabeth A; Kondo, Kayo; Olney, Laura; Luxardi, Guillaume; Miyamura, Yoshinori; Yilma, Tilahun D; Villalobos, Itzel Bustos; Bergstrom, Jennifer W; Kronenberg, Daniel G; Soulika, Athena M; Adamopoulos, Iannis E; Maverakis, Emanual

    2017-02-01

    It is widely accepted that central and effector memory CD4 + T cells originate from naïve T cells after they have encountered their cognate antigen in the setting of appropriate co-stimulation. However, if this were true the diversity of T cell receptor (TCR) sequences within the naïve T cell compartment should be far greater than that of the memory T cell compartment, which is not supported by TCR sequencing data. Here we demonstrate that aged mice with far fewer naïve T cells, respond to the model antigen, hen eggwhite lysozyme (HEL), by utilizing the same TCR sequence as their younger counterparts. CD4 + T cell repertoire analysis of highly purified T cell populations from naive animals revealed that the HEL-specific clones displayed effector and central "memory" cell surface phenotypes even prior to having encountered their cognate antigen. Furthermore, HEL-inexperienced CD4 + T cells were found to reside within the naïve, regulatory, central memory, and effector memory T cell populations at similar frequencies and the majority of the CD4 + T cells within the regulatory and memory populations were unexpanded. These findings support a new paradigm for CD4 + T cell maturation in which a specific clone can undergo a differentiation process to exhibit a "memory" or regulatory phenotype without having undergone a clonal expansion event. It also demonstrates that a foreign-specific T cell is just as likely to reside within the regulatory T cell compartment as it would the naïve compartment, arguing against the specificity of the regulatory T cell compartment being skewed towards self-reactive T cell clones. Finally, we demonstrate that the same set of foreign and autoreactive CD4 + T cell clones are repetitively generated throughout adulthood. The latter observation argues against T cell-depleting strategies or autologous stem cell transplantation as therapies for autoimmunity-as the immune system has the ability to regenerate pathogenic clones. Published by

  9. In vivo sensitivity of the embryonic and adult neural stem cell compartments to low-dose radiation

    International Nuclear Information System (INIS)

    Barazzuol, Lara; Jeggo, Penny A.

    2016-01-01

    The embryonic brain is radiation-sensitive, with cognitive deficits being observed after exposure to low radiation doses. Exposure of neonates to radiation can cause intracranial carcinogenesis. To gain insight into the basis underlying these outcomes, we examined the response of the embryonic, neonatal and adult brain to low-dose radiation, focusing on the neural stem cell compartments. This review summarizes our recent findings. At E13.5–14.5 the embryonic neocortex encompasses rapidly proliferating stem and progenitor cells. Exploiting mice with a hypomorphic mutation in DNA ligase IV (Lig4 Y288C ), we found a high level of DNA double-strand breaks (DSBs) at E14.5, which we attribute to the rapid proliferation. We observed endogenous apoptosis in Lig4 Y288C embryos and in WT embryos following exposure to low radiation doses. An examination of DSB levels and apoptosis in adult neural stem cell compartments, the subventricular zone (SVZ) and the subgranular zone (SGZ) revealed low DSB levels in Lig4 Y288C mice, comparable with the levels in differentiated neuronal tissues. We conclude that the adult SVZ does not incur high levels of DNA breakage, but sensitively activates apoptosis; apoptosis was less sensitively activated in the SGZ, and differentiated neuronal tissues did not activate apoptosis. P5/P15 mice showed intermediate DSB levels, suggesting that DSBs generated in the embryo can be transmitted to neonates and undergo slow repair. Interestingly, this analysis revealed a stage of high endogenous apoptosis in the neonatal SVZ. Collectively, these studies reveal that the adult neural stem cell compartment, like the embryonic counterpart, can sensitively activate apoptosis

  10. In vivo sensitivity of the embryonic and adult neural stem cell compartments to low-dose radiation.

    Science.gov (United States)

    Barazzuol, Lara; Jeggo, Penny A

    2016-08-01

    The embryonic brain is radiation-sensitive, with cognitive deficits being observed after exposure to low radiation doses. Exposure of neonates to radiation can cause intracranial carcinogenesis. To gain insight into the basis underlying these outcomes, we examined the response of the embryonic, neonatal and adult brain to low-dose radiation, focusing on the neural stem cell compartments. This review summarizes our recent findings. At E13.5-14.5 the embryonic neocortex encompasses rapidly proliferating stem and progenitor cells. Exploiting mice with a hypomorphic mutation in DNA ligase IV (Lig4(Y288C) ), we found a high level of DNA double-strand breaks (DSBs) at E14.5, which we attribute to the rapid proliferation. We observed endogenous apoptosis in Lig4(Y288C) embryos and in WT embryos following exposure to low radiation doses. An examination of DSB levels and apoptosis in adult neural stem cell compartments, the subventricular zone (SVZ) and the subgranular zone (SGZ) revealed low DSB levels in Lig4(Y288C) mice, comparable with the levels in differentiated neuronal tissues. We conclude that the adult SVZ does not incur high levels of DNA breakage, but sensitively activates apoptosis; apoptosis was less sensitively activated in the SGZ, and differentiated neuronal tissues did not activate apoptosis. P5/P15 mice showed intermediate DSB levels, suggesting that DSBs generated in the embryo can be transmitted to neonates and undergo slow repair. Interestingly, this analysis revealed a stage of high endogenous apoptosis in the neonatal SVZ. Collectively, these studies reveal that the adult neural stem cell compartment, like the embryonic counterpart, can sensitively activate apoptosis. © The Author 2016. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

  11. Cell kinetics in the erythroid compartment of guinea pig bone marrow: a model based on /sup 3/H-TdR studies

    Energy Technology Data Exchange (ETDEWEB)

    Prothero, J; Starling, M; Rosse, C

    1978-01-01

    A model of steady-state erythropoiesis in the guinea pig is described. The model incorporates an unidentified progenitor compartment, as well as compartments representing proerythroblasts, basophilic polychromatic and orthochromatic cells. A computer representation of the model permits a simulation of the labeling curves obtained in pulse and intermittent labeling regimes. It was found that a reasonable fit to the data can be achieved when the parameters for the various compartments are essentially identical. The results of a preliminary sensitivity analysis, carried out by perturbing the duration of S phase from the best fit value, are reported. The fit achieved to the data supports the hypothesis underlying the model that each compartment corresponds to one generation and that the flux within and between compartments is sequential.

  12. Cell kinetics in the erythroid compartment of guinea pig bone marrow: a model based on /sup 3/H-TdR studies

    Energy Technology Data Exchange (ETDEWEB)

    Prothero, J; Starling, M; Rosse, C [Washington Univ., Seattle (USA). Dept. of Biological Structure

    1978-05-01

    A model of steady state erythropoiesis in the guinea pig is described. The model incorporates an unidentified progenitor compartment, as well as compartments representing proerythroblasts, basophilic, polychromatic and orthochromatic cells. A computer representation of the model permits a simulation of the labelling curves obtained in pulse and intermittent labelling regimes. It was found that a reasonable fit to the data can be achieved when the parameters for the various compartments are essentially identical. The results of a preliminary sensitivity analysis, carried out by perturbing the duration of S phase from the best fit value, are reported. The fit achieved to the data supports the hypothesis underlying the model that each compartment corresponds to one generation and that the flux within and between compartments is sequential.

  13. Purging of the neuroblastoma stem cell compartment and tumor regression on exposure to hypoxia or cytotoxic treatment.

    Science.gov (United States)

    Marzi, Ilaria; D'Amico, Massimo; Biagiotti, Tiziana; Giunti, Serena; Carbone, Maria Vittoria; Fredducci, David; Wanke, Enzo; Olivotto, Massimo

    2007-03-15

    We worked out an experimental protocol able to purge the stem cell compartment of the SH-SY5Y neuroblastoma clone. This protocol was based on the prolonged treatment of the wild-type cell population with either hypoxia or the antiblastic etoposide. Cell fate was monitored by immunocytochemical and electrophysiologic (patch-clamp) techniques. Both treatments produced the progressive disappearance of neuronal type (N) cells (which constitute the bulk of the tumor), leaving space for a special category of epithelial-like substrate-adherent cells (S(0)). The latter represent a minimal cell component of the untreated population and are endowed with immunocytochemical markers (p75, c-kit, and CD133) and the electrophysiologic "nude" profile, typical of the neural crest stem cells. S(0) cells displayed a highly clonogenic potency and a substantial plasticity, generating both the N component and an alternative subpopulation terminally committed to the fibromuscular lineage. Unlike the N component, this lineage was highly insensitive to the apoptotic activity of hypoxia and etoposide and developed only when the neuronal option was abolished. Under these conditions, the fibromuscular progeny of S(0) expanded and progressed up to the exhaustion of the staminal compartment and to the extinction of the tumor. When combined, hypoxia and etoposide cooperated in abolishing the N cell generation and promoting the conversion of the tumor described. This synergy might mirror a natural condition in the ischemic areas occurring in cancer. These results have relevant implications for the understanding of the documented tendency of neuroblastomas to regress from a malignant to a benign phenotype, either spontaneously or on antiblastic treatment.

  14. Tapetal-Delayed Programmed Cell Death (PCD and Oxidative Stress-Induced Male Sterility of Aegilops uniaristata Cytoplasm in Wheat

    Directory of Open Access Journals (Sweden)

    Zihan Liu

    2018-06-01

    Full Text Available Cytoplasmic male sterility (CMS plays a crucial role in the utilization of hybrid vigor. Pollen development is often accompanied by oxidative metabolism responses and tapetal programmed cell death (PCD, and deficiency in these processes could lead to male sterility. Aegilops uniaristata cytoplasmic male sterility (Mu-CMS wheat is a novel male-sterile line in wheat, which possess important potential in hybrid wheat breeding. However, its CMS mechanisms remain poorly understood. In our study, U87B1-706A, with the Aegilops uniaristata cytoplasm, and the maintainer line 706B were used to explore the abortive reason. Compared with 706B, histological analysis and PCD detection of the anther demonstrated that U87B1-706A appeared as delayed tapetal PCD as well as a disorganized organelle phenotype in the early uninucleate stage. Subsequently, a shrunken microspore and disordered exine structure were exhibited in the late uninucleate stage. While the activities of antioxidase increased markedly, the nonenzymatic antioxidant contents declined obviously following overacummulation of reactive oxygen species (ROS during pollen development in U87B1-706A. Real-time quantitative PCR testified that the transcript levels of the superoxide dismutase (SOD, catalase (CAT, and ascorbate peroxidase (APX genes, encoding pivotal antioxidant enzymes, were up-regulated in early pollen development. Therefore, we deduce excess ROS as a signal may be related to the increased expression levels of enzyme genes, thereby breaking the antioxidative system balance, resulting in delayed tapetal PCD initiation, which finally led to pollen abortion and male sterility in U87B1-706A. These results provide evidence to further explore the mechanisms of abortive pollen in CMS wheat.

  15. Alanine substitution of conserved residues in the cytoplasmic tail of herpes simplex virus gB can enhance or abolish cell fusion activity and viral entry

    International Nuclear Information System (INIS)

    Ruel, Nancy; Zago, Anna; Spear, Patricia G.

    2006-01-01

    Herpes simplex virus (HSV) glycoprotein B (gB) is one of the four viral glycoproteins required for viral entry and cell fusion and is highly conserved among herpesviruses. Mutants of HSV type 2 gB were generated by substituting conserved residues in the cytoplasmic tail with alanine or by deleting 41 amino acids from the C-terminus. Some of the mutations abolished cell fusion activity and also prevented transport of gB to the cell surface, identifying residues in the gB cytoplasmic tail that are critical for intracellular transport of this glycoprotein. These mutations also prevented production of infectious virus, possibly because the mutant forms of gB were not transported to the site of envelopment. Other mutations, particularly the deletion, significantly enhanced cell fusion activity. These mutations, as well as others described previously, identify regions of the gB cytoplasmic domain that modulate cell fusion activity

  16. Equine arteritis virus is delivered to an acidic compartment of host cells via clathrin-dependent endocytosis

    International Nuclear Information System (INIS)

    Nitschke, Matthias; Korte, Thomas; Tielesch, Claudia; Ter-Avetisyan, Gohar; Tuennemann, Gisela; Cardoso, M. Cristina; Veit, Michael; Herrmann, Andreas

    2008-01-01

    Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae. Infection by EAV requires the release of the viral genome by fusion with the respective target membrane of the host cell. We have investigated the entry pathway of EAV into Baby Hamster Kindey cells (BHK). Infection of cells assessed by the plaque reduction assay was strongly inhibited by substances which interfere with clathrin-dependent endocytosis and by lysosomotropic compounds. Furthermore, infection of BHK cells was suppressed when clathrin-dependent endocytosis was inhibited by expression of antisense RNA of the clathrin-heavy chain before infection. These results strongly suggest that EAV is taken up via clathrin-dependent endocytosis and is delivered to acidic endosomal compartments

  17. Cytoplasmic influence of nucleolar development

    International Nuclear Information System (INIS)

    Ghosh, Sibdas

    1974-01-01

    The role of cytoplasmic factors on the development of nucleolus in nucleus has been investigated in Ehrlich mouse ascites tumour cells using tritiated thymidine/uridine for autoradiography. It is inferred from the observations that the cytoplasmic factors has some but not absolute control over the development of nucleolus. (M.G.B.)

  18. The Cell Wall Teichuronic Acid Synthetase (TUAS Is an Enzyme Complex Located in the Cytoplasmic Membrane of Micrococcus luteus

    Directory of Open Access Journals (Sweden)

    Lingyi Lynn Deng

    2010-01-01

    composed of disaccharide repeating units [-4-β-D-ManNAcAp-(1→6α-D-Glcp−1-]n, which is covalently anchored to the peptidoglycan on the inner cell wall and extended to the outer surface of the cell envelope. An enzyme complex responsible for the TUA chain biosynthesis was purified and characterized. The 440 kDa enzyme complex, named teichuronic acid synthetase (TUAS, is an octomer composed of two kinds of glycosyltransferases, Glucosyltransferase, and ManNAcA-transferase, which is capable of catalyzing the transfer of disaccharide glycosyl residues containing both glucose and the N-acetylmannosaminuronic acid residues. TUAS displays hydrophobic properties and is found primarily associated with the cytoplasmic membrane. The purified TUAS contains carotinoids and lipids. TUAS activity is diminished by phospholipase digestion. We propose that TUAS serves as a multitasking polysaccharide assembling station on the bacterial membrane.

  19. Actin based processes that could determine the cytoplasmic architecture of plant cells

    NARCIS (Netherlands)

    Honing, van der H.S.; Emons, A.M.C.; Ketelaar, M.J.

    2007-01-01

    Actin polymerisation can generate forces that are necessary for cell movement, such as the propulsion of a class of bacteria, including Listeria, and the protrusion of migrating animal cells. Force generation by the actin cytoskeleton in plant cells has not been studied. One process in plant cells

  20. Myc-nick: a cytoplasmic cleavage product of Myc that promotes alpha-tubulin acetylation and cell differentiation.

    Science.gov (United States)

    Conacci-Sorrell, Maralice; Ngouenet, Celine; Eisenman, Robert N

    2010-08-06

    The Myc oncoprotein family comprises transcription factors that control multiple cellular functions and are widely involved in oncogenesis. Here we report the identification of Myc-nick, a cytoplasmic form of Myc generated by calpain-dependent proteolysis at lysine 298 of full-length Myc. Myc-nick retains conserved Myc box regions but lacks nuclear localization signals and the bHLHZ domain essential for heterodimerization with Max and DNA binding. Myc-nick induces alpha-tubulin acetylation and altered cell morphology by recruiting histone acetyltransferase GCN5 to microtubules. During muscle differentiation, while the levels of full-length Myc diminish, Myc-nick and acetylated alpha-tubulin levels are increased. Ectopic expression of Myc-nick accelerates myoblast fusion, triggers the expression of myogenic markers, and permits Myc-deficient fibroblasts to transdifferentiate in response to MyoD. We propose that the cleavage of Myc by calpain abrogates the transcriptional inhibition of differentiation by full-length Myc and generates Myc-nick, a driver of cytoplasmic reorganization and differentiation. Copyright 2010 Elsevier Inc. All rights reserved.

  1. Effect of external pH on the cytoplasmic and vacuolar pHs in Mung bean root-tip cells

    International Nuclear Information System (INIS)

    Torimitsu, Keiichi; Yazaki, Yoshiaki; Nagasuka, Kinuyo; Ohta, Eiji; Sakata, Makoto

    1984-01-01

    The effect of the external pH on the intracellular pH in mung bean (Vigna mungo (L.) Hepper) root-tip cells was investigated with the 31 P nuclear magnetic resonance (NMR) method. The 31 P NMR spectra showed three peaks caused by cytoplasmic G-6-P, cytoplasmic Psub(i) and vacuolar Psub(i). The cytoplasmic and vacuolar pHs could be determined by comparing the Psub(i) chemical shifts with the titration curve. When the external pH was changed over a range from pH 3 to 10, the cytoplasmic pH showed smaller changes than the vacuolar pH, suggesting that the former is regulated more strictly than the latter. The H + -ATPase inhibitor, DCCD, caused the breakdown of the mechanism that regulates the intracellular pH. H + -ATPase appears to have an important part in the regulation of the intracellular pH. (author)

  2. S-layer and cytoplasmic membrane – exceptions from the typical archaeal cell wall with a focus on double membranes

    Directory of Open Access Journals (Sweden)

    Andreas eKlingl

    2014-11-01

    Full Text Available The common idea of typical cell wall architecture in archaea consists of a pseudo-crystalline proteinaceous surface layer (S-layer, situated upon the cytoplasmic membrane. This is true for the majority of described archaea, hitherto. Within the crenarchaea, the S-layer often represents the only cell wall component, but there are various exceptions from this wall architecture. Beside (glycosylated S-layers in (hyperthermophilic cren- and euryarchaea as well as halophilic archaea, one can find a great variety of other cell wall structures like proteoglycan-like S-layers (Halobacteria, glutaminylglycan (Natronococci, methanochondroitin (Methanosarcina or double layered cell walls with pseudomurein (Methanothermus and Methanopyrus. The presence of an outermost cellular membrane in the crenarchaeal species Ignicoccus hospitalis already gave indications for an outer membrane similar to Gram-negative bacteria. Although there is just limited data concerning their biochemistry and ultrastructure, recent studies on the euryarchaeal methanogen Methanomassiliicoccus luminyensis, cells of the ARMAN group, and the SM1 euryarchaeon delivered further examples for this exceptional cell envelope type consisting of two membranes.

  3. Cytoplasmic and nuclear anti-apoptotic roles of αB-crystallin in retinal pigment epithelial cells.

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    Woo Jin Jeong

    Full Text Available In addition to its well-characterized role in the lens, αB-crystallin performs other functions. Methylglyoxal (MGO can alter the function of the basement membrane of retinal pigment epithelial (RPE cells. Thus, if MGO is not efficiently detoxified, it can induce adverse reactions in RPE cells. In this study, we examined the mechanisms underlying the anti-apoptotic activity of αB-crystallin in the human retinal pigment epithelial cell line ARPE-19 following MGO treatment using various assays, including nuclear staining, flow cytometry, DNA electrophoresis, pulse field gel electrophoresis, western blot analysis, confocal microscopy and co-immunoprecipitation assays. To directly assess the role of phosphorylation of αB-crystallin, we used site-directed mutagenesis to convert relevant serine residues to alanine residues. Using these techniques, we demonstrated that MGO induces apoptosis in ARPE-19 cells. Silencing αB-crystallin sensitized ARPE-19 cells to MGO-induced apoptosis, indicating that αB-crystallin protects ARPE-19 cells from MGO-induced apoptosis. Furthermore, we found that αB-crystallin interacts with the caspase subtypes, caspase-2L, -2S, -3, -4, -7, -8, -9 and -12 in untreated control ARPE-19 cells and that MGO treatment caused the dissociation of these caspase subtypes from αB-crystallin; transfection of S19A, S45A or S59A mutants caused the depletion of αB-crystallin from the nuclei of untreated control RPE cells leading to the release of caspase subtypes. Additionally, transfection of these mutants enhanced MGO-induced apoptosis in ARPE-19 cells, indicating that phosphorylation of nuclear αB-crystallin on serine residues 19, 45 and 59 plays a pivotal role in preventing apoptosis in ARPE-19 cells. Taken together, these results suggest that αB-crystallin prevents caspase activation by physically interacting with caspase subtypes in the cytoplasm and nucleus, thereby protecting RPE cells from MGO-induced apoptosis.

  4. Cytoplasmic Overexpression of CD95L in Esophageal Adenocarcinoma Cells Overcomes Resistance to CD95-Mediated Apoptosis

    Directory of Open Access Journals (Sweden)

    Gregory A. Watson

    2011-03-01

    Full Text Available Introduction: The CD95/CD95L pathway plays a critical role in tissue homeostasis and immune system regulation; however, the function of this pathway in malignancy remains poorly understood. We hypothesized that CD95L expression in esophageal adenocarcinoma confers advantages to the neoplasm other than immune privilege. Methods: CD95L expression was characterized in immortalized squamous esophagus (HET-1A and Barrett esophagus (BAR-T cells; adenocarcinoma cell lines FLO-1, SEG-1, and BIC-1, and MDA468 (- control; and KFL cells (+ control. Analyses included reverse transcription-polymerase chain reaction, immunoblots of whole cell and secretory vesicle lysates, FACScan analysis, laser scanning confocal microscopy of native proteins and fluorescent constructs, and assessment of apoptosis and ERK1/2 pathways. Results: Cleaved, soluble CD95L is expressed at both the RNA and protein levels in these cell lines derived from esophageal adenocarcinoma and other human tissues. CD95L was neither trafficked to the cell membrane nor secreted into the media or within vesicles, rather the protein seems to be sequestered in the cytoplasm. CD95 and CD95L colocalize by immunofluorescence, but an interaction was not proven by immunoprecipitation. Overexpression of CD95L in the adenocarcinoma cell lines induced robust apoptosis and, under conditions of pan-caspase inhibition, resulted in activation of ERK signaling. Conclusions: CD95L localization in EA cells is inconsistent with the conference of immune privilege and is more consistent with a function that promotes tumor growth through alternative CD95 signaling. Reduced cell surface expression of CD95 affects cell sensitivity to extracellular apoptotic signals more significantly than alterations in downstream modulators of apoptosis.

  5. The human CD8β M-4 isoform dominant in effector memory T cells has distinct cytoplasmic motifs that confer unique properties.

    Directory of Open Access Journals (Sweden)

    Deepshi Thakral

    Full Text Available The CD8 co-receptor influences T cell recognition and responses in both anti-tumor and anti-viral immunity. During evolution in the ancestor of humans and chimpanzees, the CD8B gene acquired two additional exons. As a result, in humans, there are four CD8β splice variants (M1 to M4 that differ in their cytoplasmic tails. The M-1 isoform which is the equivalent of murine CD8β, is predominantly expressed in naïve T cells, whereas, the M-4 isoform is predominantly expressed in effector memory T cells. The characteristics of the M-4 isoform conferred by its unique 36 amino acid cytoplasmic tail are not known. In this study, we identified a dihydrophobic leucine-based receptor internalization motif in the cytoplasmic tail of M-4 that regulated its cell surface expression and downregulation after activation. Further the M-4 cytoplasmic tail was able to associate with ubiquitinated targets in 293T cells and mutations in the amino acids NPW, a potential EH domain binding site, either enhanced or inhibited the interaction. In addition, the M-4 tail was itself mono-ubiquitinated on a lysine residue in both 293T cells and a human T cell line. When peripheral blood human T cells expressed CD8αβ M-4, the frequency of MIP-1β secreting cells responding to antigen presenting cells was two-fold higher as compared to CD8αβ M-1 expressing T cells. Thus, the cytoplasmic tail of the CD8β M-4 isoform has unique characteristics, which likely contributed to its selective expression and function in human effector memory T cells.

  6. The presence of PHB granules in cytoplasm protects non-halophilic bacterial cells against the harmful impact of hypertonic environments.

    Science.gov (United States)

    Obruca, Stanislav; Sedlacek, Petr; Mravec, Filip; Krzyzanek, Vladislav; Nebesarova, Jana; Samek, Ota; Kucera, Dan; Benesova, Pavla; Hrubanova, Kamila; Milerova, Miluse; Marova, Ivana

    2017-10-25

    Numerous prokaryotes accumulate polyhydroxybutyrate (PHB) intracellularly as a storage material. It has also been proposed that PHB accumulation improves bacterial stress resistance. Cupriavidus necator and its PHB non-accumulating mutant were employed to investigate the protective role of PHB under hypertonic conditions. The presence of PHB granules enhanced survival of the bacteria after exposure to hypertonic conditions. Surprisingly, when coping with such conditions, the bacteria did not utilize PHB to harvest carbon or energy, suggesting that, in the osmotic upshock of C. necator, the protective mechanism of PHB granules is not associated with their hydrolysis. The presence of PHB granules influenced the overall properties of the cells, since challenged PHB-free cells underwent massive plasmolysis accompanied by damage to the cell membrane and the leakage of cytoplasm content, while no such effects were observed in PHB containing bacteria. Moreover, PHB granules demonstrated "liquid-like" properties indicating that they can partially repair and stabilize cell membranes by plugging small gaps formed during plasmolysis. In addition, the level of dehydration and changes in intracellular pH in osmotically challenged cells were less pronounced for PHB-containing cultures, demonstrating the important role of PHB for bacterial survival under hyperosmotic conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Study of biological compartments

    International Nuclear Information System (INIS)

    Rocha, A.F.G. da

    1976-01-01

    The several types of biological compartments are studied such as monocompartmental system, one-compartment balanced system irreversible fluxes, two closed compartment system, three compartment systems, catenary systems and mammilary systems [pt

  8. ATP-containing vesicles in stria vascular marginal cell cytoplasms in neonatal rat cochlea are lysosomes.

    Science.gov (United States)

    Liu, Jun; Liu, Wenjing; Yang, Jun

    2016-02-11

    We confirmed that ATP is released from cochlear marginal cells in the stria vascular but the cell organelle in which ATP stores was not identified until now. Thus, we studied the ATP-containing cell organelles and suggest that these are lysosomes. Primary cultures of marginal cells of Sprague-Dawley rats aged 1-3 days was established. Vesicles within marginal cells stained with markers were identified under confocal laser scanning microscope and transmission electron microscope (TEM). Then ATP release from marginal cells was measured after glycyl-L-phenylalanine-ß- naphthylamide (GPN) treatment using a bioluminescent assay. Quinacrine-stained granules within marginal cells were labeled with LysoTracker, a lysosome tracer, and lysosomal-associated membrane protein 1(LAMP1), but not labeled with the mitochondrial tracer MitoTracker. Furthermore, LysoTracker-labelled puncta showed accumulation of Mant-ATP, an ATP analog. Treatment with 200 μM GPN quenched fluorescently labeled puncta after incubation with LysoTracker or quinacrine, but not MitoTracker. Quinacrine-labeled organelles observed by TEM were lysosomes, and an average 27.7 percent increase in ATP luminescence was observed in marginal cells extracellular fluid after GPN treatment. ATP-containing vesicles in cochlear marginal cells of the stria vascular from neonatal rats are likely lysosomes. ATP release from marginal cells may be via Ca(2+)-dependent lysosomal exocytosis.

  9. A model for the dynamic nuclear/nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein based on live cell imaging

    International Nuclear Information System (INIS)

    You, Jae-Hwan; Howell, Gareth; Pattnaik, Asit K.; Osorio, Fernando A.; Hiscox, Julian A.

    2008-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, in common with many other positive strand RNA viruses, encodes a nucleocapsid (N) protein which can localise not only to the cytoplasm but also to the nucleolus in virus-infected cells and cells over-expressing N protein. The dynamic trafficking of positive strand RNA virus nucleocapsid proteins and PRRSV N protein in particular between the cytoplasm and nucleolus is unknown. In this study live imaging of permissive and non-permissive cell lines, in conjunction with photo-bleaching (FRAP and FLIP), was used to investigate the trafficking of fluorescent labeled (EGFP) PRRSV-N protein. The data indicated that EGFP-PRRSV-N protein was not permanently sequestered to the nucleolus and had equivalent mobility to cellular nucleolar proteins. Further the nuclear import of N protein appeared to occur faster than nuclear export, which may account for the observed relative distribution of N protein between the cytoplasm and the nucleolus

  10. Periodic harvesting of embryonic stem cells from a hollow-fiber membrane based four-compartment bioreactor.

    Science.gov (United States)

    Knöspel, Fanny; Freyer, Nora; Stecklum, Maria; Gerlach, Jörg C; Zeilinger, Katrin

    2016-01-01

    Different types of stem cells have been investigated for applications in drug screening and toxicity testing. In order to provide sufficient numbers of cells for such in vitro applications a scale-up of stem cell culture is necessary. Bioreactors for dynamic three-dimensional (3D) culture of growing cells offer the option for culturing large amounts of stem cells at high densities in a closed system. We describe a method for periodic harvesting of pluripotent stem cells (PSC) during expansion in a perfused 3D hollow-fiber membrane bioreactor, using mouse embryonic stem cells (mESC) as a model cell line. A number of 100 × 10(6) mESC were seeded in bioreactors in the presence of mouse embryonic fibroblasts (MEF) as feeder cells. Over a cultivation interval of nine days cells were harvested by trypsin perfusion and mechanical agitation every second to third culture day. A mean of 380 × 10(6) mESC could be removed with every harvest. Subsequent to harvesting, cells continued growing in the bioreactor, as determined by increasing glucose consumption and lactate production. Immunocytochemical staining and mRNA expression analysis of markers for pluripotency and the three germ layers showed a similar expression of most markers in the harvested cells and in mESC control cultures. In conclusion, successful expansion and harvesting of viable mESC from bioreactor cultures with preservation of sterility was shown. The present study is the first one showing the feasibility of periodic harvesting of adherent cells from a continuously perfused four-compartment bioreactor including further cultivation of remaining cells. © 2015 American Institute of Chemical Engineers.

  11. Assessment of the Nucleus-to-Cytoplasmic Ratio in MCF-7 Cells Using Ultra-high Frequency Ultrasound and Photoacoustics

    Science.gov (United States)

    Moore, M. J.; Strohm, E. M.; Kolios, M. C.

    2016-12-01

    The nucleus-to-cytoplasmic (N:C) ratio of a cell is often used when assessing histology for the presence of malignant disease. In this proof of concept study, we present a new, non-optical method for determination of the N:C ratio using ultra-high Frequency ultrasound (US) and photoacoustics (PA). When using transducers in the 100 MHz-500 MHz range, backscattered US pulses and emitted PA waves are encoded with information pertaining to the dimension and morphology of micron-sized objects. If biological cells are interrogated, the diameter of the scattering or absorbing structure can be assessed by fitting the power spectra of the measured US or PA signals to theoretical models for US backscatter and PA emission from a fluid sphere. In this study, the cell and nucleus diameters of 9 MCF-7 breast cancer cells were determined using a new simplified model that calculates the theoretical values of the location of the power spectra minima for both US and PA signals. These diameters were then used to calculate the N:C ratio of the measured cells. The average cell diameter determined by US pulses from a transducer with a central frequency of 375 MHz was found to be 15.5 μ m± 1.8 μ m. The PA waves emitted by the cell nuclei were used to determine an average nuclear diameter of 12.0 μ m± 1.3 μ m. The N:C ratio for these cells was calculated to be 1.9± 1.0, which agrees well with previously reported N:C values for this cell type.

  12. Extending roGFP Emission via Förster-Type Resonance Energy Transfer Relay Enables Simultaneous Dual Compartment Ratiometric Redox Imaging in Live Cells.

    Science.gov (United States)

    Norcross, Stevie; Trull, Keelan J; Snaider, Jordan; Doan, Sara; Tat, Kiet; Huang, Libai; Tantama, Mathew

    2017-11-22

    Reactive oxygen species (ROS) mediate both intercellular and intraorganellar signaling, and ROS propagate oxidative stress between cellular compartments such as mitochondria and the cytosol. Each cellular compartment contains its own sources of ROS as well as antioxidant mechanisms, which contribute to dynamic fluctuations in ROS levels that occur during signaling, metabolism, and stress. However, the coupling of redox dynamics between cellular compartments has not been well studied because of the lack of available sensors to simultaneously measure more than one subcellular compartment in the same cell. Currently, the redox-sensitive green fluorescent protein, roGFP, has been used extensively to study compartment-specific redox dynamics because it provides a quantitative ratiometric readout and it is amenable to subcellular targeting as a genetically encoded sensor. Here, we report a new family of genetically encoded fluorescent protein sensors that extend the fluorescence emission of roGFP via Förster-type resonance energy transfer to an acceptor red fluorescent protein for dual-color live-cell microscopy. We characterize the redox and optical properties of the sensor proteins, and we demonstrate that they can be used to simultaneously measure cytosolic and mitochondrial ROS in living cells. Furthermore, we use these sensors to reveal cell-to-cell heterogeneity in redox coupling between the cytosol and mitochondria when neuroblastoma cells are exposed to reductive and metabolic stresses.

  13. Cytoplasmic Z-RNA

    International Nuclear Information System (INIS)

    Zarling, D.A.; Calhoun, C.J.; Hardin, C.C.; Zarling, A.H.

    1987-01-01

    Specific immunochemical probes for Z-RNA were generated and characterized to search for possible Z-RNA-like double helices in cells. Z-RNA was detected in the cytoplasm of fixed protozoan cells by immunofluorescence microscopy using these anti-Z-RNA IgCs. In contrast, autoimmune or experimentally elicited anti-DNA antibodies, specifically reactive with B-DNA or Z-DNA, stained the nuclei. Pre-or nonimmune IgGs did not bind to the cells. RNase A or T1 digestion eliminated anti-Z-RNA IgG binding to cytoplasmic determinants; however, DNase I or mung bean nuclease had no effect. Doxorubicin and ethidium bromide prevented anti-Z-RNA antibody binding; however, actinomycin D, which does not bind double-stranded RNA, did not. Anti-Z-RNA immunofluorescence was specifically blocked in competition assays by synthetic Z-RNA but not Z-DNA, A-RNA, or single-stranded RNAs. Thus, some cytoplasmic sequences in fixed cells exist in the left-handed Z-RNA conformation

  14. Differential nuclear remodeling of mammalian somatic cells by Xenopus laevis oocyte and egg cytoplasm

    International Nuclear Information System (INIS)

    Alberio, Ramiro; Johnson, Andrew D.; Stick, Reimer; Campbell, Keith H.S.

    2005-01-01

    The mechanisms governing nuclear reprogramming have not been fully elucidated yet; however, recent studies show a universally conserved ability of both oocyte and egg components to reprogram gene expression in somatic cells. The activation of genes associated with pluripotency by oocyte/egg components may require the remodeling of nuclear structures, such that they can acquire the features of early embryos and pluripotent cells. Here, we report on the remodeling of the nuclear lamina of mammalian cells by Xenopus oocyte and egg extracts. Lamin A/C is removed from somatic cells incubated in oocyte and egg extracts in an active process that requires permeable nuclear pores. Removal of lamin A/C is specific, since B-type lamins are not changed, and it is not dependent on the incorporation Xenopus egg specific lamin III. Moreover, transcriptional activity is differentially regulated in somatic cells incubated in the extracts. Pol I and II transcriptions are maintained in cells in oocyte extracts; however, both activities are abolished in egg extracts. Our study shows that components of oocyte and egg extracts can modify the nuclear lamina of somatic cells and that this nuclear remodeling induces a structural change in the nucleus which may have implications for transcriptional activity. These experiments suggest that modifications in the nuclear lamina structure by the removal of somatic proteins and the incorporation of oocyte/egg components may contribute to the reprogramming of somatic cell nuclei and may define a characteristic configuration of pluripotent cells

  15. The cytoplasmic C-terminus of polycystin-1 increases cell proliferation in kidney epithelial cells through serum-activated and Ca2+-dependent pathway(s)

    International Nuclear Information System (INIS)

    Manzati, Elisa; Aguiari, Gianluca; Banzi, Manuela; Manzati, Michele; Selvatici, Rita; Falzarano, Sofia; Maestri, Iva; Pinton, Paolo; Rizzuto, Rosario; Senno, Laura del

    2005-01-01

    Polycystin-1 (PC1) is a large transmembrane protein important in renal differentiation and defective in most cases of autosomal dominant polycystic kidney disease (ADPKD), a common cause of renal failure in adults. Although the genetic basis of ADPKD has been elucidated, molecular and cellular mechanisms responsible for the dysregulation of epithelial cell growth in ADPKD cysts are still not well defined. We approached this issue by investigating the role of the carboxyl cytoplasmic domain of PC1 involved in signal transduction on the control of kidney cell proliferation. Therefore, we generated human HEK293 cells stably expressing the PC1 cytoplasmic tail as a membrane targeted TrkA-PC1 chimeric receptor protein (TrkPC1). We found that TrkPC1 increased cell proliferation through an increase in cytoplasmic Ca 2+ levels and activation of PKCα, thereby upregulating D1 and D3 cyclin, downregulating p21 waf1 and p27 kip1 cyclin inhibitors, and thus inducing cell cycle progression from G0/G1 to the S phase. Interestingly, TrkPC1-dependent Ca 2+ increase and PKCα activation are not constitutive, but require serum factor(s) as parallel component. In agreement with this observation, a significant increase in ERK1/2 phosphorylation was observed. Consistently, inhibitors specifically blocking either PKCα or ERK1/2 prevented the TrkPC1-dependent proliferation increase. NGF, the TrkA ligand, blocked this increase. We propose that in kidney epithelial cells the overexpression of PC1 C-terminus upregulates serum-evoked intracellular Ca 2+ by counteracting the growth-suppression activity of endogenous PC1 and leading to an increase in cell proliferation

  16. Phenotypic and functional profiling of CD4 T cell compartment in distinct populations of healthy adults with different antigenic exposure.

    Directory of Open Access Journals (Sweden)

    Sophie Roetynck

    Full Text Available Multiparameter flow cytometry has revealed extensive phenotypic and functional heterogeneity of CD4 T cell responses in mice and humans, emphasizing the importance of assessing multiple aspects of the immune response in correlation with infection or vaccination outcome. The aim of this study was to establish and validate reliable and feasible flow cytometry assays, which will allow us to characterize CD4 T cell population in humans in field studies more fully.We developed polychromatic flow cytometry antibody panels for immunophenotyping the major CD4 T cell subsets as well as broadly characterizing the functional profiles of the CD4 T cells in peripheral blood. We then validated these assays by conducting a pilot study comparing CD4 T cell responses in distinct populations of healthy adults living in either rural or urban Kenya. This study revealed that the expression profile of CD4 T cell activation and memory markers differed significantly between African and European donors but was similar amongst African individuals from either rural or urban areas. Adults from rural Kenya had, however, higher frequencies and greater polyfunctionality among cytokine producing CD4 T cells compared to both urban populations, particularly for "Th1" type of response. Finally, endemic exposure to malaria in rural Kenya may have influenced the expansion of few discrete CD4 T cell populations with specific functional signatures.These findings suggest that environmentally driven T cell activation does not drive the dysfunction of CD4 T cells but is rather associated with greater magnitude and quality of CD4 T cell response, indicating that the level or type of microbial exposure and antigenic experience may influence and shape the functionality of CD4 T cell compartment. Our data confirm that it is possible and mandatory to assess multiple functional attributes of CD4 T cell response in the context of infection.

  17. Dielectrophoretic analysis of changes in cytoplasmic ion levels due to ion channel blocker action reveals underlying differences between drug-sensitive and multidrug-resistant leukaemic cells

    International Nuclear Information System (INIS)

    Duncan, L; Shelmerdine, H; Hughes, M P; Coley, H M; Huebner, Y; Labeed, F H

    2008-01-01

    Dielectrophoresis (DEP)-the motion of particles in non-uniform AC fields-has been used in the investigation of cell electrophysiology. The technique offers the advantages of rapid determination of the conductance and capacitance of membrane and cytoplasm. However, it is unable to directly determine the ionic strengths of individual cytoplasmic ions, which has potentially limited its application in assessing cell composition. In this paper, we demonstrate how dielectrophoresis can be used to investigate the cytoplasmic ion composition by using ion channel blocking agents. By blocking key ion transporters individually, it is possible to determine their overall contribution to the free ions in the cytoplasm. We use this technique to evaluate the relative contributions of chloride, potassium and calcium ions to the cytoplasmic conductivities of drug sensitive and resistant myelogenous leukaemic (K562) cells in order to determine the contributions of individual ion channel activity in mediating multi-drug resistance in cancer. Results indicate that whilst K + and Ca 2+ levels were extremely similar between sensitive and resistant lines, levels of Cl - were elevated by three times to that in the resistant line, implying increased chloride channel activity. This result is in line with current theories of MDR, and validates the use of ion channel blockers with DEP to investigate ion channel function. (note)

  18. Cellular Subcompartments through Cytoplasmic Streaming.

    Science.gov (United States)

    Pieuchot, Laurent; Lai, Julian; Loh, Rachel Ann; Leong, Fong Yew; Chiam, Keng-Hwee; Stajich, Jason; Jedd, Gregory

    2015-08-24

    Cytoplasmic streaming occurs in diverse cell types, where it generally serves a transport function. Here, we examine streaming in multicellular fungal hyphae and identify an additional function wherein regimented streaming forms distinct cytoplasmic subcompartments. In the hypha, cytoplasm flows directionally from cell to cell through septal pores. Using live-cell imaging and computer simulations, we identify a flow pattern that produces vortices (eddies) on the upstream side of the septum. Nuclei can be immobilized in these microfluidic eddies, where they form multinucleate aggregates and accumulate foci of the HDA-2 histone deacetylase-associated factor, SPA-19. Pores experiencing flow degenerate in the absence of SPA-19, suggesting that eddy-trapped nuclei function to reinforce the septum. Together, our data show that eddies comprise a subcellular niche favoring nuclear differentiation and that subcompartments can be self-organized as a consequence of regimented cytoplasmic streaming. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Macrophage conditioned medium induced cellular network formation in MCF-7 cells through enhanced tunneling nanotube formation and tunneling nanotube mediated release of viable cytoplasmic fragments

    International Nuclear Information System (INIS)

    Patheja, Pooja; Sahu, Khageswar

    2017-01-01

    Infiltrating macrophages in tumor microenvironment, through their secreted cytokines and growth factors, regulate several processes of cancer progression such as cancer cell survival, proliferation, invasion, metastasis and angiogenesis. Recently, intercellular cytoplasmic bridges between cancer cells referred as tunneling nanotubes (TNTs) have been recognized as novel mode of intercellular communication between cancer cells. In this study, we investigated the effect of inflammatory mediators present in conditioned medium derived from macrophages on the formation of TNTs in breast adenocarcinoma cells MCF-7. Results show that treatment with macrophage conditioned medium (MφCM) not only enhanced TNT formation between cells but also stimulated the release of independently migrating viable cytoplasmic fragments, referred to as microplasts, from MCF-7 cells. Time lapse microscopy revealed that microplasts were released from parent cancer cells in extracellular space through formation of TNT-like structures. Mitochondria, vesicles and cytoplasm could be transferred from parent cell body to microplasts through connecting TNTs. The microplasts could also be resorbed into the parent cell body by retraction of the connecting TNTs. Microplast formation inhibited in presence cell migration inhibitor, cytochalasin-B. Notably by utilizing migratory machinery within microplasts, distantly located MCF-7 cells formed several TNT based intercellular connections, leading to formation of physically connected network of cells. Together, these results demonstrate novel role of TNTs in microplast formation, novel modes of TNT formation mediated by microplasts and stimulatory effect of MφCM on cellular network formation in MCF-7 cells mediated through enhanced TNT and microplast formation.

  20. Macrophage conditioned medium induced cellular network formation in MCF-7 cells through enhanced tunneling nanotube formation and tunneling nanotube mediated release of viable cytoplasmic fragments

    Energy Technology Data Exchange (ETDEWEB)

    Patheja, Pooja, E-mail: pooja.patheja8@gmail.com [Laser Biomedical Applications Section, Raja Ramanna Centre for Advanced Technology, Indore 452013, Madhya Pradesh (India); Homi Bhabha National Institute, Training School Complex, Anushaktinagar, Mumbai 400094, Maharashtra (India); Sahu, Khageswar [Laser Biomedical Applications Section, Raja Ramanna Centre for Advanced Technology, Indore 452013, Madhya Pradesh (India)

    2017-06-15

    Infiltrating macrophages in tumor microenvironment, through their secreted cytokines and growth factors, regulate several processes of cancer progression such as cancer cell survival, proliferation, invasion, metastasis and angiogenesis. Recently, intercellular cytoplasmic bridges between cancer cells referred as tunneling nanotubes (TNTs) have been recognized as novel mode of intercellular communication between cancer cells. In this study, we investigated the effect of inflammatory mediators present in conditioned medium derived from macrophages on the formation of TNTs in breast adenocarcinoma cells MCF-7. Results show that treatment with macrophage conditioned medium (MφCM) not only enhanced TNT formation between cells but also stimulated the release of independently migrating viable cytoplasmic fragments, referred to as microplasts, from MCF-7 cells. Time lapse microscopy revealed that microplasts were released from parent cancer cells in extracellular space through formation of TNT-like structures. Mitochondria, vesicles and cytoplasm could be transferred from parent cell body to microplasts through connecting TNTs. The microplasts could also be resorbed into the parent cell body by retraction of the connecting TNTs. Microplast formation inhibited in presence cell migration inhibitor, cytochalasin-B. Notably by utilizing migratory machinery within microplasts, distantly located MCF-7 cells formed several TNT based intercellular connections, leading to formation of physically connected network of cells. Together, these results demonstrate novel role of TNTs in microplast formation, novel modes of TNT formation mediated by microplasts and stimulatory effect of MφCM on cellular network formation in MCF-7 cells mediated through enhanced TNT and microplast formation.

  1. Transport and sorting of sphingolipids in polarized cells : the involvement of the sub-apical compartment

    NARCIS (Netherlands)

    IJzendoorn, Sven Christian David van

    1999-01-01

    The work described in this thesis has provided a novel insight into the process of sphingolipid transport and sorting in polarized cells. We have used HepG2 cells as a model system to study polarized traffic in hepatic cells. Under specific culture conditions, HepG2 cells acquire a polarized

  2. Dimethyl sulfoxide-inducible cytoplasmic factor involved in erythroid differentiation in mouse erythroleukemia (Friend) cells

    International Nuclear Information System (INIS)

    Watanabe, T.; Oishi, M.

    1987-01-01

    A previous report described an intracellular factor (differentiation-inducing factor I, or DIF-I) that seem to play a role in erythroid differentiation in mouse erythroleukemia (MEL) cells. The authors have detected another erythroid-inducing factor in cell-free extracts from dimethyl sulfoxide- or hexamethylenebis(acetamide)-treated MEL cells, which acts synergistically with DIF-I. The partially purified factor (termed DIF-II) triggered erythroid differentiation when introduced into undifferentiated MEL cells that had been potentiated by the induction of DIF-I. The activity in the extracts appeared in an inducible manner after addition of dimethyl sulfoxide or hexamethylenebis(acetamide), reached a maximum at 6 hr, and then rapidly decreased. The induction was inhibited by phorbol 12-myristate 13-acetate and also by cycloheximide. No induction was observed in a mutant MEL cell line defective in erythroid differentiation. These characteristics are consistent with the supposition that DIF-II is one of the putative dimethyl sulfoxide-inducible factors detected in previously reported cell-fusion and cytoplast-fusion experiments. The role of DIF-II in MEL-cell differentiation and in vitro differentiation in general is discussed

  3. A genetic polymorphism evolving in parallel in two cell compartments and in two clades

    Directory of Open Access Journals (Sweden)

    Watt Ward B

    2013-01-01

    Full Text Available Abstract Background The enzyme phosphoenolpyruvate carboxykinase, PEPCK, occurs in its guanosine-nucleotide-using form in animals and a few prokaryotes. We study its natural genetic variation in Colias (Lepidoptera, Pieridae. PEPCK offers a route, alternative to pyruvate kinase, for carbon skeletons to move between cytosolic glycolysis and mitochondrial Krebs cycle reactions. Results PEPCK is expressed in both cytosol and mitochondrion, but differently in diverse animal clades. In vertebrates and independently in Drosophila, compartment-specific paralogous genes occur. In a contrasting expression strategy, compartment-specific PEPCKs of Colias and of the silkmoth, Bombyx, differ only in their first, 5′, exons; these are alternatively spliced onto a common series of following exons. In two Colias species from distinct clades, PEPCK sequence is highly variable at nonsynonymous and synonymous sites, mainly in its common exons. Three major amino acid polymorphisms, Gly 335 ↔ Ser, Asp 503 ↔ Glu, and Ile 629 ↔ Val occur in both species, and in the first two cases are similar in frequency between species. Homology-based structural modelling shows that the variants can alter hydrogen bonding, salt bridging, or van der Waals interactions of amino acid side chains, locally or at one another′s sites which are distant in PEPCK′s structure, and thus may affect its enzyme function. We ask, using coalescent simulations, if these polymorphisms′ cross-species similarities are compatible with neutral evolution by genetic drift, but find the probability of this null hypothesis is 0.001 ≤ P ≤ 0.006 under differing scenarios. Conclusion Our results make the null hypothesis of neutrality of these PEPCK polymorphisms quite unlikely, but support an alternative hypothesis that they are maintained by natural selection in parallel in the two species. This alternative can now be justifiably tested further via studies of PEPCK genotypes′ effects

  4. Critical importance of the de novo pyrimidine biosynthesis pathway for Trypanosoma cruzi growth in the mammalian host cell cytoplasm

    International Nuclear Information System (INIS)

    Hashimoto, Muneaki; Morales, Jorge; Fukai, Yoshihisa; Suzuki, Shigeo; Takamiya, Shinzaburo; Tsubouchi, Akiko; Inoue, Syou; Inoue, Masayuki; Kita, Kiyoshi; Harada, Shigeharu; Tanaka, Akiko; Aoki, Takashi; Nara, Takeshi

    2012-01-01

    Highlights: ► We established Trypanosoma cruzi lacking the gene for carbamoyl phosphate synthetase II. ► Disruption of the cpsII gene significantly reduced the growth of epimastigotes. ► In particular, the CPSII-null mutant severely retarded intracellular growth. ► The de novo pyrimidine pathway is critical for the parasite growth in the host cell. -- Abstract: The intracellular parasitic protist Trypanosoma cruzi is the causative agent of Chagas disease in Latin America. In general, pyrimidine nucleotides are supplied by both de novo biosynthesis and salvage pathways. While epimastigotes—an insect form—possess both activities, amastigotes—an intracellular replicating form of T. cruzi—are unable to mediate the uptake of pyrimidine. However, the requirement of de novo pyrimidine biosynthesis for parasite growth and survival has not yet been elucidated. Carbamoyl-phosphate synthetase II (CPSII) is the first and rate-limiting enzyme of the de novo biosynthetic pathway, and increased CPSII activity is associated with the rapid proliferation of tumor cells. In the present study, we showed that disruption of the T. cruzicpsII gene significantly reduced parasite growth. In particular, the growth of amastigotes lacking the cpsII gene was severely suppressed. Thus, the de novo pyrimidine pathway is important for proliferation of T. cruzi in the host cell cytoplasm and represents a promising target for chemotherapy against Chagas disease.

  5. Internalization kinetics and cytoplasmic localization of functionalized diatomite nanoparticles in cancer cells by Raman imaging.

    Science.gov (United States)

    Managò, Stefano; Migliaccio, Nunzia; Terracciano, Monica; Napolitano, Michela; Martucci, Nicola M; De Stefano, Luca; Rendina, Ivo; De Luca, Anna Chiara; Lamberti, Annalisa; Rea, Ilaria

    2018-04-01

    Porous biosilica nanoparticles obtained from diatomites (DNPs) have been recently demonstrated to be non-toxic nanovectors of therapeutic agents in cancer cells. In this work, the internalization kinetics and intracellular spatial distribution of functionalized DNPs incubated with human lung epidermoid carcinoma cell line (H1355) up to 72 hours are investigated by Raman imaging. The label-free Raman results are compared with confocal fluorescence microscopy and photoluminescence (PL) data. Raman bands specifically assigned to DNPs and cellular components provide evidence that the nanovectors are internalized and co-localize with lipid environments. A considerable DNPs uptake in cells is observed within 6 hours, with equilibrium being achieved after 18 hours. The obtained data show the presence of DNPs up to 72 hours, without damage to cell viability or morphology. The PL measurements performed on DNPs not penetrating the cells at different incubation times are strongly correlated with the results obtained by Raman imaging and confocal microscopy analyses. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Induction and differentiation of human induced pluripotent stem cells into functional cardiomyocytes on a compartmented monolayer of gelatin nanofibers

    Science.gov (United States)

    Tang, Yadong; Liu, Li; Li, Junjun; Yu, Leqian; Wang, Li; Shi, Jian; Chen, Yong

    2016-07-01

    Extensive efforts have been devoted to develop new substrates for culture and differentiation of human induced pluripotent stem cells (hiPSCs) toward cardiac cell-based assays. A more exciting prospect is the construction of cardiac tissue for robust drug screening and cardiac tissue repairing. Here, we developed a patch method by electrospinning and crosslinking of monolayer gelatin nanofibers on a honeycomb frame made of poly(ethylene glycol) diacrylate (PEGDA). The monolayer of the nanofibrous structure can support cells with minimal exogenous contact and a maximal efficiency of cell-medium exchange whereas a single hiPSC colony can be uniformly formed in each of the honeycomb compartments. By modulating the treatment time of the ROCK inhibitor Y-27632, the shape of the hiPSC colony could be controlled from a flat layer to a hemisphere. Afterwards, the induction and differentiation of hiPSCs were achieved on the same patch, leading to a uniform cardiac layer with homogeneous contraction. This cardiac layer could then be used for extracellular recording with a commercial multi-electrode array, showing representative field potential waveforms of matured cardiac tissues with appropriate drug responses.Extensive efforts have been devoted to develop new substrates for culture and differentiation of human induced pluripotent stem cells (hiPSCs) toward cardiac cell-based assays. A more exciting prospect is the construction of cardiac tissue for robust drug screening and cardiac tissue repairing. Here, we developed a patch method by electrospinning and crosslinking of monolayer gelatin nanofibers on a honeycomb frame made of poly(ethylene glycol) diacrylate (PEGDA). The monolayer of the nanofibrous structure can support cells with minimal exogenous contact and a maximal efficiency of cell-medium exchange whereas a single hiPSC colony can be uniformly formed in each of the honeycomb compartments. By modulating the treatment time of the ROCK inhibitor Y-27632, the shape

  7. Methuosis: Nonapoptotic Cell Death Associated with Vacuolization of Macropinosome and Endosome Compartments

    OpenAIRE

    Maltese, William A.; Overmeyer, Jean H.

    2014-01-01

    Apoptosis is the most widely recognized form of physiological programmed cell death. During the past three decades, various nonapoptotic forms of cell death have gained increasing attention, largely because of their potential importance in pathological processes, toxicology, and cancer therapy. A recent addition to the panoply of cell death phenotypes is methuosis. The neologism is derived from the Greek methuo (to drink to intoxication) because the hallmark of this form of cell death is disp...

  8. Influence of recipient cytoplasm cell stage on transcription in bovine nucleus transfer embryos

    DEFF Research Database (Denmark)

    Smith, Steven D.; Soloy, Eva; Kanka, Jiri

    1996-01-01

    Nucleus transfer for the production of multiple embryos derived from a donor embryo relies upon the reprogramming of the donor nucleus so that it behaves similar to a zygotic nucleus. One indication of nucleus reprogramming is the RNA synthetic activity. In normal bovine embryogenesis, the embryo....... NTE were produced using either a MII phase (nonactivated) cytoplasts at 32 hr of maturation or S-phase (activated) cytoplasts activated with calcium ionophore A23187 and cycloheximide treatment approximately 8 hr prior to fusion with a blastomere from an in-vitro-produced morula stage embryo at 32 hr...... of maturation. Control in-vitro-produced embryos were 3H-uridine-labelled and fixed at the 2-, 4-, early 8-, and late 8-cell stages. NTE were similarly prepared at 1, 3, and 20 hr postfusion and at the 2-, 4-, and 8-cell stages. In the control embryos, RNA synthesis was absent in the 2-, 4-, and early 8-cell...

  9. Reconfiguration of NKT Cell Subset Compartment Is Associated with Plaque Development in Patients with Carotid Artery Stenosis.

    Science.gov (United States)

    Cai, Lun; Yu, Lei; Liu, Sa; Li, Tongxun; Zhang, Xiaoping; Cui, Wei; Du, Jie; Zhang, Qinyi

    2017-02-01

    Accumulating evidence shows that immune cells play an important role in carotid atherosclerotic plaque development. In this study, we assessed the association of 6 different natural killer T (NKT) cell subsets, based on CD57 and CD8 expression, with risk for development of carotid atherosclerotic plaque (CAP). Molecular expression by peripheral NKT cells was evaluated in 13 patients with high-risk CAP and control without carotid stenosis (n = 18). High-risk CAP patients, compared with healthy subjects, had less percentage of CD57+CD8- NKT cell subsets (8.64 ± 10.15 versus 19.62 ± 10.8 %; P = 0.01) and CD57+CD8int NKT cell subsets (4.32 ± 3.04 versus 11.87 ± 8.56 %; P = 0.002), with a corresponding increase in the CD57-CD8high NKT cell subsets (33.22 ± 11.87 versus 18.66 ± 13.68 %; P = 0.007). Intracellular cytokine staining showed that CD8+ NKT cell subset was the main cytokine-producing NKT cell. Cytokine production in plasma was measured with Bio-Plex assay. The expression levels of pro-inflammatory mediators (IFN-γ, IL-17, IP-10) were significantly higher in CAP patients as compared to that from controls. These data provide evidence that NKT cell subset compartment reconfiguration in patients with carotid stenosis seems to be associated with the occurrence of carotid atherosclerotic plaque and suggest that both pathogenic and protective NKT cell subsets exist.

  10. Identification of Cytoplasmic Proteins Interacting with the Mammary Cell Transforming Domain of Ese-1

    Science.gov (United States)

    2009-04-01

    measure auto - fluorescence , cells were incubated overnight at 4°C with blocking buffer alone. shRNA tranfected cells under- went the same procedure at 48...to a 50-GGA(A/T)-30 DNA coremotif [2]. All ETS proteins, with the exception of GA-binding protein (GABP)a, bind to DNA as a monomer and are auto ...skin, retina and other epithelia [7-10]. During mouse embryo develop- ment, Elf3 mRNA expression levels increase progressively, from embryonic day 7 to

  11. Intracellular pH (pHin) and cytosolic calcium ([Ca2+]cyt) regulation via ATPases: studies in cell populations, single cells, and subcellular compartments

    Science.gov (United States)

    Rojas, Jose D.; Sanka, Shankar C.; Gyorke, Sandor; Wesson, Donald E.; Minta, Akwasi; Martinez-Zaguilan, Raul

    1999-07-01

    Changes in pHin and (Ca2+)cyt are important in the signal transduction mechanisms leading to many physiological responses including cell growth, motility, secretion/exocytosis, etc. The concentrations of these ions are regulated via primary and secondary ion transporting mechanisms. In diabetes, specific pH and Ca2+ regulatory mechanism might be altered. To study these ions, we employ fluorescence spectroscopy, and cell imagin spectroscopy/confocal microscopy. pH and Ca2+ indicators are loaded in the cytosol with acetoxymethyl ester forms of dyes, and in endosomal/lysosomal (E/L) compartments by overnight incubation of cells with dextran- conjugated ion fluorescent probes. We focus on specific pH and Ca2+ regulatory systems: plasmalemmal vacuolar- type H+-ATPases (pm V-ATPases) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA). As experimental models, we employ vascular smooth muscle (VSM) and microvascular endothelial cells. We have chosen these cells because they are important in blood flow regulation and in angiogenesis. These processes are altered in diabetes. In many cell types, ion transport processes are dependent on metabolism of glucose for maximal activity. Our main findings are: (a) glycolysis coupling the activity of SERCA is required for cytosolic Ca2+ homeostasis in both VSM and microvascular endothelial cells; (b) E/L compartments are important for pH and Ca2+ regulation via H+-ATPases and SERCA, respectively; and (c) pm-V- ATPases are important for pHin regulation in microvascular endothelial cells.

  12. Ouabain-Induced Cytoplasmic Vesicles and Their Role in Cell Volume Maintenance

    Directory of Open Access Journals (Sweden)

    M. A. Russo

    2015-01-01

    Full Text Available Cellular swelling is controlled by an active mechanism of cell volume regulation driven by a Na+/K+-dependent ATPase and by aquaporins which translocate water along the osmotic gradient. Na+/K+-pump may be blocked by ouabain, a digitalic derivative, by inhibition of ATP, or by drastic ion alterations of extracellular fluid. However, it has been observed that some tissues are still able to control their volume despite the presence of ouabain, suggesting the existence of other mechanisms of cell volume control. In 1977, by correlating electron microscopy observation with ion and water composition of liver slices incubated in different metabolic conditions in the presence or absence of ouabain, we observed that hepatocytes were able to control their volume extruding water and recovering ion composition in the presence of ouabain. In particular, hepatocytes were able to sequester ions and water in intracellular vesicles and then secrete them at the bile canaliculus pole. We named this “vesicular mechanism of cell volume control.” Afterward, this mechanism has been confirmed by us and other laboratories in several mammalian tissues. This review summarizes evidences regarding this mechanism, problems that are still pending, and questions that need to be answered. Finally, we shortly review the importance of cell volume control in some human pathological conditions.

  13. Activation-induced proteolysis of cytoplasmic domain of zeta in T cell receptors and Fc receptors.

    Science.gov (United States)

    Taupin, J L; Anderson, P

    1994-12-01

    The CD3-T cell receptor (TCR) complex on T cells and the Fc gamma receptor type III (Fc gamma RIII)-zeta-gamma complex on natural killer cells are functionally analogous activation receptors that associate with a family of disulfide-linked dimers composed of the related subunits zeta and gamma. Immunochemical analysis of receptor complexes separated on two-dimensional diagonal gels allowed the identification of a previously uncharacterized zeta-p14 heterodimer. zeta-p14 is a component of both CD3-TCR and Fc gamma RIII-zeta-gamma. Peptide mapping analysis shows that p14 is structurally related to zeta, suggesting that it is either: (i) derived from zeta proteolytically or (ii) the product of an alternatively spliced mRNA. The observation that COS cells transformed with a cDNA encoding zeta express zeta-p14 supports the former possibility. The expression of CD3-TCR complexes including zeta-p14 increases following activation with phorbol 12-myristate 13-acetate or concanavalin A, suggesting that proteolysis of zeta may contribute to receptor modulation or desensitization.

  14. Cytoplasmic organelles determine complexity and specificity of calcium signalling in adrenal chromaffin cells

    Czech Academy of Sciences Publication Activity Database

    Garsia-Sancho, J.; Verkhratsky, Alexei

    2008-01-01

    Roč. 192, č. 2 (2008), s. 263-271 ISSN 1748-1708 Institutional research plan: CEZ:AV0Z50390512 Keywords : Ca2+ signalling * calcium microdomains * chromaffin cells Subject RIV: JE - Non-nuclear Energetics, Energy Consumption ; Use Impact factor: 2.455, year: 2008

  15. Stat5 Exerts Distinct, Vital Functions in the Cytoplasm and Nucleus of Bcr-Abl{sup +} K562 and Jak2(V617F){sup +} HEL Leukemia Cells

    Energy Technology Data Exchange (ETDEWEB)

    Weber, Axel [Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main 60596 (Germany); Borghouts, Corina [Ganymed Pharmaceuticals AG, Mainz 55131 (Germany); Brendel, Christian [Boston Children’s Hospital, Division of Hematology/Oncology, Boston, MA 02115 (United States); Moriggl, Richard [Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna 1090 (Austria); Delis, Natalia; Brill, Boris; Vafaizadeh, Vida; Groner, Bernd, E-mail: Groner@em.uni-frankfurt.de [Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main 60596 (Germany)

    2015-03-19

    Signal transducers and activators of transcription (Stats) play central roles in the conversion of extracellular signals, e.g., cytokines, hormones and growth factors, into tissue and cell type specific gene expression patterns. In normal cells, their signaling potential is strictly limited in extent and duration. The persistent activation of Stat3 or Stat5 is found in many human tumor cells and contributes to their growth and survival. Stat5 activation plays a pivotal role in nearly all hematological malignancies and occurs downstream of oncogenic kinases, e.g., Bcr-Abl in chronic myeloid leukemias (CML) and Jak2(V617F) in other myeloproliferative diseases (MPD). We defined the mechanisms through which Stat5 affects growth and survival of K562 cells, representative of Bcr-Abl positive CML, and HEL cells, representative for Jak2(V617F) positive acute erythroid leukemia. In our experiments we suppressed the protein expression levels of Stat5a and Stat5b through shRNA mediated downregulation and demonstrated the dependence of cell survival on the presence of Stat5. Alternatively, we interfered with the functional capacities of the Stat5 protein through the interaction with a Stat5 specific peptide ligand. This ligand is a Stat5 specific peptide aptamer construct which comprises a 12mer peptide integrated into a modified thioredoxin scaffold, S5-DBD-PA. The peptide sequence specifically recognizes the DNA binding domain (DBD) of Stat5. Complex formation of S5-DBD-PA with Stat5 causes a strong reduction of P-Stat5 in the nuclear fraction of Bcr-Abl-transformed K562 cells and a suppression of Stat5 target genes. Distinct Stat5 mediated survival mechanisms were detected in K562 and Jak2(V617F)-transformed HEL cells. Stat5 is activated in the nuclear and cytosolic compartments of K562 cells and the S5-DBD-PA inhibitor most likely affects the viability of Bcr-Abl{sup +} K562 cells through the inhibition of canonical Stat5 induced target gene transcription. In HEL cells

  16. Cytoplasmic superoxide dismutase and catalase activity and resistance to radiation lethality in murine tumor cells

    International Nuclear Information System (INIS)

    Davy, C.A.; Tesfay, Z.; Jones, J.; Rosenberg, R.C.; McCarthy, C.; Rosenberg, S.O.

    1986-01-01

    Reduced species of molecular oxygen are produced by the interaction of ionizing radiation with aqueous solutions containing molecular oxygen. The enzymes catalase and superoxide dismutase (SOD) are thought to function in vivo as scavengers of metabolically produced peroxide and superoxide respectively. SOD has been shown to protect against the lethal effects of ionizing radiation in vitro and in vivo. The authors have investigated the relationship between the cytosolic SOD catalase content and the sensitivity to radiation lethality of a number of murine cell lines (402AX, EL-4, MB-2T3, MB-4, MEL, P-815, SAI, SP-2, and SV-3T3). K/sub i/(CN - ) for murine Cu-Zn-SOD was determined to be 6.8 x 10 -6 M. No cytosolic Mn-SOD activity was found in any of the cell lines studied. No correlation was found between the cytosolic Cu-Zn-SOD or cytosolic catalase activity and the resistance to radiation lethality or the murine cell lines studied

  17. Ultrastructural and biochemical evidence for the presence of mature steroidogenic acute regulatory protein (StAR) in the cytoplasm of human luteal cells.

    Science.gov (United States)

    Sierralta, Walter D; Kohen, Paulina; Castro, Olga; Muñoz, Alex; Strauss, Jerome F; Devoto, Luigi

    2005-10-20

    The distribution of the steroidogenic acute regulatory protein (StAR) inside thecal and granulosa-lutein cells of human corpus luteum (CL) was assessed by immunoelectron microscopy. We found greater levels of StAR immunolabeling in steroidogenic cells from early- and mid-than in late luteal phase CL and lower levels in cells from women treated with a GnRH antagonist in the mid-luteal phase. Immunoelectron microscopy revealed significant levels of StAR antigen in the mitochondria and in the cytoplasm of luteal cells. The 30 kDa mature StAR protein was present in both mitochondria and cytosol (post-mitochondrial) fractions from homogenates of CL at different ages, whereas cytochrome c and mitochondrial HSP70 were detected only in the mitochondrial fraction. Therefore, we hypothesized that either appreciable processing of StAR 37 kDa pre-protein occurs outside the mitochondria, or mature StAR protein is selectively released into the cytoplasm after mitochondrial processing. The presence of mature StAR in the cytoplasm is consonant with the notion that StAR acts on the outer mitochondrial membrane to effect sterol import, and that StAR may interact with other cytoplasmic proteins involved in cholesterol metabolism, including hormone sensitive lipase.

  18. Deficiency in memory B cell compartment in a patient with infertility and recurrent pregnancy losses.

    Science.gov (United States)

    Sung, N; Byeon, H J; Garcia, M D Salazar; Skariah, A; Wu, L; Dambaeva, S; Beaman, K; Gilman-Sachs, A; Kwak-Kim, J

    2016-11-01

    Alterations in normal balance of B cell subsets have been reported in various rheumatic diseases. In this study, we report a woman with a history of recurrent pregnancy losses (RPL) and infertility who had low levels of memory B cells. A 35-year-old woman with a history of RPL and infertility was demonstrated to have increased peripheral blood CD19+ B cells with persistently low levels of memory B cell subsets. Prior to the frozen donor egg transfer cycle, prednisone and intravenous immunoglobulin G (IVIg) treatment was initiated and patient achieved dichorionic diamniotic twin pregnancies. During pregnancy, proportion (%) of switched memory B cells CD27+IgD- increased, while percent of total CD19+ B cells and CD27-IgD+ naive B cells were gradually decreased with a high dose IVIg treatment. She developed cervical incompetence at 20 weeks of gestation, received a Cesarean section at 32 weeks of gestation due to preterm labor, and delivered twin babies. B cell subset abnormalities may be associated with infertility, RPL and preterm labor, and further investigation is needed. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  19. Inhibitiory properties of cytoplasmic extract of Lactobacilli isolated from common carp intestine on human chronic myelocytic leukemia K562 cell line: an in vitro study

    Directory of Open Access Journals (Sweden)

    Kabiri F

    2011-03-01

    Full Text Available "n Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 st1":*{behavior:url(#ieooui } /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Arial; mso-bidi-theme-font:minor-bidi;} Background: Lactobacillus species are genetically diverse groups of Lactic Acid Bacteria (LAB that have been introduced as probiotics, because of some characteristics such as their anti-tumor properties, helping the intestinal flora balance, production of antibiotics, stimulation of host immune response, etc. The aim of this study was to investigate the effects of cytoplasmic extraction and cell wall of Lactobacillus species isolated from the intestine of common carp on human chronic myelocytic leukemia or K562 cancer cell lines."n"nMethods: The intestinal contents of 115 common carp captured from the natural resources of West Azerbaijan province in Iran were examined for LAB. After isolation, the identification of Lactobacilli was done according to traditional and molecular bacteriological tests. Subsequently, a suspension of each bacterium was prepared and the protein content of the cytoplasm was extracted. Cell wall disintegration was done by cell lysis buffer and sonication. The effects of cytoplasmic extraction and cell wall on K562 cell line proliferation were investigated by MTT assays."n"nResults: The cytoplasmic extraction of the isolated Lactobacilli had significant (p<0.05 anti

  20. Contribution of the actomyosin motor to the temperature-dependent translational diffusion of water by cytoplasmic streaming in Elodea canadensis cells.

    Science.gov (United States)

    Vorob'ev, V N; Anisimov, A V; Dautova, N R

    2004-12-01

    The extent to which the actomyosin motor responsible for cytoplasmic streaming contributes to the translational diffusion of water in Elodea canadensis cells was studied by a nuclear magnetic resonance (NMR) spin-echo technique. The relative contribution of the actomyosin motor was determined from the corresponding apparent diffusion coefficient by the Einstein-Smolukhovsky relation. It is equal to the difference between the diffusional displacements of the cytoplasmic and the bulk water (deltaX). The NMR data show that the temperature dependence of deltaX is humpshaped, which is characteristic of enzyme reactions. At the same time, the apparent diffusion coefficient of cytoplasmic water increases with an increase in temperature. The most significant contribution of the actomyosin motor to deltaX is observed at temperatures below 20 degrees C. Within the temperature range of 20 to 33 degrees C, deltaX changes only slightly, and a further increase in temperature reduces deltaX to zero.

  1. Statistical modelling of subdiffusive dynamics in the cytoplasm of living cells: A FARIMA approach

    Science.gov (United States)

    Burnecki, K.; Muszkieta, M.; Sikora, G.; Weron, A.

    2012-04-01

    Golding and Cox (Phys. Rev. Lett., 96 (2006) 098102) tracked the motion of individual fluorescently labelled mRNA molecules inside live E. coli cells. They found that in the set of 23 trajectories from 3 different experiments, the automatically recognized motion is subdiffusive and published an intriguing microscopy video. Here, we extract the corresponding time series from this video by image segmentation method and present its detailed statistical analysis. We find that this trajectory was not included in the data set already studied and has different statistical properties. It is best fitted by a fractional autoregressive integrated moving average (FARIMA) process with the normal-inverse Gaussian (NIG) noise and the negative memory. In contrast to earlier studies, this shows that the fractional Brownian motion is not the best model for the dynamics documented in this video.

  2. A MODULAR PHOTOSYNTHETIC MICROBIAL FUEL CELL WITH INTERCHANGEABLE ALGAE SOLAR COMPARTMENTS

    OpenAIRE

    Fleury, Daniel

    2017-01-01

    This project trial provides a novel small-scale solar harnessing technology which increases environmental effectiveness while maintaining optimal energy efficiency. Although modern solar panels are purposed in producing clean energy, the materials and byproducts of solar cell manufacturing are not eco-friendly. Thus, considering an organic, renewable and energy efficient solar cell model is necessary. Investigations explored multiple highly-photosynthetic algal species which were later integr...

  3. Reorganization of the Endosomal System in Salmonella-Infected Cells: The Ultrastructure of Salmonella-Induced Tubular Compartments

    Science.gov (United States)

    Krieger, Viktoria; Liebl, David; Zhang, Yuying; Rajashekar, Roopa; Chlanda, Petr; Giesker, Katrin; Chikkaballi, Deepak; Hensel, Michael

    2014-01-01

    During the intracellular life of Salmonella enterica, a unique membrane-bound compartment termed Salmonella-containing vacuole, or SCV, is formed. By means of translocated effector proteins, intracellular Salmonella also induce the formation of extensive, highly dynamic membrane tubules termed Salmonella-induced filaments or SIF. Here we report the first detailed ultrastructural analyses of the SCV and SIF by electron microscopy (EM), EM tomography and live cell correlative light and electron microscopy (CLEM). We found that a subset of SIF is composed of double membranes that enclose portions of host cell cytosol and cytoskeletal filaments within its inner lumen. Despite some morphological similarities, we found that the formation of SIF double membranes is independent from autophagy and requires the function of the effector proteins SseF and SseG. The lumen of SIF network is accessible to various types of endocytosed material and our CLEM analysis of double membrane SIF demonstrated that fluid phase markers accumulate only between the inner and outer membrane of these structures, a space continual with endosomal lumen. Our work reveals how manipulation of the endosomal membrane system by an intracellular pathogen results in a unique tubular membrane compartmentalization of the host cell, generating a shielded niche permissive for intracellular proliferation of Salmonella. PMID:25254663

  4. Cancer cell-associated cytoplasmic B7–H4 is induced by hypoxia through hypoxia-inducible factor-1α and promotes cancer cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Jeon, You-Kyoung [Department of Microbiology and Immunology, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Advanced Research Center for Multiple Myeloma, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Park, Sae-Gwang; Choi, Il-Whan [Department of Microbiology and Immunology, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Lee, Soo-Woong [Advanced Research Center for Multiple Myeloma, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Lee, Sang Min [Department of Internal Medicine, Division of Hematology/Oncology, Busan Paik Hospital, Inje University, Busan 614-735 (Korea, Republic of); Choi, Inhak, E-mail: miccih@inje.ac.kr [Department of Microbiology and Immunology, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Advanced Research Center for Multiple Myeloma, Inje University College of Medicine, Busan 614-735 (Korea, Republic of)

    2015-04-03

    Aberrant B7–H4 expression in cancer tissues serves as a novel prognostic biomarker for poor survival in patients with cancer. However, the factor(s) that induce cancer cell-associated B7–H4 remain to be fully elucidated. We herein demonstrate that hypoxia upregulates B7–H4 transcription in primary CD138{sup +} multiple myeloma cells and cancer cell lines. In support of this finding, analysis of the Multiple Myeloma Genomics Portal (MMGP) data set revealed a positive correlation between the mRNA expression levels of B7–H4 and the endogenous hypoxia marker carbonic anhydrogenase 9. Hypoxia-induced B7–H4 expression was detected in the cytoplasm, but not in cancer cell membranes. Chromatin immunoprecipitation analysis demonstrated binding of hypoxia-inducible factor-1α (HIF-1α) to proximal hypoxia-response element (HRE) sites within the B7–H4 promoter. Knockdown of HIF-1α and pharmacological inhibition of HIF-1α diminished B7–H4 expression. Furthermore, knockdown of cytoplasmic B7–H4 in MCF-7 decreased the S-phase cell population under hypoxia. Finally, MMGP analysis revealed a positive correlation between the transcript levels of B7–H4 and proliferation-related genes including MKI67, CCNA1, and Myc in several patients with multiple myeloma. Our results provide insight into the mechanisms underlying B7–H4 upregulation and its role in cancer cell proliferation in a hypoxic tumor microenvironment. - Highlights: • Hypoxia upregulates B7–H4 transcription and protein expression. • Hypoxia-induced B7–H4 is detected in the cytoplasm, but not on membrane. • ChIP assay reveals a binding of HIF-1α to B7–H4 promoter at HRE site. • Knockdown and pharmacological inhibition of HIF-1α reduce B7–H4 expression. • B7–H4 knockdown decrease the number of cells in S-phase of cell cycle.

  5. F4/80 inhibits osteoclast differentiation via downregulation of nuclear factor of activated T cells, cytoplasmic 1.

    Science.gov (United States)

    Kang, Ju-Hee; Sim, Jung-Sun; Zheng, Ting; Yim, Mijung

    2017-04-01

    Osteoclastogenesis is an essential process in bone metabolism, which can be induced by RANKL stimulation. The F4/80 glycoprotein is a member of the EGF-transmembrane 7 (TM7) family and has been established as a specific cell-surface marker for murine macrophages. This study aimed to identify the role of F4/80 in osteoclastogenesis. Using mouse bone marrow-derived macrophages (BMMs), we observed that the mRNA level of F4/80 was dramatically reduced as these cells differentiated into osteoclasts. Furthermore, osteoclastogenesis was decreased in F4/80 high BMMs compared to F4/80 -/low BMMs. The inhibitory effect of F4/80 was associated with decreased expression of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1). Ectopic overexpression of a constitutively active form of NFATc1 rescued the anti-osteoclastogenic effect of F4/80 completely, suggesting that the anti-osteoclastogenic effect of F4/80 was mainly due to reduction in NFATc1 expression. As an underlying mechanism, we demonstrated that the presence of F4/80 abrogated the effect of RANKL on the phosphorylation of CREB and activated the expression of IFN-β, which are restored by cyclic AMP. Collectively, our results demonstrate that the presence of F4/80 suppresses RANKL-induced osteoclastogenesis by impairing the expression of NFATc1 via CREB and IFN-β. Therefore, F4/80 may hold therapeutic potential for bone destructive diseases.

  6. Spindle orientation bias in gut epithelial stem cell compartments is lost in precancerous tissue

    NARCIS (Netherlands)

    Quyn, A.J.; Appleton, P.L.; Carey, F.A.; Steele, R.J.; Barker, N.; Clevers, H.; Ridgway, R.A.; Sansom, O.J.; Nathke, I.S.

    2010-01-01

    The importance of asymmetric divisions for stem cell function and maintenance is well established in the developing nervous system and the skin; however, its role in gut epithelium and its importance for tumorigenesis is still debated. We demonstrate alignment of mitotic spindles perpendicular to

  7. Repopulation of the Stem-Cell Compartment in Haemopoietic and Lymphatic Tissues of Mice after X-Irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Vos, O. [Medical Biological Laboratory of the National Defense Organization TNO, Rijswijk Z.H. (Netherlands)

    1968-08-15

    Repopulation in haemopoietic tissues of mice was studied at various times after irradiation with a sub-lethal X-ray dose of 460 R and after exposure to a lethal X-ray dose of 800 R followed by transplantation of 4 x 10{sup 6} syngeneic bone-marrow cells. The number of stem cells was determined with the exogenous and endogenous spleen colony technique in the bone-marrow of the femur and in the spleen. Repopulation in lymphatic tissues was investigated at various times after sub-lethal irradiation with 460 R, sub-lethal irradiation with 460 R followed by allogeneic skin transplantation, lethal irradiation with 800 R followed by transplantation of 2 x 10{sup 6} syngeneic bone-marrow cells, and irradiation with 800 R followed by transplantation of 2 x 10{sup 6} and 5 x 10{sup 6} syngeneic bone-marrow and lymph node cells, respectively. The number of stem cells was estimated in the spleen and in lymph nodes with a technique based on a graft-versus- host reaction. The results showed that a rapid repopulation occurred in haemopoietic tissues under all circumstances. In the spleen an overshoot was found during a period beginning shortly after the 7th day and lasting for some weeks. In lymphatic tissues repopulation by stem cells was slow. During the first one or two weeks some decrease was even observed. This was followed by a slow repopulation that around 100 days after irradiation reached a level slightly below that found in control animals. Small differences of repopulation in the various organs and under different circumstances are discussed. It was postulated that immuno-competent lymphatic stem cells (antigen-sensitive cells or PC{sub 1} cells) do not proliferate or do so only to a limited extent. This compartment must be replenished by repopulation of differentiating stem cells from the bone marrow. It seems probable that stem-cell depletion or antigeneic stimulation has only a marginal effect upon repopulation. (author)

  8. Digital image analysis supports a nuclear-to-cytoplasmic ratio cutoff value of 0.5 for atypical urothelial cells.

    Science.gov (United States)

    Hang, Jen-Fan; Charu, Vivek; Zhang, M Lisa; VandenBussche, Christopher J

    2017-09-01

    An elevated nuclear-to-cytoplasmic (N:C) ratio of ≥0.5 is a required criterion for the diagnosis of atypical urothelial cells (AUC) in The Paris System for Reporting Urinary Cytology. To validate the N:C ratio cutoff value and its predictive power for high-grade urothelial carcinoma (HGUC), the authors retrospectively reviewed the urinary tract cytology specimens of 15 cases of AUC with HGUC on follow-up (AUC-HGUC) and 33 cases of AUC without HGUC on follow-up (AUC-N-HGUC). The number of atypical cells in each case was recorded, and each atypical cell was photographed and digitally examined to calculate the nuclear size and N:C ratio. On average, the maximum N:C ratios of atypical cells were significantly different between the AUC-HGUC and AUC-N-HGUC cohorts (0.53 vs 0.43; P =.00009), whereas the maximum nuclear sizes of atypical cells (153.43 μM 2 vs 201.47 μM 2 ; P = .69) and the number of atypical cells per case (10.13 vs 7.88; P = .12) were not found to be significantly different. Receiver operating characteristic analysis demonstrated that the maximum N:C ratio alone had high discriminatory capacity (area under the curve, 79.19%; 95% confidence interval, 64.19%-94.19%). The optimal maximum N:C ratio threshold was 0.486, giving a sensitivity of 73.3% and a specificity of 84.8% for predicting HGUC on follow-up. The identification of AUC with an N:C ratio >0.486 has a high predictive power for HGUC on follow-up in AUC specimens. This justifies using the N:C ratio as a required criterion for the AUC category. Individual laboratories using different cytopreparation methods may require independent validation of the N:C ratio cutoff value. Cancer Cytopathol 2017;125:710-6. © 2017 American Cancer Society. © 2017 American Cancer Society.

  9. Compartment-specific immunity in the human gut: properties and functions of dendritic cells in the colon versus the ileum.

    Science.gov (United States)

    Mann, Elizabeth R; Bernardo, David; English, Nicholas R; Landy, Jon; Al-Hassi, Hafid O; Peake, Simon T C; Man, Ripple; Elliott, Timothy R; Spranger, Henning; Lee, Gui Han; Parian, Alyssa; Brant, Steven R; Lazarev, Mark; Hart, Ailsa L; Li, Xuhang; Knight, Stella C

    2016-02-01

    Dendritic cells (DC) mediate intestinal immune tolerance. Despite striking differences between the colon and the ileum both in function and bacterial load, few studies distinguish between properties of immune cells in these compartments. Furthermore, information of gut DC in humans is scarce. We aimed to characterise human colonic versus ileal DC. Human DC from paired colonic and ileal samples were characterised by flow cytometry, electron microscopy or used to stimulate T cell responses in a mixed leucocyte reaction. A lower proportion of colonic DC produced pro-inflammatory cytokines (tumour necrosis factor-α and interleukin (IL)-1β) compared with their ileal counterparts and exhibited an enhanced ability to generate CD4(+)FoxP3(+)IL-10(+) (regulatory) T cells. There were enhanced proportions of CD103(+)Sirpα(-) DC in the colon, with increased proportions of CD103(+)Sirpα(+) DC in the ileum. A greater proportion of colonic DC subsets analysed expressed the lymph-node-homing marker CCR7, alongside enhanced endocytic capacity, which was most striking in CD103(+)Sirpα(+) DC. Expression of the inhibitory receptor ILT3 was enhanced on colonic DC. Interestingly, endocytic capacity was associated with CD103(+) DC, in particular CD103(+)Sirpα(+) DC. However, expression of ILT3 was associated with CD103(-) DC. Colonic and ileal DC differentially expressed skin-homing marker CCR4 and small-bowel-homing marker CCR9, respectively, and this corresponded to their ability to imprint these homing markers on T cells. The regulatory properties of colonic DC may represent an evolutionary adaptation to the greater bacterial load in the colon. The colon and the ileum should be regarded as separate entities, each comprising DC with distinct roles in mucosal immunity and imprinting. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  10. Hemopoietic stem-cell compartment of the SCID mouse: Double-exponential survival curve after γ irradiation

    International Nuclear Information System (INIS)

    Taniguchi, Satoshi; Hirabayashi, Yoko; Inoue, Tohru; Kanisawa, Masayoshi; Sasaki, Hideki; Komatsu, Kenshi; Mori, K.J.

    1993-01-01

    It has been reported that SCID (severe combined immunodeficiency, scid/scid) mice are more radiosensitive than normal mice. In the present studies, graded doses of radiation were given to bone marrow cells from SCID mice, and double-exponential survival curves were observed for day-9 and day-12 colony-forming units in the spleen (CFU-S). Single-exponential curves were found for SCID CFU in in vitro assays for granulocyte/macrophage-CFUs and erythroid burst-forming units, as reported elsewhere. Since the size of this more resistant fraction seems to decrease with stem-cell maturation, the finding implies that this fraction is a primitive subpopulation of the stem-cell compartment. The mean lethal dose (D 0 ), however, of this less sensitive SCID CFU-S is much less than the D 0 of regular CFU-S in normal littermates. Spleen colonies produced by SCID bone marrow were relatively small and abortive. The size of these colonies decreased nearly exponentially with increasing doses of radiation. These colonies produced by the sensitive fraction have disappeared, being killed by a relatively low dose of radiation. This observation might account for the high lymphomagenesis arising from primitive hemopoietic stem cells in SCID mice, because the smallness of the colonies suggests that there is unrepaired or misrepaired damage. Furthermore, this less sensitive fraction might be a source of the open-quotes leakyclose quotes change of T and B cells, possibly due to the induction of an equivocal repair system which appears in the later stages of life in the SCID mice. 34 refs., 5 figs., 3 tabs

  11. Changes in protein metabolism after irradiation. Pt. 1. Protease activity, protease pattern, protein and free amino acids in cytoplasm and cell organelles of the rat spleen after 600 R whole body x irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Valet, G [Max-Planck-Institut fuer Biochemie, Muenchen (F.R. Germany). Abt. fuer Experimentelle Medizin

    1975-12-01

    The protease activity of cytoplasm and cell organelles of the rat spleen against spleen protein and hemoglobin as a substrate increases during a initial reaction phase of the organism on the first day after 600 R whole body X-irradiation. The alkaline protease in the cytoplasm and the acid protease in the cell organelles increase, whereas the protease activity against externally added hemoglobin as substrate decreases below the initial values. The protein, the protease activity and the free amino acids of the cytoplasm and the cell organelles decrease during the disease phase on day 3 and 4 after irradiation. The protein loss of the spleen is therefore not explained by an increased protease activity. Acid proteases appear in the cytoplasm which derive probably from the cell organelles. The protease activity and the free amino acids are increased in the cytoplasm and the cell organelles during the regeneration phase of the organism between day 15 and 18 after irradiation.

  12. Mouse mammary tumor virus uses mouse but not human transferrin receptor 1 to reach a low pH compartment and infect cells

    International Nuclear Information System (INIS)

    Wang Enxiu; Obeng-Adjei, Nyamekye; Ying Qihua; Meertens, Laurent; Dragic, Tanya; Davey, Robert A.; Ross, Susan R.

    2008-01-01

    Mouse mammary tumor virus (MMTV) is a pH-dependent virus that uses mouse transferrin receptor 1 (TfR1) for entry into cells. Previous studies demonstrated that MMTV could induce pH 5-dependent fusion-from-with of mouse cells. Here we show that the MMTV envelope-mediated cell-cell fusion requires both the entry receptor and low pH (pH 5). Although expression of the MMTV envelope and TfR1 was sufficient to mediate low pH-dependent syncytia formation, virus infection required trafficking to a low pH compartment; infection was independent of cathepsin-mediated proteolysis. Human TfR1 did not support virus infection, although envelope-mediated syncytia formation occurred with human cells after pH 5 treatment and this fusion depended on TfR1 expression. However, although the MMTV envelope bound human TfR1, virus was only internalized and trafficked to a low pH compartment in cells expressing mouse TfR1. Thus, while human TfR1 supported cell-cell fusion, because it was not internalized when bound to MMTV, it did not function as an entry receptor. Our data suggest that MMTV uses TfR1 for all steps of entry: cell attachment, induction of the conformational changes in Env required for membrane fusion and internalization to an appropriate acidic compartment

  13. Macrophage conditioned medium induced cellular network formation in MCF-7 cells through enhanced tunneling nanotube formation and tunneling nanotube mediated release of viable cytoplasmic fragments.

    Science.gov (United States)

    Patheja, Pooja; Sahu, Khageswar

    2017-06-15

    Infiltrating macrophages in tumor microenvironment, through their secreted cytokines and growth factors, regulate several processes of cancer progression such as cancer cell survival, proliferation, invasion, metastasis and angiogenesis. Recently, intercellular cytoplasmic bridges between cancer cells referred as tunneling nanotubes (TNTs) have been recognized as novel mode of intercellular communication between cancer cells. In this study, we investigated the effect of inflammatory mediators present in conditioned medium derived from macrophages on the formation of TNTs in breast adenocarcinoma cells MCF-7. Results show that treatment with macrophage conditioned medium (MɸCM) not only enhanced TNT formation between cells but also stimulated the release of independently migrating viable cytoplasmic fragments, referred to as microplasts, from MCF-7 cells. Time lapse microscopy revealed that microplasts were released from parent cancer cells in extracellular space through formation of TNT-like structures. Mitochondria, vesicles and cytoplasm could be transferred from parent cell body to microplasts through connecting TNTs. The microplasts could also be resorbed into the parent cell body by retraction of the connecting TNTs. Microplast formation inhibited in presence cell migration inhibitor, cytochalasin-B. Notably by utilizing migratory machinery within microplasts, distantly located MCF-7 cells formed several TNT based intercellular connections, leading to formation of physically connected network of cells. Together, these results demonstrate novel role of TNTs in microplast formation, novel modes of TNT formation mediated by microplasts and stimulatory effect of MɸCM on cellular network formation in MCF-7 cells mediated through enhanced TNT and microplast formation. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Pregnancy alters the circulating B cell compartment in atopic asthmatic women, and transitional B cells are positively associated with the development of allergy manifestations in their progeny.

    Science.gov (United States)

    Martins, Catarina; Lima, Jorge; Nunes, Glória; Borrego, Luís Miguel

    2016-12-01

    Maternal atopy is a risk factor for allergy. B cells are poorly studied in reproduction and atopy. We aimed to assess how pregnancy affects B cells in atopic women and whether B cells relate to allergic manifestations in offspring. Women with and without atopic asthma, pregnant and non-pregnant were enrolled for the study, and circulating B cells were evaluated by flow cytometry, using CD19, CD27, CD38, IgD, and IgM. Compared to healthy non-pregnant, atopic asthmatic non-pregnant (ANP) women presented increased B cell counts, enlarged memory subsets, less transitional cells, and plasmablasts. Atopic asthmatic pregnant (AP) and healthy pregnant (HP) women showed similarities: reduced B cell counts and percentages, fewer memory cells, especially switched, and higher plasmablast percentages. Transitional B cell percentages were increased in AP women with allergic manifestations in their progeny. Atopic asthmatic non-pregnant women have a distinctive B cell compartment. B cells change in pregnancy, similarly in AP and HP women. The recognition that AP women with allergy in their progeny have a typical immune profile may help, in the future, the adoption of preventive measures to avoid the manifestation of allergic diseases in their newborns. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. Cell polarity and patterning by PIN trafficking through early endosomal compartments in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Hirokazu Tanaka

    2013-05-01

    Full Text Available PIN-FORMED (PIN proteins localize asymmetrically at the plasma membrane and mediate intercellular polar transport of the plant hormone auxin that is crucial for a multitude of developmental processes in plants. PIN localization is under extensive control by environmental or developmental cues, but mechanisms regulating PIN localization are not fully understood. Here we show that early endosomal components ARF GEF BEN1 and newly identified Sec1/Munc18 family protein BEN2 are involved in distinct steps of early endosomal trafficking. BEN1 and BEN2 are collectively required for polar PIN localization, for their dynamic repolarization, and consequently for auxin activity gradient formation and auxin-related developmental processes including embryonic patterning, organogenesis, and vasculature venation patterning. These results show that early endosomal trafficking is crucial for cell polarity and auxin-dependent regulation of plant architecture.

  16. Cell specificity of the cytoplasmic Ca2+ response to tolbutamide is impaired in beta-cells from hyperglycemic mice

    DEFF Research Database (Denmark)

    Gustavsson, Natalia; Larsson-Nyrén, Gerd; Lindström, Per

    2006-01-01

    We recently reported that the timing and magnitude of the nutrient-induced Ca(2+) response are specific and reproducible for each isolated beta-cell. We have now used tolbutamide and arginine to test if the cell specificity exists also for the response to non-nutrient stimulation of beta-cells an...

  17. Mechanisms for cytoplasmic organization: an overview.

    Science.gov (United States)

    Pagliaro, L

    2000-01-01

    One of the basic characteristics of life is the intrinsic organization of cytoplasm, yet we know surprisingly little about the manner in which cytoplasmic macromolecules are arranged. It is clear that cytoplasm is not the homogeneous "soup" it was once envisioned to be, but a comprehensive model for cytoplasmic organization is not available in modern cell biology. The premise of this volume is that phase separation in cytoplasm may play a role in organization at the subcellular level. Other mechanisms for non-membrane-bounded intracellular organization have previously been proposed. Some of these will be reviewed in this chapter. Multiple mechanisms, involving phase separation, specific intracellular targeting, formation of macromolecular complexes, and channeling, all could well contribute to cytoplasmic organization. Temporal and spatial organization, as well as composition, are likely to be important in defining the characteristics of cytoplasm.

  18. Feasibility of using sodium chloride as a tracer for the characterization of the distribution of matter in complex multi-compartment 3D bioreactors for stem cell culture.

    Science.gov (United States)

    Gerlach, Jörg C; Witaschek, Tom; Strobel, Catrin; Brayfield, Candace A; Bornemann, Reinhard; Catapano, Gerardo; Zeilinger, Katrin

    2010-06-01

    The experimental characterization of the distribution of matter in complex multi-compartment three-dimensional membrane bioreactors for human cell culture is complicated by tracer interactions with the membranes and other bioreactor constituents. This is due to the fact that membranes with a high specific surface area often feature a hydrophobic chemical backbone that may adsorb tracers often used to this purpose, such as proteins and dyes. Membrane selectivity, and its worsening caused by protein adsorption, may also hinder tracer transfer across neighboring compartments, thus preventing effective characterization of the distribution of matter in the whole bioreactor. Tracer experiments with sodium chloride (NaCl) may overcome some of these limitations and be effectively used to characterize the distribution of matter in complex 3D multi-compartments membrane bioreactors for stem cell culture. NaCl freely permeates most used membranes, it does not adsorb on uncharged membranes, and its concentration may be accurately measured in terms of solution conductivity. In this preliminary study, the feasibility of complex multi-compartment membrane bioreactors was investigated with a NaCl concentration pulse challenge to characterize how their distribution of matter changes when they are operated under different conditions. In particular, bioreactors consisting of three different membrane types stacked on top of one another to form a 3D network were characterized under different feed conditions.

  19. Contribution of Mesenteric Lymph Nodes and GALT to the Intestinal Foxp3+ Regulatory T-Cell CompartmentSummary

    Directory of Open Access Journals (Sweden)

    Duke Geem

    2016-05-01

    Full Text Available Background & Aims: Foxp3+ regulatory T cells (Tregs in the intestine promote immune tolerance to enteric antigens. Previous studies have shown that C-C chemokine receptor 7 (CCR7-dependent migration of intestinal dendritic cells to the mesenteric lymph nodes (mLN is involved in peripheral Foxp3+ Treg accumulation in the intestine and the establishment of oral tolerance. However, the relative contribution of this CCR7+ dendritic cell–mLN–Treg axis to the total intestinal Foxp3+ Treg pool during the steady-state remains unclear. In this study, the contribution of CCR7, as well as the mLN and gut-associated lymphoid tissue (GALT, to the intestinal Foxp3+ Treg compartment in the small intestine (SI and large intestine (LI was assessed. Methods: Intestinal Foxp3+ Tregs were quantitated in Ccr7-/- mice and in mice devoid of secondary lymphoid organs—including mLN and GALT—owing to a deficiency in lymphotoxin (LT signaling. Specific analyses of Foxp3+Helios+ thymically derived (tTregs and Foxp3+Helios- peripherally derived (pTregs in the SI and LI, as well as the role for the mLN in supporting Foxp3+ pTreg development using the B6.Cg-Tg(TcraTcrb425Cbn/J/ovalbumin (OVA feeding system, were performed. Results: Foxp3+ Tregs were enriched in the intestine relative to the mLN, independent of CCR7. In the absence of the mLN and GALT, normal frequency and numbers of Foxp3+ Tregs were observed in LTα-deficient (Lta-/- mice. However, Foxp3+Helios- pTregs were decreased in the SI of Lta-/- mice, corresponding with defective Foxp3+ pTreg expansion to OVA. In the LI, however, the proportion of Foxp3+Helios- pTregs and Foxp3+ pTreg induction to OVA was comparable between Lta-/- and Lta+/+ mice, which coincided with preferential expression of Treg-inducing/immunoregulatory cytokines. Conclusions: The overall size of the intestinal Foxp3+Treg pool is not impacted significantly by CCR7, mLN, or GALT during the steady-state. However, m

  20. Compartment-specific distribution of human intestinal innate lymphoid cells is altered in HIV patients under effective therapy.

    Directory of Open Access Journals (Sweden)

    Benjamin Krämer

    2017-05-01

    Full Text Available Innate lymphocyte cells (ILCs, a novel family of innate immune cells are considered to function as key orchestrators of immune defences at mucosal surfaces and to be crucial for maintaining an intact intestinal barrier. Accordingly, first data suggest depletion of ILCs to be involved in human immunodeficiency virus (HIV-associated damage of the intestinal mucosa and subsequent microbial translocation. However, although ILCs are preferentially localized at mucosal surfaces, only little is known regarding distribution and function of ILCs in the human gastrointestinal tract. Here, we show that in HIV(- individuals composition and functional capacity of intestinal ILCs is compartment-specific with group 1 ILCs representing the major fraction in the upper gastrointestinal (GI tract, whereas ILC3 are the predominant population in ileum and colon, respectively. In addition, we present first data indicating that local cytokine concentrations, especially that of IL-7, might modulate composition of gut ILCs. Distribution of intestinal ILCs was significantly altered in HIV patients, who displayed decreased frequency of total ILCs in ileum and colon owing to reduced numbers of both CD127(+ILC1 and ILC3. Of note, frequency of colonic ILC3 was inversely correlated with serum levels of I-FABP and sCD14, surrogate markers for loss of gut barrier integrity and microbial translocation, respectively. Both expression of the IL-7 receptor CD127 on ILCs as well as mucosal IL-7 mRNA levels were decreased in HIV(+ patients, especially in those parts of the GI tract with reduced ILC frequencies, suggesting that impaired IL-7 responses of ILCs might contribute to incomplete reconstitution of ILCs under effective anti-retroviral therapy. This is the first report comparing distribution and function of ILCs along the intestinal mucosa of the entire human gastrointestinal tract in HIV(+ and HIV(- individuals.

  1. The Decrease of Peripheral Blood CD4+ T Cells Indicates Abdominal Compartment Syndrome in Severe Acute Pancreatitis.

    Directory of Open Access Journals (Sweden)

    Yao Liu

    Full Text Available Few data are available on the role of T lymphocytes and inflammatory cytokines in abdominal compartment syndrome (ACS in severe acute pancreatitis (SAP. We conducted a retrospective study to assess the risk factors associated with ACS in SAP.A total of 76 SAP patients who were admitted within 24 hours after symptom onset in our study. There were 36 patients suffering from ACS and 40 from intra-abdominal hypertension (IAH. On the 1st, 3rd and 7th days after hospital admission, the following variables were assessed: serum value of C-reactive protein (CRP, and the proportions of peripheral CD4+ and CD8+ T lymphocytes. Acute Physiology and Chronic Health Evaluation II (APACHE II score, and computed tomography severity index (CTSI score were assessed on days 1 and 7 after hospitalization.Compared with the patients with IAH, ACS patients showed statistically higher CRP value on 7th day after hospital admission, proportions of CD4+ T cells on days 1, 3, 7 and CD4+/CD8+ ratio on day 1 were significantly lower (P < 0.05, respectively. A CD4+ T cell proportion of 30.3% on the 1st day indicated ACS with an area under the curve (AUC of 0.774, a sensitivity with 82.5% and specificity with 72.0%, respectively. Sensitivity/specificity for predicting ACS in SAP patients on day 1 was 70.0%/68.0% for CD4+/CD8+ ratio, 72.2%/65.0% for APACHE II score.The reduction of peripheral blood CD4+ T lymphocytes is associated with ACS in SAP, and may act as a potential predictor of ACS in SAP.

  2. Cell biology of anaerobic ammonium-oxidizing bacteria

    NARCIS (Netherlands)

    Niftrik, L.A.M.P. van

    2008-01-01

    Anammox bacteria perform anaerobic ammonium oxidation to dinitrogen gas and belong to the phylum Planctomycetes. Whereas most Prokaryotes consist of one compartment, the cytoplasm bounded by the cytoplasmic membrane and cell wall, the species within this phylum are compartmentalized by intracellular

  3. Sequestration of highly expressed mRNAs in cytoplasmic granules, P-bodies, and stress granules enhances cell viability.

    Directory of Open Access Journals (Sweden)

    Anna Lavut

    Full Text Available Transcriptome analyses indicate that a core 10%-15% of the yeast genome is modulated by a variety of different stresses. However, not all the induced genes undergo translation, and null mutants of many induced genes do not show elevated sensitivity to the particular stress. Elucidation of the RNA lifecycle reveals accumulation of non-translating mRNAs in cytoplasmic granules, P-bodies, and stress granules for future regulation. P-bodies contain enzymes for mRNA degradation; under stress conditions mRNAs may be transferred to stress granules for storage and return to translation. Protein degradation by the ubiquitin-proteasome system is elevated by stress; and here we analyzed the steady state levels, decay, and subcellular localization of the mRNA of the gene encoding the F-box protein, UFO1, that is induced by stress. Using the MS2L mRNA reporter system UFO1 mRNA was observed in granules that colocalized with P-bodies and stress granules. These P-bodies stored diverse mRNAs. Granules of two mRNAs transported prior to translation, ASH1-MS2L and OXA1-MS2L, docked with P-bodies. HSP12 mRNA that gave rise to highly elevated protein levels was not observed in granules under these stress conditions. ecd3, pat1 double mutants that are defective in P-body formation were sensitive to mRNAs expressed ectopically from strong promoters. These highly expressed mRNAs showed elevated translation compared with wild-type cells, and the viability of the mutants was strongly reduced. ecd3, pat1 mutants also exhibited increased sensitivity to different stresses. Our interpretation is that sequestration of highly expressed mRNAs in P-bodies is essential for viability. Storage of mRNAs for future regulation may contribute to the discrepancy between the steady state levels of many stress-induced mRNAs and their proteins. Sorting of mRNAs for future translation or decay by individual cells could generate potentially different phenotypes in a genetically identical

  4. Three major nucleolar proteins migrate from nucleolus to nucleoplasm and cytoplasm in root tip cells of Vicia faba L. exposed to aluminum.

    Science.gov (United States)

    Qin, Rong; Zhang, Huaning; Li, Shaoshan; Jiang, Wusheng; Liu, Donghua

    2014-09-01

    Results from our previous investigation indicated that Al could affect the nucleolus and induce extrusion of silver-staining nucleolar particles containing argyrophilic proteins from the nucleolus into the cytoplasm in root tip cells of Vicia faba L. So far, the nucleolar proteins involved have not been identified. It is well known that nucleophosmin (B23), nucleolin (C23), and fibrillarin are three major and multifunctional nucleolar proteins. Therefore, effects of Al on B23, C23, and fibrillarin in root tip cells of V. faba exposed to 100 μM Al for 48 h were observed and analyzed using indirect immunofluorescence microscopy and Western blotting. The results from this work demonstrated that after 100 μM of Al treatment for 48 h, B23 and C23 migrated from the nucleolus to the cytoplasm and fibrillarin from the nucleolus to the nucleoplasm. In some cells, fibrillarin was present only in the cytoplasm. Western blotting data revealed higher expression of the three major nucleolar proteins in Al-treated roots compared with the control and that the B23 content increased markedly. These findings confirmed our previous observations.

  5. Infection of human and non-human cells by a highly fusogenic primary CD4-independent HIV-1 isolate with a truncated envelope cytoplasmic tail

    International Nuclear Information System (INIS)

    Saha, Kunal; Yan Hui; Nelson, Julie A.E.; Zerhouni-Layachi, Bouchra

    2005-01-01

    Truncation of the envelope cytoplasmic tail has enabled FIV, SIV, and some laboratory HIV-1 strains to acquire broader cellular tropism and enhanced fusogenicity. Here we have characterized a primary CD4-independent HIV-1 isolate (92UG046-T8) with a truncated cytoplasmic tail that was able to infect and induce syncytia in primary lymphocytes from human, chimpanzee, and monkey, as well as CD4-negative cell lines from human and monkey. Increased syncytia were also noticeable with 293 cells expressing the cloned envelope from the 92UG046-T8 isolate suggesting envelope-mediated cellular fusion. Except pooled serum from HIV-1-infected individuals, monoclonal anti-envelope antibodies or antibodies/antagonists against CD4, CXCR4, and CCR5 were not able to prevent infection by the 92UG046-T8 isolate. This is the first report showing a primary HIV-1 variant with truncated cytoplasmic tail which is highly fusogenic and can infect a broad range of cells from human and non-human origins. In vivo evolution of similar HIV-1 mutants may have important implications in AIDS pathogenesis

  6. Reconstitution of the myeloid and lymphoid compartments after the transplantation of autologous and genetically modified CD34+ bone marrow cells, following gamma irradiation in cynomolgus macaques

    Directory of Open Access Journals (Sweden)

    Auregan Gwenaelle

    2008-06-01

    Full Text Available Abstract Background Prolonged, altered hematopoietic reconstitution is commonly observed in patients undergoing myeloablative conditioning and bone marrow and/or mobilized peripheral blood-derived stem cell transplantation. We studied the reconstitution of myeloid and lymphoid compartments after the transplantation of autologous CD34+ bone marrow cells following gamma irradiation in cynomolgus macaques. Results The bone marrow cells were first transduced ex vivo with a lentiviral vector encoding eGFP, with a mean efficiency of 72% ± 4%. The vector used was derived from the simian immunodeficiency lentivirus SIVmac251, VSV-g pseudotyped and encoded eGFP under the control of the phosphoglycerate kinase promoter. After myeloid differentiation, GFP was detected in colony-forming cells (37% ± 10%. A previous study showed that transduction rates did not differ significantly between colony-forming cells and immature cells capable of initiating long-term cultures, indicating that progenitor cells and highly immature hematopoietic cells were transduced with similar efficiency. Blood cells producingeGFP were detected as early as three days after transplantation, and eGFP-producing granulocyte and mononuclear cells persisted for more than one year in the periphery. Conclusion The transplantation of CD34+ bone marrow cells had beneficial effects for the ex vivo proliferation and differentiation of hematopoietic progenitors, favoring reconstitution of the T- and B-lymphocyte, thrombocyte and red blood cell compartments.

  7. In vitro differentiation of bone marrow stromal cells into neurons and glial cells and differential protein expression in a two-compartment bone marrow stromal cell/neuron co-culture system.

    Science.gov (United States)

    Qi, Xu; Shao, Ming; Peng, Haisheng; Bi, Zhenggang; Su, Zhiqiang; Li, Hulun

    2010-07-01

    This study was performed to establish a bone marrow stromal cell (BMSC)/neuron two-compartment co-culture model in which differentiation of BMSCs into neurons could occur without direct contact between the two cell types, and to investigate protein expression changes during differentiation of this entirely BMSC-derived population. Cultured BMSCs isolated from Wistar rats were divided into three groups: BMSC culture, BMSC/neuron co-culture and BMSC/neuron two-compartment co-culture. Cells were examined for neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) expression. The electrophysiological behavior of the BMSCs was examined using patch clamping. Proteins that had significantly different expression levels in BMSCs cultured alone and co-cultured with neurons were studied using a protein chip-mass spectroscopy technique. Expression of NSE and GFAP were significantly higher in co-culture cells than in two-compartment co-culture cells, and significantly higher in both co-culture groups than in BMSCs cultured alone. Five proteins showed significant changes in expression during differentiation: TIP39_RAT and CALC_RAT underwent increases, and INSL6_RAT, PNOC_RAT and PCSK1_RAT underwent decreases in expression. We conclude that BMSCs can differentiate into neurons during both contact co-culture with neurons and two-compartment co-culture with neurons. The rate at which BMSCs differentiated into neurons was higher in contact co-culture than in non-contact co-culture.

  8. Protein synthesis and the recovery of both survival and cytoplasmic "petite" mutation in ultraviolet-treated yeast cells. II. Mitochondrial protein synthesis.

    Science.gov (United States)

    Heude, M; Chanet, R

    1975-04-01

    The contribution of mitochondrial proteins in the repair of UV-induced lethal and cytoplasmic genetic damages was studied in dark liquid held exponential and stationary phase yeast cells. This was performed by using the specific inhibitors, erythromycin (ER) anc chloramphenicol (CAP). It was shown that mitochondrial proteins are involved in the recovery of stationary phase cells. Mitochondrial proteins are partly implicated in the mechanisms leading to the restoration of the (see article) genotype in UV-irradiated dark liquid held exponential phase cells. Here again, in stationary phase cells, mitochondrial enzymes do not seem to participate in the negative liquid holding (NLH) process for the (see article) induction, as shown by inhibiting mitochondrial protein synthesis or both mitochondrial and nuclear protein synthesis. When cells are grown in glycerol, the response after dark liquid holding of UV-treated cells in the different growth stages are similar to that found for glucose-grown cells. In other words, the fate of cytoplasmic genetic damage, in particular, is not correlated with the repressed or derepressed state of the mitochondria.

  9. Epidermal Growth Factor Cytoplasmic Domain Affects ErbB Protein Degradation by the Lysosomal and Ubiquitin-Proteasome Pathway in Human Cancer Cells

    Directory of Open Access Journals (Sweden)

    Aleksandra Glogowska

    2012-05-01

    Full Text Available The cytoplasmic domains of EGF-like ligands, including EGF cytoplasmic domain (EGFcyt, have important biological functions. Using specific constructs and peptides of human EGF cytoplasmic domain, we demonstrate that EGFcyt facilitates lysosomal and proteasomal protein degradation, and this coincided with growth inhibition of human thyroid and glioma carcinoma cells. EGFcyt and exon 22–23-encoded peptide (EGF22.23 enhanced procathepsin B (procathB expression and procathB-mediated lysosomal degradation of EGFR/ErbB1 as determined by inhibitors for procathB and the lysosomal ATPase inhibitor BafA1. Presence of mbEGFctF, EGFcyt, EGF22.23, and exon 23-encoded peptides suppressed the expression of the deubiqitinating enzyme ubiquitin C-terminal hydrolase-L1 (UCH-L1. This coincided with hyperubiquitination of total cellular proteins and ErbB1/2 and reduced proteasome activity. Upon small interfering RNA-mediated silencing of endogenously expressed UCH-L1, a similar hyperubiquitinylation phenotype, reduced ErbB1/2 content, and attenuated growth was observed. The exon 23-encoded peptide region of EGFcyt was important for these biologic actions. Structural homology modeling of human EGFcyt showed that this molecular region formed an exposed surface loop. Peptides derived from this EGFcyt loop structure may aid in the design of novel peptide therapeutics aimed at inhibiting growth of cancer cells.

  10. From lymphopoiesis to plasma cells differentiation, the age-related modifications of B cell compartment are influenced by "inflamm-ageing".

    Science.gov (United States)

    Bulati, Matteo; Caruso, Calogero; Colonna-Romano, Giuseppina

    2017-07-01

    Ageing is a complex process characterized by a general decline in physiological functions with increasing morbidity and mortality. The most important aspect of ageing is the chronic inflammatory status, named "inflamm-ageing", strictly associated with the deterioration of the immune function, termed "immunosenescence". Both are causes of increased susceptibility of elderly to infectious diseases, cancer, dementia, cardiovascular diseases and autoimmunity, and of a decreased response to vaccination. It has been widely demonstrated that ageing has a strong impact on the remodelling of the B cell branch of immune system. The first evident effect is the significant decrease in circulating B cells, primarily due to the reduction of new B cell coming from bone marrow (BM) progenitors, as inflammation directly impacts on B lymphopoiesis. Besides, in aged individuals, there is a shift from naïve to memory immunoglobulins production, accompanied by the impaired ability to produce high affinity protective antibodies against newly encountered antigens. This is accompanied by the increase of expanded clones of B cells, which correlates with poor health status. Age-related modifications also occur in naïve/memory B cells subsets. Indeed, in the elderly, there is a reduction of naïve B cells, accompanied by the expansion of memory B cells that show a senescence-associated phenotype. Finally, elderly show the impaired ability of memory B cells to differentiate into plasma cells. It can be concluded that inflammation is the leading cause of the age-related impairment of B cell compartment, which play certainly a key role in the development of age-related diseases. This makes study of B cells in the aged an important tool for monitoring immunosenescence, chronic inflammatory disorders and the effectiveness of vaccines or pharmacological therapies. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Cell-Free and Cell-Based Approaches to Explore the Roles of Host Membranes and Lipids in the Formation of Viral Replication Compartment Induced by Tombusviruses.

    Science.gov (United States)

    Nagy, Peter D; Pogany, Judit; Xu, Kai

    2016-03-03

    Plant positive strand RNA viruses are intracellular infectious agents that take advantage of cellular lipids and membranes to support replication and protect viral RNA from degradation by host antiviral responses. In this review, we discuss how Tomato bushy stunt virus (TBSV) co-opts lipid transfer proteins and modulates lipid metabolism and transport to facilitate the assembly of the membrane-bound viral replicase complexes within intricate replication compartments. Identification and characterization of the proviral roles of specific lipids and proteins involved in lipid metabolism based on results from yeast (Saccharomyces cerevisiae) model host and cell-free approaches are discussed. The review also highlights the advantage of using liposomes with chemically defined composition to identify specific lipids required for TBSV replication. Remarkably, all the known steps in TBSV replication are dependent on cellular lipids and co-opted membranes.

  12. Arrest of cytoplasmic streaming induces algal proliferation in green paramecia.

    Directory of Open Access Journals (Sweden)

    Toshiyuki Takahashi

    Full Text Available A green ciliate Paramecium bursaria, bearing several hundreds of endosymbiotic algae, demonstrates rotational microtubule-based cytoplasmic streaming, in which cytoplasmic granules and endosymbiotic algae flow in a constant direction. However, its physiological significance is still unknown. We investigated physiological roles of cytoplasmic streaming in P. bursaria through host cell cycle using video-microscopy. Here, we found that cytoplasmic streaming was arrested in dividing green paramecia and the endosymbiotic algae proliferated only during the arrest of cytoplasmic streaming. Interestingly, arrest of cytoplasmic streaming with pressure or a microtubule drug also induced proliferation of endosymbiotic algae independently of host cell cycle. Thus, cytoplasmic streaming may control the algal proliferation in P. bursaria. Furthermore, confocal microscopic observation revealed that a division septum was formed in the constricted area of a dividing paramecium, producing arrest of cytoplasmic streaming. This is a first report to suggest that cytoplasmic streaming controls proliferation of eukaryotic cells.

  13. Sepsis reveals compartment-specific responses in intestinal proliferation and apoptosis in transgenic mice whose enterocytes re-enter the cell cycle.

    Science.gov (United States)

    Lyons, John D; Klingensmith, Nathan J; Otani, Shunsuke; Mittal, Rohit; Liang, Zhe; Ford, Mandy L; Coopersmith, Craig M

    2017-12-01

    Cell production and death are tightly regulated in the rapidly renewing gut epithelium, with proliferation confined to crypts and apoptosis occurring in villi and crypts. This study sought to determine how stress alters these compartmentalized processes. Wild-type mice made septic via cecal ligation and puncture had decreased crypt proliferation and increased crypt and villus apoptosis. Fabpi -TAg mice expressing large T-antigen solely in villi had ectopic enterocyte proliferation with increased villus apoptosis in unmanipulated animals. Septic fabpi -TAg mice had an unexpected increase in villus proliferation compared with unmanipulated littermates, whereas crypt proliferation was decreased. Cell cycle regulators cyclin D1 and cyclin D2 were decreased in jejunal tissue in septic transgenic mice. In contrast, villus and crypt apoptosis were increased in septic fabpi -TAg mice. To examine the relationship between apoptosis and proliferation in a compartment-specific manner, fabpi -TAg mice were crossed with fabpl -Bcl-2 mice, resulting in expression of both genes in the villus but Bcl-2 alone in the crypt. Septic bi-transgenic animals had decreased crypt apoptosis but had a paradoxical increase in villus apoptosis compared with septic fabpi -TAg mice, associated with decreased proliferation in both compartments. Thus, sepsis unmasks compartment-specific proliferative and apoptotic regulation that is not present under homeostatic conditions.-Lyons, J. D., Klingensmith, N. J., Otani, S., Mittal, R., Liang, Z., Ford, M. L., Coopersmith, C. M. Sepsis reveals compartment-specific responses in intestinal proliferation and apoptosis in transgenic mice whose enterocytes re-enter the cell cycle. © FASEB.

  14. The female lower genital tract is a privileged compartment with IL-10 producing dendritic cells and poor Th1 immunity following Chlamydia trachomatis infection.

    Directory of Open Access Journals (Sweden)

    Ellen Marks

    Full Text Available While a primary genital tract infection with C. trachomatis stimulates partial-protection against re-infection, it may also result in severe inflammation and tissue destruction. Here we have dissected whether functional compartments exist in the genital tract that restrict Th1-mediated protective immunity. Apart from the Th1-subset, little is known about the role of other CD4(+ T cell subsets in response to a genital tract chlamydial infection. Therefore, we investigated CD4(+ T cell subset differentiation in the genital tract using RT-PCR for expression of critical transcription factors and cytokines in the upper (UGT and lower genital tract (LGT of female C57BL/6 mice in response to C. trachomatis serovar D infection. We found that the Th1 subset dominated the UGT, as IFN-γ and T-bet mRNA expression were high, while GATA-3 was low following genital infection with C. trachomatis serovar D. By contrast, IL-10 and GATA-3 mRNA dominated the LGT, suggesting the presence of Th2 cells. These functional compartments also attracted regulatory T cells (Tregs differently as increased FoxP3 mRNA expression was seen primarily in the UGT. Although IL-17A mRNA was somewhat up-regulated in the LGT, no significant change in RORγ-t mRNA expression was observed, suggesting no involvement of Th17 cells. The dichotomy between the LGT and UGT was maintained during infection by IL-10 because in IL-10-deficient mice the distinction between the two compartments was completely lost and a dramatic shift to the predominance of Th1 cells in the LGT occurred. Unexpectedly, the major source of IL-10 was CD11c(+ CD11b(+ DC, probably creating an anti-inflammatory privileged site in the LGT.

  15. Cytoplasmic Streaming - Skylab Student Experiment ED-63

    Science.gov (United States)

    1973-01-01

    This chart describes the Skylab student experiment (ED-63), Cytoplasmic Streaming, proposed by Cheryl A. Peitz of Arapahoe High School, Littleton, Colorado. Experiment ED-63 was to observe the effect of zero-gravity on cytoplasmic streaming in the aquatic plant named Elodea, commonly called water weed or water thyme. The phenomenon of cytoplasmic streaming is not well understood, but it is recognized as the circulation mechanism of the internal materials or cytoplasm of a cell. Cytoplasm is a gelatinous substance that has the ability to change its viscosity and flow, carrying various cell materials with it. The activity can be stimulated by sunlight or heat. In March 1972, NASA and the National Science Teachers Association selected 25 experiment proposals for flight on Skylab. Science advisors from the Marshall Space Flight Center aided and assisted the students in developing the proposals for flight on Skylab.

  16. Hydrodynamic property of the cytoplasm is sufficient to mediate cytoplasmic streaming in the Caenorhabiditis elegans embryo

    Science.gov (United States)

    Niwayama, Ritsuya; Shinohara, Kyosuke; Kimura, Akatsuki

    2011-01-01

    Cytoplasmic streaming is a type of intracellular transport widely seen in nature. Cytoplasmic streaming in Caenorhabditis elegans at the one-cell stage is bidirectional; the flow near the cortex (“cortical flow”) is oriented toward the anterior, whereas the flow in the central region (“cytoplasmic flow”) is oriented toward the posterior. Both cortical flow and cytoplasmic flow depend on non-muscle-myosin II (NMY-2), which primarily localizes in the cortex. The manner in which NMY-2 proteins drive cytoplasmic flow in the opposite direction from remote locations has not been fully understood. In this study, we demonstrated that the hydrodynamic properties of the cytoplasm are sufficient to mediate the forces generated by the cortical myosin to drive bidirectional streaming throughout the cytoplasm. We quantified the flow velocities of cytoplasmic streaming using particle image velocimetry (PIV) and conducted a three-dimensional hydrodynamic simulation using the moving particle semiimplicit method. Our simulation quantitatively reconstructed the quantified flow velocity distribution resolved through PIV analysis. Furthermore, our PIV analyses detected microtubule-dependent flows during the pronuclear migration stage. These flows were reproduced via hydrodynamic interactions between moving pronuclei and the cytoplasm. The agreement of flow dynamics in vivo and in simulation indicates that the hydrodynamic properties of the cytoplasm are sufficient to mediate cytoplasmic streaming in C. elegans embryos. PMID:21730185

  17. Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

    Directory of Open Access Journals (Sweden)

    Q. Sun

    2012-10-01

    Full Text Available The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3 can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.

  18. p38α phosphorylates serine 258 within the cytoplasmic domain of tissue factor and prevents its incorporation into cell-derived microparticles.

    Science.gov (United States)

    Ettelaie, Camille; Elkeeb, Azza M; Maraveyas, Anthony; Collier, Mary Elizabeth W

    2013-03-01

    We previously showed that the phosphorylation of Ser253 within the cytoplasmic domain of human tissue factor (TF) initiates the incorporation and release of this protein into cell-derived microparticles. Furthermore, subsequent phosphorylation of Ser258 terminates this process. However, the identity of the kinase responsible for the phosphorylation of Ser258 and mode of action of this enzyme remain unknown. In this study, p38α was identified as the proline-directed kinase capable of phosphorylating Ser258 specifically, and without any detectable activity towards Ser253. Furthermore, using synthetic peptides, it was shown that the Km for the reaction decreased by approximately 10 fold on substitution of Ser253 with phospho-Ser253. Either inhibition of p38 using SB202190 or knockdown of p38α expression in coronary artery endothelial cells overexpressing wild-type TF, resulted in decreased phosphorylation of Ser258, following activation of cells with PAR2-agonist peptide (PAR2-AP). In agreement with our previous data, inhibition of phosphorylation of this residue maintained the release of TF. Activation of PAR2 in cells transfected to overexpress TF, resulted in two separate peaks of p38 activity at approximately 40 and 120 min post-activation. Furthermore, overexpression of Ala253-substituted TF enhanced the second p38 activation peak. However, the second peak was absent in cells devoid of TF or in cells overexpressing the Asp253-substituted TF. Our data clearly identifies p38α as a kinase capable of phosphorylating Ser258 within the cytoplasmic domain of TF. Moreover, it appears that the presence of TF within the cells regulates the late activation of p38 and consequently the termination of TF release into microparticles. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

    International Nuclear Information System (INIS)

    Sun, Q.; Xiong, J.; Lu, J.; Xu, S.; Li, Y.; Zhong, X.P.; Gao, G.K.; Liu, H.Q.

    2012-01-01

    The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy

  20. Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Q. [Department of Hyperbaric Medicine, No. 401 Hospital of PLA, Qingdao (China); Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China); Xiong, J. [Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China); Lu, J. [Office of Medical Education, Training Department, Second Military Medical University, Shanghai (China); Xu, S. [Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China); Li, Y. [State Food and Drug Administration of China,Huangdao Branch, Qingdao (China); Zhong, X.P.; Gao, G.K. [Department of Hyperbaric Medicine, No. 401 Hospital of PLA, Qingdao (China); Liu, H.Q. [2Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China)

    2012-06-22

    The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.

  1. Heavy subunit of cell surface Gal/GalNAc lectin (Hgl) undergoes degradation via endo-lysosomal compartments in Entamoeba histolytica.

    Science.gov (United States)

    Verma, Kuldeep; Datta, Sunando

    2017-06-14

    The human gut parasite Entamoeba histolytica uses a multifunctional virulence factor, Hgl, a cell surface transmembrane receptor subunit of Gal/GalNAc lectin that contributes to adhesion, invasion, cytotoxicity and immune response in the host. At present, the physiologic importance of Hgl receptor is mostly known for pathogenicity of E. histolytica. However, the molecular mechanisms of Hgl trafficking events and their association with the intracellular membrane transport machinery are largely unknown. We used biochemical and microscopy-based assays to understand the Hgl trafficking in the amoebic trophozoites. Our results suggest that the Hgl is constitutively degraded through delivery into amoebic lysosome-like compartments. Further, we also observed that the Hgl was significantly colocalized with amoebic Rab GTPases such as EhRab5, EhRab7A, and EhRab11B. While, we detected association of Hgl with all these Rab GTPases in early vacuolar compartments, only EhRab7A remains associated with Hgl till its transport to amoebic lysosome-like compartments.

  2. TB-IRIS and remodelling of the T cell compartment in highly immunosuppressed HIV+ patients with TB: the CAPRI T (ANRS-12614) study

    Science.gov (United States)

    Haridas, V.; Pean, P.; Jasenosky, L.D.; Madec, Y.; Laureillard, D.; Sok, T.; Sath, S.; Borand, L.; Marcy, O.; Chan, S.; Tsitsikov, E.; Delfraissy, J.-F.; Blanc, F.-X.; Goldfeld, A.E.

    2015-01-01

    Objective To investigate the impact of tuberculosis (TB)-associated immune reconstitution syndrome (IRIS) upon immunological recovery and the T cell compartment after initiation of TB and antiretroviral therapy (ART). Design and methods We prospectively evaluated T cell immunophenotypes by flow cytometry and cytokines by Luminex assays in a subset (n=154) of highly immunosuppressed HIV+ patients with TB from the CAMELIA randomized clinical trial. We compared findings from patients who developed TB-IRIS to findings from patients who did not develop TB-IRIS. Data were evaluated with mixed effect linear regression, Kaplan-Meier estimates, and Wilcoxon rank sum tests, and q-values were calculated to control for multiple comparisons. Results Development of TB-IRIS was associated with significantly greater pre-ART frequencies of HLA-DR+CD45RO+CD4+, CCR5+CD4+, OX40+CD4+, and Fas+ effector memory (EM) CD8+ T cells, and significantly elevated levels of plasma IL-6, IL-1β, IL-8, and IL-10 and viral load. Post-ART initiation, EM CD4+ and Fas+ EM CD4+ T cell frequencies significantly expanded, and central memory (CM) CD4+ T cell frequencies significantly contracted in patients who experienced TB-IRIS. By week 34 post-TB treatment initiation, EM/CM CD4+ T cell ratios were markedly higher in TB-IRIS versus non-TB-IRIS patients. Conclusions A distinct pattern of pre-ART T cell and cytokine markers appear to poise the immune response to develop TB-IRIS. Experience of TB-IRIS is then associated with long-term remodeling of the CD4+ T cell memory compartment towards an EM-dominated phenotype. We speculate that these pre- and post-ART TB-IRIS-associated immune parameters may contribute to superior immune control of TB/HIV co-infection and better clinical outcome. PMID:25486415

  3. Increased infectivity in human cells and resistance to antibody-mediated neutralization by truncation of the SIV gp41 cytoplasmic tail

    Directory of Open Access Journals (Sweden)

    Takeo eKuwata

    2013-05-01

    Full Text Available The role of antibodies in protecting the host from human immunodeficiency virus type 1 (HIV-1 infection is of considerable interest, particularly because the RV144 trial results suggest that antibodies contribute to protection. Although infection of nonhuman primates with simian immunodeficiency virus (SIV is commonly used as an animal model of HIV-1 infection, the viral epitopes that elicit potent and broad neutralizing antibodies to SIV have not been identified. We isolated a monoclonal antibody (MAb B404 that potently and broadly neutralizes various SIV strains. B404 targets a conformational epitope comprising the V3 and V4 loops of Env that intensely exposed when Env binds CD4. B404-resistant variants were obtained by passaging viruses in the presence of increasing concentration of B404 in PM1/CCR5 cells. Genetic analysis revealed that the Q733stop mutation, which truncates the cytoplasmic tail of gp41, was the first major substitution in Env during passage. The maximal inhibition by B404 and other MAbs were significantly decreased against a recombinant virus with a gp41 truncation compared with the parental SIVmac316. This indicates that the gp41 truncation was associated with resistance to antibody-mediated neutralization. The infectivities of the recombinant virus with the gp41 truncation were 7900-fold, 1000-fold, and 140-fold higher than those of SIVmac316 in PM1, PM1/CCR5, and TZM-bl cells, respectively. Immunoblotting analysis revealed that the gp41 truncation enhanced the incorporation of Env into virions. The effect of the gp41 truncation on infectivity was not obvious in the HSC-F macaque cell line, although the resistance of viruses harboring the gp41 truncation to neutralization was maintained. These results suggest that viruses with a truncated gp41 cytoplasmic tail were selected by increased infectivity in human cells and by acquiring resistance to neutralizing antibody.

  4. Quantitative proteomic view on secreted, cell surface-associated, and cytoplasmic proteins of the methicillin-resistant human pathogen Staphylococcus aureus under iron-limited conditions.

    Science.gov (United States)

    Hempel, Kristina; Herbst, Florian-Alexander; Moche, Martin; Hecker, Michael; Becher, Dörte

    2011-04-01

    Staphylococcus aureus is capable of colonizing and infecting humans by its arsenal of surface-exposed and secreted proteins. Iron-limited conditions in mammalian body fluids serve as a major environmental signal to bacteria to express virulence determinants. Here we present a comprehensive, gel-free, and GeLC-MS/MS-based quantitative proteome profiling of S. aureus under this infection-relevant situation. (14)N(15)N metabolic labeling and three complementing approaches were combined for relative quantitative analyses of surface-associated proteins. The surface-exposed and secreted proteome profiling approaches comprise trypsin shaving, biotinylation, and precipitation of the supernatant. By analysis of the outer subproteomic and cytoplasmic protein fraction, 1210 proteins could be identified including 221 surface-associated proteins. Thus, access was enabled to 70% of the predicted cell wall-associated proteins, 80% of the predicted sortase substrates, two/thirds of lipoproteins and more than 50% of secreted and cytoplasmic proteins. For iron-deficiency, 158 surface-associated proteins were quantified. Twenty-nine proteins were found in altered amounts showing particularly surface-exposed proteins strongly induced, such as the iron-regulated surface determinant proteins IsdA, IsdB, IsdC and IsdD as well as lipid-anchored iron compound-binding proteins. The work presents a crucial subject for understanding S. aureus pathophysiology by the use of methods that allow quantitative surface proteome profiling.

  5. ACUTE COMPARTMENT SYNDROME

    African Journals Online (AJOL)

    muscle destruction, muscle fibrosis, contractures and permanent disability and at worst case scenario of amputation (3,4). As reported by Frink et al (3) on their study on acute compartment syndrome it can occur even when there is no fracture. Also general surgeons have reported acute compartment syndrome.

  6. Clinical implications in the shift of syndecan-1 expression from the cell membrane to the cytoplasm in bladder cancer

    International Nuclear Information System (INIS)

    Miyake, Makito; Lawton, Adrienne; Dai, Yunfeng; Chang, Myron; Mengual, Lourdes; Alcaraz, Antonio; Goodison, Steve; Rosser, Charles J

    2014-01-01

    To determine the diagnostic and prognostic capability of urinary and tumoral syndecan-1 (SDC-1) levels in patients with cancer of the urinary bladder. SDC-1 levels were quantitated by enzyme-linked immunosorbent assay (ELISA) in 308 subjects (102 cancer subjects and 206 non-cancer subjects) to assess its diagnostic capabilities in voided urine. The performance of SDC-1 was evaluated using the area under the curve of a receiver operating characteristic curve. In addition, immunohistochemical (IHC) staining assessed SDC-1 protein expression in 193 bladder specimens (185 cancer subjects and 8 non-cancer subjects). Outcomes were correlated to SDC-1 levels. Mean urinary levels of SDC-1 did not differ between the cancer subjects and the non-cancer subjects, however, the mean urinary levels of SDC-1 were reduced in high-grade compared to low-grade disease (p < 0.0001), and in muscle invasive bladder cancer (MIBC) compared to non-muscle invasive bladder cancer (NMIBC) (p = 0.005). Correspondingly, preliminary data note a shift from a membranous cellular localization of SDC-1 in normal tissue, low-grade tumors and NMIBC, to a distinctly cytoplasmic localization in high-grade tumors and MIBC was observed in tissue specimens. Alone urinary SDC-1 may not be a diagnostic biomarker for bladder cancer, but its urinary levels and cellular localization were associated with the differentiation status of patients with bladder tumors. Further studies are warranted to define the potential role for SDC-1 in bladder cancer progression

  7. Tumor-Derived Microvesicles Modulate Antigen Cross-Processing via Reactive Oxygen Species-Mediated Alkalinization of Phagosomal Compartment in Dendritic Cells

    Directory of Open Access Journals (Sweden)

    Federico Battisti

    2017-09-01

    Full Text Available Dendritic cells (DCs are the only antigen-presenting cells able to prime naïve T cells and cross-prime antigen-specific CD8+ T cells. Their functionality is a requirement for the induction and maintenance of long-lasting cancer immunity. Albeit intensively investigated, the in vivo mechanisms underlying efficient antigen cross-processing and presentation are not fully understood. Several pieces of evidence indicate that antigen transfer to DCs mediated by microvesicles (MVs enhances antigen immunogenicity. This mechanism is also relevant for cross-presentation of those tumor-associated glycoproteins such as MUC1 that are blocked in HLA class II compartment when internalized by DCs as soluble molecules. Here, we present pieces of evidence that the internalization of tumor-derived MVs modulates antigen-processing machinery of DCs. Employing MVs derived from ovarian cancer ascites fluid and established tumor cell lines, we show that MV uptake modifies DC phagosomal microenvironment, triggering reactive oxygen species (ROS accumulation and early alkalinization. Indeed, tumor MVs carry radical species and the MV uptake by DCs counteracts the chemically mediated acidification of the phagosomal compartment. Further pieces of evidence suggest that efficacious antigen cross-priming of the MUC1 antigen carried by the tumor MVs results from the early signaling induced by MV internalization and the function of the antigen-processing machinery of DCs. These results strongly support the hypothesis that tumor-derived MVs impact antigen immunogenicity by tuning the antigen-processing machinery of DCs, besides being carrier of tumor antigens. Furthermore, these findings have important implications for the exploitation of MVs as antigenic cell-free immunogen for DC-based therapeutic strategies.

  8. The Protective Effect of Cell Wall and Cytoplasmic Fraction of Selenium Enriched Yeast on 1, 2-Dimethylhydrazine-induced Damage in Liver

    Directory of Open Access Journals (Sweden)

    Mitra Dadrass

    2014-02-01

    Full Text Available Background: 1, 2-Dimethylhydrazine (DMH enhances lipid peroxidation rate by tumor mitochondria than normal tissue counterpart and causes many disorders in antioxidant system in liver. It also increases the level of enzymes that metabolize toxin in liver and colon. The aim of this study was to evaluate the alteration of liver and its enzymes after DMH injection and evaluate protective effect of cell wall and cytoplasmic fractions of Saccharomyces cereviseae enriched with selenium (Se on these tissues. Materials and Methods: Forty eight female rats were prepared and acclimatized to the laboratory conditions for two weeks, and all animals received 1, 2- dimethyl hydrazine chloride (40 mg/kg body weight twice a week for 4 weeks except healthy control. At first colon carcinoma (aberrant crypt foci confirmed by light microscope. Then the changes resulting from injection of DMH on liver of animals in initial and advanced stages of colon cancer were examined. In addition, the protective effect of cell wall and cytoplasmic fractions of Selenium-enriched S. cerevisiae were investigated in two phases. First phase in initial stage and second phase in advanced stage of colon cancer were performed respectively. Forty weeks following the first DMH injection, all survived animals were sacrificed. Then, colon and liver removed and exsanguinated by heart puncture. For measuring the levels of enzymes (AST, ALT, and ALP, a commercial kit (Parsazmoon, Iran and an autoanalyzer (BT 3000 Pluse, Italy were used. Results: The results showed that subcutaneous injection of DMH increased the ALT, AST, and ALP levels up to 78.5, 161.38, and 275.88 U/L compared to the control, respectively. Moreover, statistical analysis in both phases of experiment revealed that the enzyme levels were decreased in the treated groups in comparison with the DMH-injected group, while the levels of these enzymes were lower in the control group. Conclusion: It should be concluded that

  9. A role for complexes of survival of motor neurons (SMN) protein with gemins and profilin in neurite-like cytoplasmic extensions of cultured nerve cells

    International Nuclear Information System (INIS)

    Sharma, Aarti; Lambrechts, Anja; Le thi Hao; Le, Thanh T.; Sewry, Caroline A.; Ampe, Christophe; Burghes, Arthur H.M.; Morris, Glenn E.

    2005-01-01

    Spinal muscular atrophy (SMA) is caused by reduced levels of SMN (survival of motor neurons protein) and consequent loss of motor neurons. SMN is involved in snRNP transport and nuclear RNA splicing, but axonal transport of SMN has also been shown to occur in motor neurons. SMN also binds to the small actin-binding protein, profilin. We now show that SMN and profilin II co-localise in the cytoplasm of differentiating rat PC12 cells and in neurite-like extensions, especially at their growth cones. Many components of known SMN complexes were also found in these extensions, including gemin2 (SIP-1), gemin6, gemin7 and unrip (unr-interacting protein). Coilin p80 and Sm core protein immunoreactivity, however, were seen only in the nucleus. SMN is known to associate with β-actin mRNA and specific hnRNPs in axons and in neurite extensions of cultured nerve cells, and SMN also stimulates neurite outgrowth in cultures. Our results are therefore consistent with SMN complexes, rather than SMN alone, being involved in the transport of actin mRNPs along the axon as in the transport of snRNPs into the nucleus by similar SMN complexes. Antisense knockdown of profilin I and II isoforms inhibited neurite outgrowth of PC12 cells and caused accumulation of SMN and its associated proteins in cytoplasmic aggregates. BIAcore studies demonstrated a high affinity interaction of SMN with profilin IIa, the isoform present in developing neurons. Pathogenic missense mutations in SMN, or deletion of exons 5 and 7, prevented this interaction. The interaction is functional in that SMN can modulate actin polymerisation in vitro by reducing the inhibitory effect of profilin IIa. This suggests that reduced SMN in SMA might cause axonal pathfinding defects by disturbing the normal regulation of microfilament growth by profilins

  10. The D Domain of LRRC4 anchors ERK1/2 in the cytoplasm and competitively inhibits MEK/ERK activation in glioma cells

    Directory of Open Access Journals (Sweden)

    Zeyou Wang

    2016-11-01

    Full Text Available Abstract Background As a well-characterized key player in various signal transduction networks, extracellular-signal-regulated kinase (ERK1/2 has been widely implicated in the development of many malignancies. We previously found that Leucine-rich repeat containing 4 (LRRC4 was a tumor suppressor and a negative regulator of the ERK/MAPK pathway in glioma tumorigenesis. However, the precise molecular role of LRRC4 in ERK signal transmission is unclear. Methods The interaction between LRRC4 and ERK1/2 was assessed by co-immunoprecipitation and GST pull-down assays in vivo and in vitro. We also investigated the interaction of LRRC4 and ERK1/2 and the role of the D domain in ERK activation in glioma cells. Results Here, we showed that LRRC4 and ERK1/2 interact via the D domain and CD domain, respectively. Following EGF stimuli, the D domain of LRRC4 anchors ERK1/2 in the cytoplasm and abrogates ERK1/2 activation and nuclear translocation. In glioblastoma cells, ectopic LRRC4 expression competitively inhibited the interaction of endogenous mitogen-activated protein kinase (MEK and ERK1/2. Mutation of the D domain decreased the LRRC4-mediated inhibition of MAPK signaling and its anti-proliferation and anti-invasion roles. Conclusions Our results demonstrated that the D domain of LRRC4 anchors ERK1/2 in the cytoplasm and competitively inhibits MEK/ERK activation in glioma cells. These findings identify a new mechanism underlying glioblastoma progression and suggest a novel therapeutic strategy by restoring the activity of LRRC4 to decrease MAPK cascade activation.

  11. On the entry of an emerging arbovirus into host cells: Mayaro virus takes the highway to the cytoplasm through fusion with early endosomes and caveolae-derived vesicles

    Directory of Open Access Journals (Sweden)

    Carlos A.M. Carvalho

    2017-04-01

    Full Text Available Mayaro virus (MAYV is an emergent sylvatic alphavirus in South America, related to sporadic outbreaks of a chikungunya-like human febrile illness accompanied by severe arthralgia. Despite its high potential for urban emergence, MAYV is still an obscure virus with scarce information about its infection cycle, including the corresponding early events. Even for prototypical alphaviruses, the cell entry mechanism still has some rough edges to trim: although clathrin-mediated endocytosis is quoted as the putative route, alternative paths as distinct as direct virus genome injection through the cell plasma membrane seems to be possible. Our aim was to clarify crucial details on the entry route exploited by MAYV to gain access into the host cell. Tracking the virus since its first contact with the surface of Vero cells by fluorescence microscopy, we show that its entry occurs by a fast endocytic process and relies on fusion with acidic endosomal compartments. Moreover, blocking clathrin-mediated endocytosis or depleting cholesterol from the cell membrane leads to a strong inhibition of viral infection, as assessed by plaque assays. Following this clue, we found that early endosomes and caveolae-derived vesicles are both implicated as target membranes for MAYV fusion. Our findings unravel the very first events that culminate in a productive infection by MAYV and shed light on potential targets for a rational antiviral therapy, besides providing a better comprehension of the entry routes exploited by alphaviruses to get into the cell.

  12. Creatine-creatine phosphate shuttle modeled as two-compartment system at different levels of creatine kinase activity

    DEFF Research Database (Denmark)

    Fedosov, Sergey

    1994-01-01

    In order to characterize ADP-ATP and creatine-creatine phosphate (Cr-CrP) shuttles a minimal mathematical model with two compartments and cyclic turnover of matter was designed. The 'mitochondrial' compartment contained 'ATP-synthase' and 'mitochondrial ereatine kinase' (mitCK). The 'cytoplasmic......' compartment consisted of 'ATPase', 'cytoplasmic creatine kinase' (cytCK) and an 'ADP-binding structure'. The exchange of metabolites between these compartments was limited. Different levels of cytCK and mitCK expression as welt as different exchange rate constants between the compartments were assigned...

  13. The Instrumentation of a Microfluidic Analyzer Enabling the Characterization of the Specific Membrane Capacitance, Cytoplasm Conductivity, and Instantaneous Young's Modulus of Single Cells.

    Science.gov (United States)

    Wang, Ke; Zhao, Yang; Chen, Deyong; Huang, Chengjun; Fan, Beiyuan; Long, Rong; Hsieh, Chia-Hsun; Wang, Junbo; Wu, Min-Hsien; Chen, Jian

    2017-06-19

    This paper presents the instrumentation of a microfluidic analyzer enabling the characterization of single-cell biophysical properties, which includes seven key components: a microfluidic module, a pressure module, an imaging module, an impedance module, two LabVIEW platforms for instrument operation and raw data processing, respectively, and a Python code for data translation. Under the control of the LabVIEW platform for instrument operation, the pressure module flushes single cells into the microfluidic module with raw biophysical parameters sampled by the imaging and impedance modules and processed by the LabVIEW platform for raw data processing, which were further translated into intrinsic cellular biophysical parameters using the code developed in Python. Based on this system, specific membrane capacitance, cytoplasm conductivity, and instantaneous Young's modulus of three cell types were quantified as 2.76 ± 0.57 μF/cm², 1.00 ± 0.14 S/m, and 3.79 ± 1.11 kPa for A549 cells ( n cell = 202); 1.88 ± 0.31 μF/cm², 1.05 ± 0.16 S/m, and 3.74 ± 0.75 kPa for 95D cells ( n cell = 257); 2.11 ± 0.38 μF/cm², 0.87 ± 0.11 S/m, and 5.39 ± 0.89 kPa for H460 cells ( n cell = 246). As a semi-automatic instrument with a throughput of roughly 1 cell per second, this prototype instrument can be potentially used for the characterization of cellular biophysical properties.

  14. The Fanconi anemia proteins FAA and FAC function in different cellular compartments to protect against cross-linking agent cytotoxicity.

    Science.gov (United States)

    Kruyt, F A; Youssoufian, H

    1998-10-01

    Fanconi anemia (FA) is an autosomal recessive disease characterized by chromosomal instability, bone marrow failure, and a high risk of developing malignancies. Although the disorder is genetically heterogeneous, all FA cells are defined by their sensitivity to the apoptosis-inducing effect of cross-linking agents, such as mitomycin C (MMC). The cloned FA disease genes, FAC and FAA, encode proteins with no homology to each other or to any known protein. We generated a highly specific antibody against FAA and found the protein in both the cytoplasm and nucleus of mammalian cells. By subcellular fractionation, FAA is also associated with intracellular membranes. To identify the subcellular compartment that is relevant for FAA activity, we appended nuclear export and nuclear localization signals to the carboxy terminus of FAA and enriched its localization in either the cytoplasm or the nucleus. Nuclear localization of FAA was both necessary and sufficient to correct MMC sensitivity in FA-A cells. In addition, we found no evidence for an interaction between FAA and FAC either in vivo or in vitro. Together with a previous finding that FAC is active in the cytoplasm but not in the nucleus, our results indicate that FAA and FAC function in separate subcellular compartments. Thus, FAA and FAC, if functionally linked, are more likely to be in a linear pathway rather than form a macromolecular complex to protect against cross-linker cytotoxicity.

  15. Modified expression of cytoplasmic isocitrate dehydrogenase electrophoretic isoforms in seminal plasma of men with sertoli-cell-only syndrome and seminoma.

    Science.gov (United States)

    Starita-Geribaldi, Mireille; Samson, Michel; Guigonis, Jean-Marie; Pointis, Georges; Fenichel, Patrick

    2008-06-01

    Two isoforms of human cytoplasmic isocitrate dehydrogenase (IDPc) of close molecular weights and different isoelectric points were identified in human seminal plasma (SP) by two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (MS). These two isoforms were detected in the normospermic men SP and their expressions were markedly altered in patients with testicular seminoma, the most frequent testicular germ cell cancer (TGCC): increase of the more acidic spot and decrease of the more basic one. Since oligospermia has been considered as a high risk pathological condition for developing a testicular cancer, the two IDPc isoforms were analyzed in SP of a group of secretory azoospermic patients. In this group the two spots displayed similar variations of expression to those observed in testicular seminoma. These results propose IDPc as a promising SP biomarker of testicular seminoma. Whether IDPc alteration in secretory azoospermia is predictive of testicular seminoma remains to be elucidated. (c) 2007 Wiley-Liss, Inc.

  16. Changes in protein metabolism after irradiation. Pt. 2. Protease activity, protease pattern, protein and free amino acids in cytoplasm and cell organelles of the rat liver after 600 R whole body X-irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Valet, G [Max-Planck-Institut fuer Biochemie, Muenchen (F.R. Germany). Abt. fuer Experimentelle Medizin

    1976-01-01

    The protease activity of cytoplasm and cell organelles of the rat liver against liver protein and hemoglobin as a substrate increases during an initial reaction phase on the first day after 600 R whole body x irradiation. This is probably a consequence of the degradation of cellular debris. The protein, the protease activity and the free amino acids of the cytoplasm and the cell organelles decrease during the disease phase on day 3 and 4 after irradiation. The protein loss of the liver is therefore not explained by an increased protease activity. The protease activity and the free amino acids are increased in the cytoplasm and the cell organelles during the regeneration phase of the organism between day 15 and 18 after irradiation.

  17. [THE PHYSICAL CHEMICAL, BIOLOGICAL BASICS OF CELLS ABSORPTION OF UNESTERIFIED FATTY ACIDS; ALBUMIN, CAVEOLIN, CLATHRIN AND LIPID-BINDING PROTEINS OF CYTOPLASM (THE LECTURE)].

    Science.gov (United States)

    Titov, V N; Shoibonov, B B

    2016-03-01

    From aposition of phylogenetic theory of general pathology, obesity and metabolic syndrome are pathology of fatty cells. However, the first is a pathology of phylogenetically early visceral fatty cells of omentum. They supply with substratum of energy realization of biologic function of trophology, homeostasis, endoecology and adaptation. The visceral fatty cells of omentum have no receptors to insulin and synthesize adaptively insulin and they are not characterized by biologic reaction of proliferation. The obesity is a pathology of late in phylogenesis subcutaneous adpocytes. They are insulin-dependent and supply with substratum of energy realization of one biologic function of locomotion--movement at the expense of constriction of cross-striated miocytes. The adipocytes in terms of adaptation synthesize humoral mediator adponectin and actively implement biologic function of proliferation. Under both aphysiologic conditions increases passive by gradient of concentration, absorption by cells albumin-unbound free fatty acids in unionized form in micellae's composition. The passive aphysiologic absorption of free fatty acids by cells which under intracellular compartmentalization don't oxidize mitochondria results in synthesis, accumulation of triglycerides in cytoplasm of cells which don't implement it physiologically. The aphysiologic absorption of free fatty acids by cells, their etherification in triglyceride, in particular, in phylogenetically late β-cells of islets and either late cardiomyocytes which fatty acids don't synthesize de novo results in development of aphysiologic processes and disorder of function. From position of biology, these cells in vivo are subjected to loss similar to apoptosis. The formation of corpuscles of apoptosis compromise biologic function of endoecology activating biologic reaction of inflammation.

  18. An easy compartment-less biofuel cell construction based on the physical co-inclusion of enzyme and mediator redox within pressed graphite discs

    Energy Technology Data Exchange (ETDEWEB)

    Cosnier, Serge [Department de Chimie Moleculaire UMR-5250, ICMG FR-2607, CNRS Universite Joseph Fourier, BP-53, 38041 Grenoble (France); Shan, Dan [Department de Chimie Moleculaire UMR-5250, ICMG FR-2607, CNRS Universite Joseph Fourier, BP-53, 38041 Grenoble (France); School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002 (China); Ding, Shou-Nian [Department de Chimie Moleculaire UMR-5250, ICMG FR-2607, CNRS Universite Joseph Fourier, BP-53, 38041 Grenoble (France); School of Chemistry and Chemical Engineering, Shouthest University, Nanjing 211189 (China)

    2010-02-15

    We report on the easy and fast immobilization of glucose oxidase (GOD) and laccase by mechanical compression with graphite particles to form disc electrodes. The electrical wiring of GOD and laccase was efficiently carried out by their co-inclusion with ferrocene (Fc) and 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonate) diammonium salt (ABTS) respectively. A glucose/air compartment-less biofuel cell was constructed based on the association of GOD-ferrocene-graphite disc and laccase-ABTS - graphite disc electrodes as bioanode and biocathode respectively. Such biofuel cell yielded a power density of 23 {mu}W cm{sup -2} at 0.33 V as well as an open-circuit voltage and a short-circuit current of 0.63 V and 166 {mu}A, respectively. (author)

  19. Live Cell Imaging Reveals Differential Modifications to Cytoplasmic Dynein Properties by Phospho- and Dephospho-mimic Mutations of the Intermediate Chain 2C S84

    Science.gov (United States)

    Blasier, Kiev R.; Humsi, Michael K.; Ha, Junghoon; Ross, Mitchell W.; Smiley, W. Russell; Inamdar, Nirja A.; Mitchell, David J.; Lo, Kevin W.-H.; Pfister, K. Kevin

    2014-01-01

    Cytoplasmic dynein is a multi-subunit motor protein responsible for intracellular cargo transport toward microtubule minus ends. There are multiple isoforms of the dynein intermediate chain (DYNC1I, IC) which is encoded by two genes. One way to regulate cytoplasmic dynein is by IC phosphorylation. The IC-2C isoform is expressed in all cells and the functional significance of phosphorylation on IC-2C serine 84 was investigated using live cell imaging of fluorescent protein-tagged wild type IC-2C (WT) and phospho- and dephospho-mimic mutant isoforms in axonal transport model systems. Both mutations modulated dynein functional properties. The dephospho-mimic mutant IC-2C S84A had greater co-localization with mitochondria than IC-2C wild-type (WT) or the phospho-mimic mutant IC-2C S84D. The dephospho-mimic mutant IC-2C S84A was also more likely to be motile than the phospho-mimic mutant IC-2C S84D or IC-2C WT. In contrast, the phospho-mimic mutant IC-2C S84D mutant was more likely to move in the retrograde direction than was the IC-2C S84A mutant. The phospho-mimic IC-2C S84D was also as likely as IC-2C WT to co-localize with mitochondria. Both the S84D phospho- and S84A, dephospho-mimic mutants were found to be capable of microtubule minus end directed (retrograde) movement in axons. They were also observed to be passively transported in the anterograde direction. These data suggest that the IC-2C S84 has a role in modulating dynein properties. PMID:24798412

  20. Characterization of Elements Regulating the Nuclear-to-Cytoplasmic Translocation of ICP0 in Late Herpes Simplex Virus 1 Infection.

    Science.gov (United States)

    Samrat, Subodh Kumar; Ha, Binh L; Zheng, Yi; Gu, Haidong

    2018-01-15

    Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It targets several host factors for proteasomal degradation and subsequently activates viral expression. ICP0 has a nuclear localization sequence and functions in the nucleus early during infection. However, later in infection, ICP0 is found solely in the cytoplasm. The molecular mechanism and biological function of the ICP0 nuclear-to-cytoplasmic translocation are not well understood. In this study, we sought to characterize elements important for this translocation. We found that (i) in human embryonic lung fibroblast (HEL) cells, ICP0 C-terminal residues 741 to 775 were necessary but not sufficient for the nuclear-to-cytoplasmic translocation; (ii) the loss of ICP0 E3 ubiquitin ligase activity, which led to defective viral replication in nonpermissive cells, also caused mutant ICP0 to be retained in the nucleus of HEL cells; (iii) in permissive U2OS cells, however, ICP0 lacking E3 ligase activity was translocated to the cytoplasm at a pace faster than that of wild-type ICP0, suggesting that nuclear retention of ICP0 occurs in an ICP0 E3 ligase-dependent manner; and (iv) the ICP0 C terminus and late viral proteins cooperate in order to overcome nuclear retention and stimulate ICP0 cytoplasmic translocation. Taken together, less ICP0 nuclear retention may contribute to the permissiveness of U2OS cells to HSV-1 in the absence of functional ICP0. IMPORTANCE A distinct characteristic for eukaryotes is the compartmentalization of cell metabolic pathways, which allows greater efficiency and specificity of cellular functions. ICP0 of HSV-1 is a multifunctional viral protein that travels through different compartments as infection progresses. Its main regulatory functions are carried out in the nucleus, but it is translocated to the cytoplasm late during HSV-1 infection. To understand the biological significance of cytoplasmic ICP0 in

  1. UV or X-irradiation increases the cytoplasmic accumulation of rhodamine 123 in various cancer cell lines

    International Nuclear Information System (INIS)

    Dumitriu, I.E.; Beyer, T.D.; Gaipl, U.S.; Kalden, J.R.; Herrmann, M.; Roedel, F.

    2003-01-01

    Purpose: Previous studies indicated that ATP-binding cassette (ABC) membrane transporters protect against UV-induced apoptosis. We investigated the effect of UVB and X-ray irradiation on the export function of these ABC transporters in primary lymphocytes and various cancer cell lines. Material and Methods: We used rhodamine accumulation assays in various human malignant cell lines and peripheral blood lymphocytes (PBL). Cells were irradiated with up to 960 mJ/cm 2 and up to 50 Gy of UVB and X-ray, respectively. Results: We demonstrated that UVB as well as X-ray irradiation inhibit the export function of the ABC transporters in a dose-dependent fashion. For PBL, this effect did not correlate with an apoptotic phenotype. In the case of the tumor cell lines, even though the irradiation-induced inhibition of membrane transporters was accompanied by phosphatidylserine exposure, only a minority of cells had lost their mitochondrial membrane potential during the observation period. Furthermore, we demonstrated that the inhibition of membrane transporters is not a general feature of apoptosis. Conclusion: Irradiation inhibits the export function of ABC transporters. Although some of the irradiated cells undergo apoptosis following irradiation, the inhibition is an unique feature accompanying irradiation and not a general hallmark of apoptotic cell death. The inhibition of drug export by irradiation may offer new potential for reverting multidrug resistance of cancer cells. (orig.)

  2. UV or X-irradiation increases the cytoplasmic accumulation of rhodamine 123 in various cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Dumitriu, I.E.; Beyer, T.D.; Gaipl, U.S.; Kalden, J.R.; Herrmann, M. [Inst. for Clinical Immunology, Dept. of Medicine III, Friedrich Alexander Univ. of Erlangen-Nuremberg, Erlangen (Germany); Roedel, F. [Dept. of Radiooncology, Univ. of Erlangen-Nuremberg, Erlangen (Germany)

    2003-08-01

    Purpose: Previous studies indicated that ATP-binding cassette (ABC) membrane transporters protect against UV-induced apoptosis. We investigated the effect of UVB and X-ray irradiation on the export function of these ABC transporters in primary lymphocytes and various cancer cell lines. Material and Methods: We used rhodamine accumulation assays in various human malignant cell lines and peripheral blood lymphocytes (PBL). Cells were irradiated with up to 960 mJ/cm{sup 2} and up to 50 Gy of UVB and X-ray, respectively. Results: We demonstrated that UVB as well as X-ray irradiation inhibit the export function of the ABC transporters in a dose-dependent fashion. For PBL, this effect did not correlate with an apoptotic phenotype. In the case of the tumor cell lines, even though the irradiation-induced inhibition of membrane transporters was accompanied by phosphatidylserine exposure, only a minority of cells had lost their mitochondrial membrane potential during the observation period. Furthermore, we demonstrated that the inhibition of membrane transporters is not a general feature of apoptosis. Conclusion: Irradiation inhibits the export function of ABC transporters. Although some of the irradiated cells undergo apoptosis following irradiation, the inhibition is an unique feature accompanying irradiation and not a general hallmark of apoptotic cell death. The inhibition of drug export by irradiation may offer new potential for reverting multidrug resistance of cancer cells. (orig.)

  3. Abdominal Compartment Syndrome

    Directory of Open Access Journals (Sweden)

    Pınar Zeyneloğlu

    2015-04-01

    Full Text Available Intraabdominal hypertension and Abdominal compartment syndrome are causes of morbidity and mortality in critical care patients. Timely diagnosis and treatment may improve organ functions. Intra-abdominal pressure monitoring is vital during evaluation of the patients and in the management algorithms. The incidence, definition and risk factors, clinical presentation, diagnosis and management of intraabdominal hypertension and Abdominal compartment syndrome were reviewed here.

  4. Protein synthesis and the recovery of both survival and cytoplasmic ''petite'' mutation in ultraviolet-treated yeast cells

    International Nuclear Information System (INIS)

    Heude, M.; Chanet, R.; Moustacchi, E.

    1975-01-01

    The contribution of nuclear-directed protein synthesis in the repair of lethal and mitochondrial genetic damage after UV-irradiation of exponential and stationary phase haploid yeast cells was examined. This was carried out using cycloheximide, a specific inhibitor of nuclear protein synthesis. It appears that nuclear protein synthesis is required for the increase in survival seen after the liquid-holding of cells at both stages, as well as for the ''petite'' recovery seen after the liquid-holding of exponential phase cells. The characteristic negative liquid-holding effect observed for the UV induction of ''petites'' in stationary phase cells (increase of the frequency of ''petites'' during storage) remained, following all the treatments which inhibited nuclear protein synthesis. However, the application of photoreactivating light following dark-holding with cycloheximide indicates that some steps of the repair of both nuclear and mitochondrial damage are performed in the absence of protein synthesis

  5. Modulation pf pulmonary surfactant secretion from alveolar type II cells by cytoplasmic free calcium ([Ca2+]/sub i/)

    International Nuclear Information System (INIS)

    Sano, K.; Voelker, D.R.; Mason, R.J.

    1986-01-01

    Ca 2+ is regulator of a variety of cellular functions including exocytosis. TPA and terbutaline have been shown to stimulate surfactant secretion from alveolar type II cells. The authors examined changes in [Ca 2+ ]/sub i/ and surfactant secretion by secretagogues in primary culture of alveolar type II cells. Cells were isolated from adult rats and were cultured for 24 h with 3 H-choline to label phosphatidylcholine. Percent secretion was determined by counting the lipids of cells and medium; cytotoxicity was excluded by measuring lactate dehydrogenase as cells and medium. [Ca 2+ ]/sub i/ was determined by measuring quin2 fluroescence of cells cultured on a glass coverslip. Ionomycin increased secretion as well as [Ca 2+ ] in dose dependent manner at the concentration from 25 to 400 nM. Ionomycin (50 nM) increased terbutaline-induced secretion in a synergistic manner but only increased TPA-induced secretion in an additive manner. Terbutaline mobilized [Ca 2+ ]/sub i/ from intracellular stores and increased [Ca 2+ ]/sub i/ by 20% from a basal level of 140 nM. TPA itself did not change [Ca 2+ ]/sub i/ but inhibited the effect of terbutaline on [Ca 2+ ]/sub i/. Loading of quin2 in the absence of extracellular calcium lowered [Ca 2+ ]/sub i/ from 143 nM to 31 nM. Lowering [Ca 2+ ]/sub i/ inhibited TPA- or terbutaline-induced secretion by 22% and 40% respectively. These results indicate that [Ca 2+ ]/sub i/ effects cAMp-induced secretion more than protein kinase C-mediated secretion in alveolar type II cells

  6. A quantitative method to track protein translocation between intracellular compartments in real-time in live cells using weighted local variance image analysis.

    Directory of Open Access Journals (Sweden)

    Guillaume Calmettes

    Full Text Available The genetic expression of cloned fluorescent proteins coupled to time-lapse fluorescence microscopy has opened the door to the direct visualization of a wide range of molecular interactions in living cells. In particular, the dynamic translocation of proteins can now be explored in real time at the single-cell level. Here we propose a reliable, easy-to-implement, quantitative image processing method to assess protein translocation in living cells based on the computation of spatial variance maps of time-lapse images. The method is first illustrated and validated on simulated images of a fluorescently-labeled protein translocating from mitochondria to cytoplasm, and then applied to experimental data obtained with fluorescently-labeled hexokinase 2 in different cell types imaged by regular or confocal microscopy. The method was found to be robust with respect to cell morphology changes and mitochondrial dynamics (fusion, fission, movement during the time-lapse imaging. Its ease of implementation should facilitate its application to a broad spectrum of time-lapse imaging studies.

  7. Cytoplasmic Domain of MscS Interacts with Cell Division Protein FtsZ: A Possible Non-Channel Function of the Mechanosensitive Channel in Escherichia Coli.

    Directory of Open Access Journals (Sweden)

    Piotr Koprowski

    Full Text Available Bacterial mechano-sensitive (MS channels reside in the inner membrane and are considered to act as emergency valves whose role is to lower cell turgor when bacteria enter hypo-osmotic environments. However, there is emerging evidence that members of the Mechano-sensitive channel Small (MscS family play additional roles in bacterial and plant cell physiology. MscS has a large cytoplasmic C-terminal region that changes its shape upon activation and inactivation of the channel. Our pull-down and co-sedimentation assays show that this domain interacts with FtsZ, a bacterial tubulin-like protein. We identify point mutations in the MscS C-terminal domain that reduce binding to FtsZ and show that bacteria expressing these mutants are compromised in growth on sublethal concentrations of β-lactam antibiotics. Our results suggest that interaction between MscS and FtsZ could occur upon inactivation and/or opening of the channel and could be important for the bacterial cell response against sustained stress upon stationary phase and in the presence of β-lactam antibiotics.

  8. Cytoplasmic Domain of MscS Interacts with Cell Division Protein FtsZ: A Possible Non-Channel Function of the Mechanosensitive Channel in Escherichia Coli.

    Science.gov (United States)

    Koprowski, Piotr; Grajkowski, Wojciech; Balcerzak, Marcin; Filipiuk, Iwona; Fabczak, Hanna; Kubalski, Andrzej

    2015-01-01

    Bacterial mechano-sensitive (MS) channels reside in the inner membrane and are considered to act as emergency valves whose role is to lower cell turgor when bacteria enter hypo-osmotic environments. However, there is emerging evidence that members of the Mechano-sensitive channel Small (MscS) family play additional roles in bacterial and plant cell physiology. MscS has a large cytoplasmic C-terminal region that changes its shape upon activation and inactivation of the channel. Our pull-down and co-sedimentation assays show that this domain interacts with FtsZ, a bacterial tubulin-like protein. We identify point mutations in the MscS C-terminal domain that reduce binding to FtsZ and show that bacteria expressing these mutants are compromised in growth on sublethal concentrations of β-lactam antibiotics. Our results suggest that interaction between MscS and FtsZ could occur upon inactivation and/or opening of the channel and could be important for the bacterial cell response against sustained stress upon stationary phase and in the presence of β-lactam antibiotics.

  9. Cytoplasmic Streaming in the Drosophila Oocyte.

    Science.gov (United States)

    Quinlan, Margot E

    2016-10-06

    Objects are commonly moved within the cell by either passive diffusion or active directed transport. A third possibility is advection, in which objects within the cytoplasm are moved with the flow of the cytoplasm. Bulk movement of the cytoplasm, or streaming, as required for advection, is more common in large cells than in small cells. For example, streaming is observed in elongated plant cells and the oocytes of several species. In the Drosophila oocyte, two stages of streaming are observed: relatively slow streaming during mid-oogenesis and streaming that is approximately ten times faster during late oogenesis. These flows are implicated in two processes: polarity establishment and mixing. In this review, I discuss the underlying mechanism of streaming, how slow and fast streaming are differentiated, and what we know about the physiological roles of the two types of streaming.

  10. Radiation dose to trabecular bone marrow stem cells from 3H, 14C and selected α-emitters incorporated in a bone remodeling compartment

    International Nuclear Information System (INIS)

    Nie Huiling; Richardson, Richard B

    2009-01-01

    A Monte Carlo simulation of repeated cubic units representing trabecular bone cavities in adult bone was employed to determine absorbed dose fractions evaluated for 3 H, 14 C and a set of α-emitters incorporated within a bone remodeling compartment (BRC). The BRC consists of a well-oxygenated vascular microenvironment located within a canopy of bone-lining cells. The International Commission on Radiological Protection (ICRP) considers that an important target for radiation-induced bone cancer is the endosteum marrow layer adjacent to bone surface where quiescent bone stem cells reside. It is proposed that the active stem cells and progenitor cells located above the BRC canopy, the 'BRC stem cell niche', is a more important radiation-induced cancer target volume. Simulation results from a static model, where no remodeling occurs, indicate that the mean dose from bone and bone surface to the 50 μm quiescent bone stem cell niche, the current ICRP target, was substantially lower (two to three times lower) than that to the narrower and hypoxic 10 μm endosteum for 3 H, 14 C and α-particles with energy range 0.5-10 MeV. The results from a dynamic model indicate that the temporal α-radiation dose to active stem/progenitor cells located in the BRC stem cell niche from the material incorporated in and buried by forming bone was 9- to 111-fold greater than the dose to the quiescent bone stem cell niche. This work indicates that the remodeling portion of the bone surface, rather than the quiescent (endosteal) surface, has the greatest risk of radiation-induced bone cancer, particularly from short-range radiation, due to the elevated dose and the radiosensitizing oxygen effect.

  11. Monitoring the systemic human memory B cell compartment of melanoma patients for anti-tumor IgG antibodies.

    Directory of Open Access Journals (Sweden)

    Amy E Gilbert

    Full Text Available Melanoma, a potentially lethal skin cancer, is widely thought to be immunogenic in nature. While there has been much focus on T cell-mediated immune responses, limited knowledge exists on the role of mature B cells. We describe an approach, including a cell-based ELISA, to evaluate mature IgG antibody responses to melanoma from human peripheral blood B cells. We observed a significant increase in antibody responses from melanoma patients (n = 10 to primary and metastatic melanoma cells compared to healthy volunteers (n = 10 (P<0.0001. Interestingly, we detected a significant reduction in antibody responses to melanoma with advancing disease stage in our patient cohort (n = 21 (P<0.0001. Overall, 28% of melanoma patient-derived B cell cultures (n = 1,800 compared to 2% of cultures from healthy controls (n = 600 produced antibodies that recognized melanoma cells. Lastly, a patient-derived melanoma-specific monoclonal antibody was selected for further study. This antibody effectively killed melanoma cells in vitro via antibody-mediated cellular cytotoxicity. These data demonstrate the presence of a mature systemic B cell response in melanoma patients, which is reduced with disease progression, adding to previous reports of tumor-reactive antibodies in patient sera, and suggesting the merit of future work to elucidate the clinical relevance of activating humoral immune responses to cancer.

  12. Monitoring the Systemic Human Memory B Cell Compartment of Melanoma Patients for Anti-Tumor IgG Antibodies

    Science.gov (United States)

    Gilbert, Amy E.; Karagiannis, Panagiotis; Dodev, Tihomir; Koers, Alexander; Lacy, Katie; Josephs, Debra H.; Takhar, Pooja; Geh, Jenny L. C.; Healy, Ciaran; Harries, Mark; Acland, Katharine M.; Rudman, Sarah M.; Beavil, Rebecca L.; Blower, Philip J.; Beavil, Andrew J.; Gould, Hannah J.; Spicer, James; Nestle, Frank O.; Karagiannis, Sophia N.

    2011-01-01

    Melanoma, a potentially lethal skin cancer, is widely thought to be immunogenic in nature. While there has been much focus on T cell-mediated immune responses, limited knowledge exists on the role of mature B cells. We describe an approach, including a cell-based ELISA, to evaluate mature IgG antibody responses to melanoma from human peripheral blood B cells. We observed a significant increase in antibody responses from melanoma patients (n = 10) to primary and metastatic melanoma cells compared to healthy volunteers (n = 10) (P<0.0001). Interestingly, we detected a significant reduction in antibody responses to melanoma with advancing disease stage in our patient cohort (n = 21) (P<0.0001). Overall, 28% of melanoma patient-derived B cell cultures (n = 1,800) compared to 2% of cultures from healthy controls (n = 600) produced antibodies that recognized melanoma cells. Lastly, a patient-derived melanoma-specific monoclonal antibody was selected for further study. This antibody effectively killed melanoma cells in vitro via antibody-mediated cellular cytotoxicity. These data demonstrate the presence of a mature systemic B cell response in melanoma patients, which is reduced with disease progression, adding to previous reports of tumor-reactive antibodies in patient sera, and suggesting the merit of future work to elucidate the clinical relevance of activating humoral immune responses to cancer. PMID:21559411

  13. Gold nanoparticles administration induces disarray of heart muscle, hemorrhagic, chronic inflammatory cells infiltrated by small lymphocytes, cytoplasmic vacuolization and congested and dilated blood vessels

    Directory of Open Access Journals (Sweden)

    Abdelhalim Mohamed Anwar K

    2011-12-01

    Full Text Available Abstract Background Despite significant research efforts on cancer therapy, diagnostics and imaging, many challenges remain unsolved. There are many unknown details regarding the interaction of nanoparticles (NPs and biological systems. The structure and properties of gold nanoparticles (GNPs make them useful for a wide array of biological applications. However, for the application of GNPs in therapy and drug delivery, knowledge regarding their bioaccumulation and associated local or systemic toxicity is necessary. Information on the biological fate of NPs, including distribution, accumulation, metabolism, and organ specific toxicity is still minimal. Studies specifically dealing with the toxicity of NPs are rare. The aim of the present study was to investigate the effects of intraperitoneal administration of GNPs on histological alterations of the heart tissue of rats in an attempt to identify and understand the toxicity and the potential role of GNPs as a therapeutic and diagnostic tool. Methods A total of 40 healthy male Wistar-Kyoto rats received 50 μl infusions of 10, 20 and 50 nm GNPs for 3 or 7 days. Animals were randomly divided into groups: 6 GNP-treated rats groups and one control group (NG. Groups 1, 2 and 3 received infusions of 50 μl GNPs of size 10 nm (3 or 7 days, 20 nm (3 or 7 days and 50 nm (3 or 7 days, respectively. Results In comparison with the respective control rats, exposure to GNPs doses produced heart muscle disarray with a few scattered chronic inflammatory cells infiltrated by small lymphocytes, foci of hemorrhage with extravasation of red blood cells, some scattered cytoplasmic vacuolization and congested and dilated blood vessels. None of the above alterations were observed in the heart muscle of any member of the control group. Conclusions The alterations induced by intraperitoneal administration of GNPs were size-dependent, with smaller ones inducing greater affects, and were also related to the time exposure to

  14. Physics and (patho)physiology in confined flows: from colloidal patterns to cytoplasmic rheology and sickle cell anemia

    Science.gov (United States)

    Mahadevan, L.

    2015-03-01

    I will discuss a few problems that involve the interaction of fluids and solids in confined spaces. (i) Jamming in pressure-driven suspension flows that show a transition from Stokes flows to Darcy flows as the solids start to lock, as in evaporative patterning in colloids (e.g. coffee stain formation) .(ii) Jamming and clogging of red blood cells, as in sickle-cell pathophysiology, with implications for other diseases that involve jamming. (iii) The mechanical response of crowded networks of filaments bathed in a fluid, as in the cytoskeleton, that can be described by poroelasticity theory. In each case, I will show how simple theories of multiphase flow and deformation can be used to explain a range of experimental observations, while failing to account for others, along with some thoughts on how to improve them.

  15. p38 MAPK and JNK antagonistically control senescence and cytoplasmic p16INK4A expression in doxorubicin-treated endothelial progenitor cells.

    Directory of Open Access Journals (Sweden)

    Paolo Spallarossa

    Full Text Available Patients treated with low-dose anthracyclines often show late onset cardiotoxicity. Recent studies suggest that this form of cardiotoxicity is the result of a progenitor cell disease. In this study we demonstrate that Cord Blood Endothelial Progenitor Cells (EPCs exposed to low, sub-apoptotic doses of doxorubicin show a senescence phenotype characterized by increased SA-b-gal activity, decreased TRF2 and chromosomal abnormalities, enlarged cell shape, and disarrangement of F-actin stress fibers accompanied by impaired migratory ability. P16( INK4A localizes in the cytoplasm of doxorubicin-induced senescent EPCs and not in the nucleus as is the case in EPCs rendered senescent by different stimuli. This localization together with the presence of an arrest in G2, and not at the G1 phase boundary, which is what usually occurs in response to the cell cycle regulatory activity of p16(INK4A, suggests that doxorubicin-induced p16( INK4A does not regulate the cell cycle, even though its increase is closely associated with senescence. The effects of doxorubicin are the result of the activation of MAPKs p38 and JNK which act antagonistically. JNK attenuates the senescence, p16( INK4A expression and cytoskeleton remodeling that are induced by activated p38. We also found that conditioned medium from doxorubicin-induced senescent cardiomyocytes does not attract untreated EPCs, unlike conditioned medium from apoptotic cardiomyocytes which has a strong chemoattractant capacity. In conclusion, this study provides a better understanding of the senescence of doxorubicin-treated EPCs, which may be helpful in preventing and treating late onset cardiotoxicity.

  16. GLYCINE-RICH RNA-BINDING PROTEIN1 interacts with RECEPTOR-LIKE CYTOPLASMIC PROTEIN KINASE1 and suppresses cell death and defense responses in pepper (Capsicum annuum).

    Science.gov (United States)

    Kim, Dae Sung; Kim, Nak Hyun; Hwang, Byung Kook

    2015-01-01

    Plants use a variety of innate immune regulators to trigger cell death and defense responses against pathogen attack. We identified pepper (Capsicum annuum) GLYCINE-RICH RNA-BINDING PROTEIN1 (CaGRP1) as a RECEPTOR-LIKE CYTOPLASMIC PROTEIN KINASE1 (CaPIK1)-interacting partner, based on bimolecular fluorescence complementation and coimmunoprecipitation analyses as well as gene silencing and transient expression analysis. CaGRP1 contains an N-terminal RNA recognition motif and a glycine-rich region at the C-terminus. The CaGRP1 protein had DNA- and RNA-binding activity in vitro. CaGRP1 interacted with CaPIK1 in planta. CaGRP1 and CaGRP1-CaPIK1 complexes were localized to the nucleus in plant cells. CaPIK1 phosphorylated CaGRP1 in vitro and in planta. Transient coexpression of CaGRP1 with CaPIK1 suppressed the CaPIK1-triggered cell death response, accompanied by a reduced CaPIK1-triggered reactive oxygen species (ROS) burst. The RNA recognition motif region of CaGRP1 was responsible for the nuclear localization of CaGRP1 as well as the suppression of the CaPIK1-triggered cell death response. CaGRP1 silencing in pepper conferred enhanced resistance to Xanthomonas campestris pv vesicatoria (Xcv) infection; however, CaPIK1-silenced plants were more susceptible to Xcv. CaGRP1 interacts with CaPIK1 and negatively regulates CaPIK1-triggered cell death and defense responses by suppressing ROS accumulation. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

  17. Deficient CD4+ T cell priming and regression of CD8+ T cell functionality in virus-infected mice lacking a normal B cell compartment

    DEFF Research Database (Denmark)

    Christensen, Jan Pravsgaard; Kauffmann, Susanne Ørding; Thomsen, Allan Randrup

    2003-01-01

    of virus-specific CD4(+) T cells was markedly impaired in B(-/-) mice infected with either virus strain. Thus, our results indicate that B cells play an important role in antiviral immunity not only as Ab producers, but also in promoting an optimal and sustained T cell response. The T cell defects......In this study, we investigate the state of T cell-mediated immunity in B cell-deficient (B(-/-)) mice infected with two strains of lymphocytic choriomeningitis virus known to differ markedly in their capacity to persist. In B(-/-) C57BL mice infected with the more persisting virus, virus......-specific CD8(+) T cells are initially generated that are qualitatively similar to those in wild-type mice. However, although cell numbers are well sustained over time, the capacity to produce cytokines is rapidly impaired. In similarly infected B(-/-) BALB/c mice, virus-specific CD8(+) T cells are completely...

  18. Distribution of elements in rat peripheral axons and nerve cell bodies determined by x-ray microprobe analysis

    Energy Technology Data Exchange (ETDEWEB)

    LoPachin, R.M. Jr.; Lowery, J.; Eichberg, J.; Kirkpatrick, J.B.; Cartwright, J. Jr.; Saubermann, A.J.

    1988-09-01

    X-ray microprobe analysis was used to determine concentrations (millimoles of element per kilogram dry weight) of Na, P, Cl, K, and Ca in cellular compartments of frozen, unfixed sections of rat sciatic and tibial nerves and dorsal root ganglion (DRG). Five compartments were examined in peripheral nerve (axoplasm, mitochondria, myelin, extraaxonal space, and Schwann cell cytoplasm), and four were analyzed in DRG nerve cell bodies (cytoplasm, mitochondria, nucleus, and nucleolus). Each morphological compartment exhibited characteristic concentrations of elements. The extraaxonal space contained high concentrations of Na, Cl, and Ca, whereas intraaxonal compartments exhibited lower concentrations of these elements but relatively high K contents. Nerve axoplasm and axonal mitochondria had similar elemental profiles, and both compartments displayed proximodistal gradients of decreasing levels of K, Cl, and, to some extent, Na. Myelin had a selectively high P concentration with low levels of other elements. The elemental concentrations of Schwann cell cytoplasm and DRG were similar, but both were different from that of axoplasm, in that K and Cl were markedly lower whereas P was higher. DRG cell nuclei contained substantially higher K levels than cytoplasm. The subcellular distribution of elements was clearly shown by color-coded images generated by computer-directed digital x-ray imaging. The results of this study demonstrate characteristic elemental distributions for each anatomical compartment, which doubtless reflect nerve cell structure and function.

  19. Juliprosopine and juliprosine from prosopis juliflora leaves induce mitochondrial damage and cytoplasmic vacuolation on cocultured glial cells and neurons.

    Science.gov (United States)

    Silva, Victor Diogenes A; Pitanga, Bruno P S; Nascimento, Ravena P; Souza, Cleide S; Coelho, Paulo Lucas C; Menezes-Filho, Noélio; Silva, André Mário M; Costa, Maria de Fátima D; El-Bachá, Ramon S; Velozo, Eudes S; Costa, Silvia L

    2013-12-16

    Prosopis juliflora is a shrub largely used for animal and human consumption. However, ingestion has been shown to induce intoxication in animals, which is characterized by neuromuscular alterations induced by mechanisms that are not yet well understood. In this study, we investigated the cytotoxicity of a total alkaloid extract (TAE) and one alkaloid fraction (F32) obtained from P. juliflora leaves to rat cortical neurons and glial cells. Nuclear magnetic resonance characterization of F32 showed that this fraction is composed of a mixture of two piperidine alkaloids, juliprosopine (majority constituent) and juliprosine. TAE and F32 at concentrations between 0.3 and 45 μg/mL were tested for 24 h on neuron/glial cell primary cocultures. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test revealed that TAE and F32 were cytotoxic to cocultures, and their IC50 values were 31.07 and 7.362 μg/mL, respectively. Exposure to a subtoxic concentration of TAE or F32 (0.3-3 μg/mL) induced vacuolation and disruption of the astrocyte monolayer and neurite network, ultrastructural changes, characterized by formation of double-membrane vacuoles, and mitochondrial damage, associated with changes in β-tubulin III and glial fibrillary acidic protein expression. Microglial proliferation was also observed in cultures exposed to TAE or F32, with increasing levels of OX-42-positive cells. Considering that F32 was more cytotoxic than TAE and that F32 reproduced in vitro the main morphologic and ultrastructural changes of "cara torta" disease, we can also suggest that piperidine alkaloids juliprosopine and juliprosine are primarily responsible for the neurotoxic damage observed in animals after they have consumed the plant.

  20. How crowded is the prokaryotic cytoplasm?

    NARCIS (Netherlands)

    Spitzer, Jan; Poolman, Bert; Ferguson, Stuart

    2013-01-01

    We consider biomacromolecular crowding within the cytoplasm of prokaryotic cells as a two-phase system of 'supercrowded' cytogel and 'dilute' cytosol; we simplify and quantify this model for a coccoid cell over a wide range of biomacromolecular crowding. The key result shows that the supercrowded

  1. Maintenance of the marginal-zone B cell compartment specifically requires the RNA-binding protein ZFP36L1.

    Science.gov (United States)

    Newman, Rebecca; Ahlfors, Helena; Saveliev, Alexander; Galloway, Alison; Hodson, Daniel J; Williams, Robert; Besra, Gurdyal S; Cook, Charlotte N; Cunningham, Adam F; Bell, Sarah E; Turner, Martin

    2017-06-01

    RNA-binding proteins of the ZFP36 family are best known for inhibiting the expression of cytokines through binding to AU-rich elements in the 3' untranslated region and promoting mRNA decay. Here we identified an indispensable role for ZFP36L1 as the regulator of a post-transcriptional hub that determined the identity of marginal-zone B cells by promoting their proper localization and survival. ZFP36L1 controlled a gene-expression program related to signaling, cell adhesion and locomotion; it achieved this in part by limiting expression of the transcription factors KLF2 and IRF8, which are known to enforce the follicular B cell phenotype. These mechanisms emphasize the importance of integrating transcriptional and post-transcriptional processes by RNA-binding proteins for maintaining cellular identity among closely related cell types.

  2. Maintenance of the marginal zone B cell compartment specifically requires the RNA-binding protein ZFP36L1

    Science.gov (United States)

    Newman, Rebecca; Ahlfors, Helena; Saveliev, Alexander; Galloway, Alison; Hodson, Daniel J; Williams, Robert; Besra, Gurdyal S.; Cook, Charlotte N; Cunningham, Adam F; Bell, Sarah E; Turner, Martin

    2017-01-01

    RNA binding proteins (RBP) of the ZFP36 family are best known for inhibiting the expression of cytokines through binding to AU rich elements in the 3’UTR and promoting mRNA decay. Here we show an indispensible role for ZFP36L1 as the regulator of a post-transcriptional hub that determined the identity of marginal zone (MZ) B cells by promoting their proper localization and survival. ZFP36L1 controlled a gene expression program related to signaling, cell-adhesion and locomotion, in part by limiting the expression of the transcription factors KLF2 and IRF8, which are known to enforce the follicular B cell phenotype. These mechanisms emphasize the importance of integrating transcriptional and post-transcriptional processes by RBP for maintaining cellular identity between closely related cell types. PMID:28394372

  3. HIV-1 Envelope Glycoprotein Trafficking through the Endosomal Recycling Compartment Is Required for Particle Incorporation.

    Science.gov (United States)

    Kirschman, Junghwa; Qi, Mingli; Ding, Lingmei; Hammonds, Jason; Dienger-Stambaugh, Krista; Wang, Jaang-Jiun; Lapierre, Lynne A; Goldenring, James R; Spearman, Paul

    2018-03-01

    The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) encodes specific trafficking signals within its long cytoplasmic tail (CT) that regulate incorporation into HIV-1 particles. Rab11-family interacting protein 1C (FIP1C) and Rab14 are host trafficking factors required for Env particle incorporation, suggesting that Env undergoes sorting from the endosomal recycling compartment (ERC) to the site of particle assembly on the plasma membrane. We disrupted outward sorting from the ERC by expressing a C-terminal fragment of FIP1C (FIP1C 560-649 ) and examined the consequences on Env trafficking and incorporation into particles. FIP1C 560-649 reduced cell surface levels of Env and prevented its incorporation into HIV-1 particles. Remarkably, Env was trapped in an exaggerated perinuclear ERC in a CT-dependent manner. Mutation of either the Yxxϕ endocytic motif or the YW 795 motif in the CT prevented Env trapping in the ERC and restored incorporation into particles. In contrast, simian immunodeficiency virus SIVmac239 Env was not retained in the ERC, while substitution of the HIV-1 CT for the SIV CT resulted in SIV Env retention in this compartment. These results provide the first direct evidence that Env traffics through the ERC and support a model whereby HIV-1 Env is specifically targeted to the ERC prior to FIP1C- and CT-dependent outward sorting to the particle assembly site on the plasma membrane. IMPORTANCE The HIV envelope protein is an essential component of the viral particle. While many aspects of envelope protein structure and function have been established, the pathway it follows in the cell prior to reaching the site of particle assembly is not well understood. The envelope protein has a very long cytoplasmic tail that interacts with the host cell trafficking machinery. Here, we utilized a truncated form of the trafficking adaptor FIP1C protein to arrest the intracellular transport of the envelope protein, demonstrating that it becomes

  4. A novel approach to tracking antigen-experienced CD4 T cells into functional compartments via tandem deep and shallow TCR clonotyping.

    Science.gov (United States)

    Estorninho, Megan; Gibson, Vivienne B; Kronenberg-Versteeg, Deborah; Liu, Yuk-Fun; Ni, Chester; Cerosaletti, Karen; Peakman, Mark

    2013-12-01

    Extensive diversity in the human repertoire of TCRs for Ag is both a cornerstone of effective adaptive immunity that enables host protection against a multiplicity of pathogens and a weakness that gives rise to potential pathological self-reactivity. The complexity arising from diversity makes detection and tracking of single Ag-specific CD4 T cells (ASTs) involved in these immune responses challenging. We report a tandem, multistep process to quantify rare TCRβ-chain variable sequences of ASTs in large polyclonal populations. The approach combines deep high-throughput sequencing (HTS) within functional CD4 T cell compartments, such as naive/memory cells, with shallow, multiple identifier-based HTS of ASTs identified by activation marker upregulation after short-term Ag stimulation in vitro. We find that clonotypes recognizing HLA class II-restricted epitopes of both pathogen-derived Ags and self-Ags are oligoclonal and typically private. Clonotype tracking within an individual reveals private AST clonotypes resident in the memory population, as would be expected, representing clonal expansions (identical nucleotide sequence; "ultraprivate"). Other AST clonotypes share CDR3β amino acid sequences through convergent recombination and are found in memory populations of multiple individuals. Tandem HTS-based clonotyping will facilitate studying AST dynamics, epitope spreading, and repertoire changes that arise postvaccination and following Ag-specific immunotherapies for cancer and autoimmune disease.

  5. Pediatric Solid-Organ Transplant Recipients Carry Chronic Loads of Epstein-Barr Virus Exclusively in the Immunoglobulin D-Negative B-Cell Compartment

    Science.gov (United States)

    Rose, Camille; Green, Michael; Webber, Steven; Ellis, Demetrius; Reyes, Jorges; Rowe, David

    2001-01-01

    Solid-organ transplant recipients are at risk for development of lymphoproliferative diseases. The purpose of this study was to examine the distribution of Epstein-Barr virus (EBV) load in the peripheral blood of pediatric transplant recipients who had become chronic viral load carriers (>8 copies/105 lymphocytes for >2 months). A total of 19 patients with viral loads ranging from 20 to 5,000 viral genome copies/105 lymphocytes were studied. Ten patients had no previous diagnosis of posttransplant lymphoproliferative disease (PT-LPD), while nine had recovered from a diagnosed case of PT-LPD. No portion of the peripheral blood viral load was detected in the cell-free plasma fraction. Viral DNA was found in a population of cells characterized as CD19hi and immunoglobulin D negative, a phenotype that is consistent with the virus being carried exclusively in the memory B-cell compartment of the peripheral blood. There was no difference in the compartmentalization based upon either the level of the viral load or the past diagnosis of an episode of PT-LPD. These results have implications for the design of tests to detect EBV infection and for the interpretation and use of positive EBV PCR assays in the management of transplant recipients. PMID:11283064

  6. Aging of the T cell compartment in mice and humans: from no naïve expectations to foggy memories

    Science.gov (United States)

    Nikolich-Žugich, Janko

    2014-01-01

    Until the mid-20th century, infectious diseases were the major cause of morbidity and mortality in humans. Massive vaccination campaigns, antibiotics, antivirals and advanced public health measures drastically reduced sickness and death of infections in children and younger adults. Older adults (>65yr of age), however, remain vulnerable to infections, and to date infectious diseases remain amongst the top 5–10 causes of death in this population. The aging of the immune system, often referred to as immune senescence, is the key phenomenon underlying this vulnerability. This review centers on age-related changes in T cells, which are dramatically and reproducibly altered with aging. I will discuss changes in T cell production, maintenance, function and response to latent persistent infection, particularly against the cytomegalovirus (CMV), that exerts profound influence on the aging T cell pool, concluding with a brief list of measures to improve immune function in older adults. PMID:25193936

  7. Cytoplasmic protein binding to highly conserved sequences in the 3' untranslated region of mouse protamine 2 mRNA, a translationally regulated transcript of male germ cells

    International Nuclear Information System (INIS)

    Kwon, Y.K.; Hecht, N.B.

    1991-01-01

    The expression of the protamines, the predominant nuclear proteins of mammalian spermatozoa, is regulated translationally during male germ-cell development. The 3' untranslated region (UTR) of protamine 1 mRNA has been reported to control its time of translation. To understand the mechanisms controlling translation of the protamine mRNAs, we have sought to identify cis elements of the 3' UTR of protamine 2 mRNA that are recognized by cytoplasmic factors. From gel retardation assays, two sequence elements are shown to form specific RNA-protein complexes. Protein binding sites of the two complexes were determined by RNase T1 mapping, by blocking the putative binding sites with antisense oligonucleotides, and by competition assays. The sequences of these elements, located between nucleotides + 537 and + 572 in protamine 2 mRNA, are highly conserved among postmeiotic translationally regulated nuclear proteins of the mammalian testis. Two closely linked protein binding sites were detected. UV-crosslinking studies revealed that a protein of about 18 kDa binds to one of the conserved sequences. These data demonstrate specific protein binding to a highly conserved 3' UTR of translationally regulated testicular mRNA

  8. The nuclear protein Sam68 is cleaved by the FMDV 3C protease redistributing Sam68 to the cytoplasm during FMDV infection of host cells

    International Nuclear Information System (INIS)

    Lawrence, Paul; Schafer, Elizabeth A.; Rieder, Elizabeth

    2012-01-01

    Picornavirus infection can lead to disruption of nuclear pore traffic, shut-off of cell translation machinery, and cleavage of proteins involved in cellular signal transduction and the innate response to infection. Here, we demonstrated that the FMDV 3C pro induced the cleavage of nuclear RNA-binding protein Sam68 C-terminus containing the nuclear localization sequence (NLS). Consequently, it stimulated the redistribution of Sam68 to the cytoplasm. The siRNA knockdown of Sam68 resulted in a 1000-fold reduction in viral titers, which prompted us to study the effect of Sam68 on FMDV post-entry events. Interestingly, Sam68 interacts with the internal ribosomal entry site within the 5′ non-translated region of the FMDV genome, and Sam68 knockdown decreased FMDV IRES-driven activity in vitro suggesting that it could modulate translation of the viral genome. The results uncover a novel role for Sam68 in the context of picornaviruses and the proteolysis of a new cellular target of the FMDV 3C pro .

  9. Role of ER stress response in photodynamic therapy: ROS generated in different subcellular compartments trigger diverse cell death pathways

    Czech Academy of Sciences Publication Activity Database

    Moserová, Irena; Králová, Jarmila

    2012-01-01

    Roč. 7, č. 3 (2012), e32972 E-ISSN 1932-6203 R&D Projects: GA MŠk(CZ) LC06077; GA ČR GA203/09/1311; GA ČR(CZ) GAP303/11/1291 Institutional research plan: CEZ:AV0Z50520514 Keywords : photodynamic therapy * porphyrin derivatives * cell death * ER stress Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.730, year: 2012

  10. Wide-range high-resolution transmission electron microscopy reveals morphological and distributional changes of endomembrane compartments during log to stationary transition of growth phase in tobacco BY-2 cells.

    Science.gov (United States)

    Toyooka, Kiminori; Sato, Mayuko; Kutsuna, Natsumaro; Higaki, Takumi; Sawaki, Fumie; Wakazaki, Mayumi; Goto, Yumi; Hasezawa, Seiichiro; Nagata, Noriko; Matsuoka, Ken

    2014-09-01

    Rapid growth of plant cells by cell division and expansion requires an endomembrane trafficking system. The endomembrane compartments, such as the Golgi stacks, endosome and vesicles, are important in the synthesis and trafficking of cell wall materials during cell elongation. However, changes in the morphology, distribution and number of these compartments during the different stages of cell proliferation and differentiation have not yet been clarified. In this study, we examined these changes at the ultrastructural level in tobacco Bright yellow 2 (BY-2) cells during the log and stationary phases of growth. We analyzed images of the BY-2 cells prepared by the high-pressure freezing/freeze substitution technique with the aid of an auto-acquisition transmission electron microscope system. We quantified the distribution of secretory and endosomal compartments in longitudinal sections of whole cells by using wide-range gigapixel-class images obtained by merging thousands of transmission electron micrographs. During the log phase, all Golgi stacks were composed of several thick cisternae. Approximately 20 vesicle clusters (VCs), including the trans-Golgi network and secretory vesicle cluster, were observed throughout the cell. In the stationary-phase cells, Golgi stacks were thin with small cisternae, and only a few VCs were observed. Nearly the same number of multivesicular body and small high-density vesicles were observed in both the stationary and log phases. Results from electron microscopy and live fluorescence imaging indicate that the morphology and distribution of secretory-related compartments dramatically change when cells transition from log to stationary phases of growth. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  11. Changes in compartments of hemospoietic and stromal marrow progenitor cells after continuous low dose gamma-irradiation

    Science.gov (United States)

    Domaratskaya, E.; Starostin, V.

    The low dose continuous gamma-irradiation chosen corresponded with that affected the organisms onboard a spacecraft (Mitrikas, Tsetlin, 2000). F1 (CBAxC57Bl/6) male and female mice were used at 3 4 months of age. Experimental mice were- irradiated during 10 days to a total dose of 15 mGy (Co60 gamma-sources, mean dose rate of 1.5-2.0 mGy/day). Another group of intact mice served as control. Younger and advanced hemopoietic progenitors measured at day 11 (i.e. CFU -S-11) and day 7 (i.e. CFU-S-7), respectively, after transplantation of test donor cells were assayed by the method of Till and McCulloch (1961). Stromal changes were evaluated by estimation of in vitro fibroblastic colony-forming units (CFU -F ) content and by the ability of ectopically grafted (under renal capsule) stroma to regenerate the new bone marrow organ. CFU-S-11 number increased of 40% as compared with control and almost 2-fold higher than that of CFU-S-7. The CFU-F content increased almost of 3-fold. Size of ectopic marrow transplants was estimated at day 70 following grafting by counting myelokariocyte and CFU -S number that repopulated the newly formed bone marrow organ. It was found more than 2-fold increase of myelokariocytes in transplants produced by marrow stroma of irradiated donors. CFU -S contents in transplants increased strikingly in comparison to control level. CFU-S-7 and CFU-S-11 increased of 7.5- and of 3.7-fold, respectively, i.e. the rate of advanced CFU - S predominated. It should be noted a good correlation between number of stromal progenitor cells (CFU-F) and ectopic transplant sizes evaluated as myelokaryocyte counts when irradiated donors used. In the same time, if sizes of transplants was measured as CFU-S-7 and CFU - S-11 numbers, their increases were more pronounced. Therefore, continuous low dose gamma- irradiation augments significantly both hemopoietic and stromal progenitor cell number in bone marrow. Additionally, the ratio of distinct CFU -S subpopulations

  12. Extraction of Carbon Dioxide and Hydrogen from Seawater by an Electrochemical Acidification Cell. Part 4. Electrode Compartments of Cell Modified and Tested in Scaled-Up Mobile Unit

    Science.gov (United States)

    2013-09-03

    The catholyte is the water fed to the cathode compartment and it must be free from hardness ions such as calcium (Ca +2 ) and magnesium (Mg +2...4NaOH + 2H2 (6) Overall: 6H2O + 4NaCl  4HClIX + 4 NaOH  + 2H2Cathode + O2Anode (7) The amount of H + generated by the cathode...electrode that is operating as the cathode. Ions that impart hardness to seawater include calcium (Ca +2 ) and magnesium (Mg +2 ) ions, and their total

  13. Picornaviruses and nuclear functions: targeting a cellular compartment distinct from the replication site of a positive-strand RNA virus

    Directory of Open Access Journals (Sweden)

    Dylan eFlather

    2015-06-01

    Full Text Available The compartmentalization of DNA replication and gene transcription in the nucleus and protein production in the cytoplasm is a defining feature of eukaryotic cells. The nucleus functions to maintain the integrity of the nuclear genome of the cell and to control gene expression based on intracellular and environmental signals received through the cytoplasm. The spatial separation of the major processes that lead to the expression of protein-coding genes establishes the necessity of a transport network to allow biomolecules to translocate between these two regions of the cell. The nucleocytoplasmic transport network is therefore essential for regulating normal cellular functioning. The Picornaviridae virus family is one of many viral families that disrupt the nucleocytoplasmic trafficking of cells to promote viral replication. Picornaviruses contain positive-sense, single-stranded RNA genomes and replicate in the cytoplasm of infected cells. As a result of the limited coding capacity of these viruses, cellular proteins are required by these intracellular parasites for both translation and genomic RNA replication. Being of messenger RNA polarity, a picornavirus genome can immediately be translated upon entering the cell cytoplasm. However, the replication of viral RNA requires the activity of RNA-binding proteins, many of which function in host gene expression, and are consequently localized to the nucleus. As a result, picornaviruses disrupt nucleocytoplasmic trafficking to exploit protein functions normally localized to a different cellular compartment from which they translate their genome to facilitate efficient replication. Furthermore, picornavirus proteins are also known to enter the nucleus of infected cells to limit host-cell transcription and down-regulate innate antiviral responses. The interactions of picornavirus proteins and host-cell nuclei are extensive, required for a productive infection, and are the focus of this review.

  14. Biocompatible click chemistry enabled compartment-specific pH measurement inside E. coli.

    Science.gov (United States)

    Yang, Maiyun; Jalloh, Abubakar S; Wei, Wei; Zhao, Jing; Wu, Peng; Chen, Peng R

    2014-09-19

    Bioorthogonal reactions, especially the Cu(I)-catalysed azide-alkyne cycloaddition, have revolutionized our ability to label and manipulate biomolecules under living conditions. The cytotoxicity of Cu(I) ions, however, has hindered the application of this reaction in the internal space of living cells. By systematically surveying a panel of Cu(I)-stabilizing ligands in promoting protein labelling within the cytoplasm of Escherichia coli, we identify a highly efficient and biocompatible catalyst for intracellular modification of proteins by azide-alkyne cycloaddition. This reaction permits us to conjugate an environment-sensitive fluorophore site specifically onto HdeA, an acid-stress chaperone that adopts pH-dependent conformational changes, in both the periplasm and cytoplasm of E. coli. The resulting protein-fluorophore hybrid pH indicators enable compartment-specific pH measurement to determine the pH gradient across the E. coli cytoplasmic membrane. This construct also allows the measurement of E. coli transmembrane potential, and the determination of the proton motive force across its inner membrane under normal and acid-stress conditions.

  15. Consequences of cytoplasmic irradiation. Studies from microbeam

    International Nuclear Information System (INIS)

    Zhou, Hongning; Hong, Mei; Chai, Yunfei; Hei, Tom K.

    2009-01-01

    The prevailing dogma for radiation biology is that genotoxic effects of ionizing radiation such as mutations and carcinogenesis are attributed mainly to direct damage to the nucleus. However, with the development of microbeam that can target precise positions inside the cells, accumulating evidences have shown that energy deposit by radiation in nuclear DNA is not required to trigger the damage, extra-nuclear or extra-cellular radiation could induce the similar biological effects as well. This review will summarize the biological responses after cytoplasm irradiated by microbeam, and the possible mechanisms involved in cytoplasmic irradiation. (author)

  16. Fanconi Anemia: A DNA repair disorder characterized by accelerated decline of the hematopoietic stem cell compartment and other features of aging.

    Science.gov (United States)

    Brosh, Robert M; Bellani, Marina; Liu, Yie; Seidman, Michael M

    2017-01-01

    Fanconi Anemia (FA) is a rare autosomal genetic disorder characterized by progressive bone marrow failure (BMF), endocrine dysfunction, cancer, and other clinical features commonly associated with normal aging. The anemia stems directly from an accelerated decline of the hematopoietic stem cell compartment. Although FA is a complex heterogeneous disease linked to mutations in 19 currently identified genes, there has been much progress in understanding the molecular pathology involved. FA is broadly considered a DNA repair disorder and the FA gene products, together with other DNA repair factors, have been implicated in interstrand cross-link (ICL) repair. However, in addition to the defective DNA damage response, altered epigenetic regulation, and telomere defects, FA is also marked by elevated levels of inflammatory mediators in circulation, a hallmark of faster decline in not only other hereditary aging disorders but also normal aging. In this review, we offer a perspective of FA as a monogenic accelerated aging disorder, citing the latest evidence for its multi-factorial deficiencies underlying its unique clinical and cellular features. Published by Elsevier B.V.

  17. One-shot flow injection spectrophotometric simultaneous determination of copper, iron and zinc in patients' sera with newly developed multi-compartment flow cell

    International Nuclear Information System (INIS)

    Teshima, Norio; Gotoh, Shingo; Ida, Kazunori; Sakai, Tadao

    2006-01-01

    We propose here an affordable flow injection method for simultaneous spectrophotometric determination of copper, iron and zinc in patients' sera. The use of a newly designed multi-compartment flow cell allowed the simultaneous determination of the three metals with a single injection ('one-shot') and a double beam spectrophotometer. The chemistry relied on the reactions of these metals with 2-(5-nitro-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)amino]phenol (nitro-PAPS) to form corresponding colored complexes. At pH 3.8, only copper-nitro-PAPS complex was formed in the presence of pyrophosphate as a masking agent for iron, and then the copper and iron(II) complexes were formed in the presence of reductant (ascorbic acid) at the same pH, and finally all three metals reacted with nitro-PAPS at pH 8.6. The characteristics were introduced into the flow system to determine each metal selectively and sensitively. Under the optimum conditions, linear calibration curves for the three metals were obtained in the range of 0.01-1 mg L -1 with a sample throughput rate of 20 h -1 . The limits of detection (3σ) were 3.9 μg L -1 for copper, 4.1 μg L -1 for iron and 4.0 μg L -1 for zinc. The proposed method was applied to analysis of some patients' sera

  18. Ontogeny of human natural killer (NK) cells: fetal NK cells mediate cytolytic function and express cytoplasmic CD3 epsilon,delta proteins

    NARCIS (Netherlands)

    Phillips, J. H.; Hori, T.; Nagler, A.; Bhat, N.; Spits, H.; Lanier, L. L.

    1992-01-01

    Natural killer (NK) cells have been defined as CD3 epsilon-, CD16+ and/or CD56+ lymphocytes that mediate major histocompatibility complex (MHC)-unrestricted cytotoxicity against certain tumors and virus-infected cells. Unlike T lymphocytes, NK cells do not rearrange or productively express T cell

  19. Endocytosis of desmosomal plaques depends on intact actin filaments and leads to a nondegradative compartment

    DEFF Research Database (Denmark)

    Holm, Pernille K.; Hansen, Steen H.; Sandvig, Kirsten

    1993-01-01

    Cellebiologi, human epithelial cell line, growth inhibition, desmosomes, clathrin-independent endocytosis, cytoskeleton, nondegradative compartment......Cellebiologi, human epithelial cell line, growth inhibition, desmosomes, clathrin-independent endocytosis, cytoskeleton, nondegradative compartment...

  20. Cytoplasmic Estrogen Receptor in breast cancer

    Science.gov (United States)

    Welsh, Allison W.; Lannin, Donald R.; Young, Gregory S.; Sherman, Mark E.; Figueroa, Jonine D.; Henry, N. Lynn; Ryden, Lisa; Kim, Chungyeul; Love, Richard R.; Schiff, Rachel; Rimm, David L.

    2011-01-01

    Purpose In addition to genomic signaling, it is accepted that ERα has non-nuclear signaling functions, which correlate with tamoxifen resistance in preclinical models. However, evidence for cytoplasmic ER localization in human breast tumors is less established. We sought to determine the presence and implications of non-nuclear ER in clinical specimens. Experimental Design A panel of ERα-specific antibodies (SP1, MC20, F10, 60c, 1D5) were validated by western blot and quantitative immunofluorescent (QIF) analysis of cell lines and patient controls. Then eight retrospective cohorts collected on tissue microarrays were assessed for cytoplasmic ER. Four cohorts were from Yale (YTMA 49, 107, 130, 128) and four others (NCI YTMA 99, South Swedish Breast Cancer Group SBII, NSABP B14, and a Vietnamese Cohort) from other sites around the world. Results Four of the antibodies specifically recognized ER by western and QIF, showed linear increases in amounts of ER in cell line series with progressively increasing ER, and the antibodies were reproducible on YTMA 49 with pearson’s correlations (r2 values)ranging from 0.87-0.94. One antibody with striking cytoplasmic staining (MC20) failed validation. We found evidence for specific cytoplasmic staining with the other 4 antibodies across eight cohorts. The average incidence was 1.5%, ranging from 0 to 3.2%. Conclusions Our data shows ERα present in the cytoplasm in a number of cases using multiple antibodies, while reinforcing the importance of antibody validation. In nearly 3,200 cases, cytoplasmic ER is present at very low incidence, suggesting its measurement is unlikely to be of routine clinical value. PMID:21980134

  1. The molecular mechanism and physiological role of cytoplasmic streaming.

    Science.gov (United States)

    Tominaga, Motoki; Ito, Kohji

    2015-10-01

    Cytoplasmic streaming occurs widely in plants ranging from algae to angiosperms. However, the molecular mechanism and physiological role of cytoplasmic streaming have long remained unelucidated. Recent molecular genetic approaches have identified specific myosin members (XI-2 and XI-K as major and XI-1, XI-B, and XI-I as minor motive forces) for the generation of cytoplasmic streaming among 13 myosin XIs in Arabidopsis thaliana. Simultaneous knockout of these myosin XI members led to a reduced velocity of cytoplasmic streaming and marked defects of plant development. Furthermore, the artificial modifications of myosin XI-2 velocity changed plant and cell sizes along with the velocity of cytoplasmic streaming. Therefore, we assume that cytoplasmic streaming is one of the key regulators in determining plant size. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  2. Late-assembly of human ribosomal protein S20 in the cytoplasm is essential for the functioning of the small subunit ribosome

    International Nuclear Information System (INIS)

    Tai, Lin-Ru; Chou, Chang-Wei; Wu, Jing-Ying; Kirby, Ralph; Lin, Alan

    2013-01-01

    Using immuno-fluorescent probing and Western blotting analysis, we reveal the exclusive cytoplasm nature of the small subunit ribosomal protein S20. To illustrate the importance of the cellular compartmentation of S20 to the function of small subunit 40S, we created a nuclear resident S20 NLS mutant gene and examined polysome profile of cells that had been transfected with the S20 NLS gene. As a result, we observed the formation of recombinant 40S carried S20 NLS but this recombinant 40S was never found in the polysome, suggesting such a recombinant 40S was translation incompetent. Moreover, by the tactic of the energy depletion and restoration, we were able to restrain the nuclear-resided S20 NLS in the cytoplasm. Yet, along a progressive energy restoration, we observed the presence of recombinant 40S subunits carrying the S20 NLS in the polysome. This proves that S20 needs to be cytoplasmic in order to make a functional 40S subunit. Furthermore, it also implies that the assembly order of ribosomal protein in eukaryote is orderly regulated. - Highlights: • The step of S20 assembled on 40S is happened in the cytoplasm. • A small subunit assembled with a nuclear S20 NLS is translational incompetence. • Using energy depletion and recovery to manipulate the cellular compartment of S20 NLS . • Cytoplasm-retained S20 NLS is crucial for creating a functional small subunit

  3. Late-assembly of human ribosomal protein S20 in the cytoplasm is essential for the functioning of the small subunit ribosome

    Energy Technology Data Exchange (ETDEWEB)

    Tai, Lin-Ru [Institute of Genome Sciences, School of Life Sciences, National Yang-Ming University, Taipei, Taiwan, ROC (China); Chou, Chang-Wei [Department of Dentistry, School of Dentistry, National Yang-Ming University, Taipei, Taiwan, ROC (China); Wu, Jing-Ying; Kirby, Ralph [Institute of Genome Sciences, School of Life Sciences, National Yang-Ming University, Taipei, Taiwan, ROC (China); Lin, Alan, E-mail: alin@ym.edu.tw [Institute of Genome Sciences, School of Life Sciences, National Yang-Ming University, Taipei, Taiwan, ROC (China); Department of Dentistry, School of Dentistry, National Yang-Ming University, Taipei, Taiwan, ROC (China)

    2013-11-15

    Using immuno-fluorescent probing and Western blotting analysis, we reveal the exclusive cytoplasm nature of the small subunit ribosomal protein S20. To illustrate the importance of the cellular compartmentation of S20 to the function of small subunit 40S, we created a nuclear resident S20{sub NLS} mutant gene and examined polysome profile of cells that had been transfected with the S20{sub NLS} gene. As a result, we observed the formation of recombinant 40S carried S20{sub NLS} but this recombinant 40S was never found in the polysome, suggesting such a recombinant 40S was translation incompetent. Moreover, by the tactic of the energy depletion and restoration, we were able to restrain the nuclear-resided S20{sub NLS} in the cytoplasm. Yet, along a progressive energy restoration, we observed the presence of recombinant 40S subunits carrying the S20{sub NLS} in the polysome. This proves that S20 needs to be cytoplasmic in order to make a functional 40S subunit. Furthermore, it also implies that the assembly order of ribosomal protein in eukaryote is orderly regulated. - Highlights: • The step of S20 assembled on 40S is happened in the cytoplasm. • A small subunit assembled with a nuclear S20{sub NLS} is translational incompetence. • Using energy depletion and recovery to manipulate the cellular compartment of S20{sub NLS}. • Cytoplasm-retained S20{sub NLS} is crucial for creating a functional small subunit.

  4. The intermediate filament protein vimentin binds specifically to a recombinant integrin α2/β1 cytoplasmic tail complex and co-localizes with native α2/β1 in endothelial cell focal adhesions

    International Nuclear Information System (INIS)

    Kreis, Stephanie; Schoenfeld, Hans-Joachim; Melchior, Chantal; Steiner, Beat; Kieffer, Nelly

    2005-01-01

    Integrin receptors are crucial players in cell adhesion and migration. Identification and characterization of cellular proteins that interact with their short α and β cytoplasmic tails will help to elucidate the molecular mechanisms by which integrins mediate bi-directional signaling across the plasma membrane. Integrin α2β1 is a major collagen receptor but to date, only few proteins have been shown to interact with the α2 cytoplasmic tail or with the α2β1 complex. In order to identify novel binding partners of a α2β1cytoplasmic domain complex, we have generated recombinant GST-fusion proteins, incorporating the leucine zipper heterodimerization cassettes of Jun and Fos. To ascertain proper functionality of the recombinant proteins, interaction with natural binding partners was tested. GST-α2 and GST-Jun α2 bound His-tagged calreticulin while GST-β1 and GST-Fos β1 proteins bound talin. In screening assays for novel binding partners, the immobilized GST-Jun α2/GST-Fos β1 heterodimeric complex, but not the single subunits, interacted specifically with endothelial cell-derived vimentin. Vimentin, an abundant intermediate filament protein, has previously been shown to co-localize with αvβ3-positive focal contacts. Here, we provide evidence that this interaction also occurs with α2β1-enriched focal adhesions and we further show that this association is lost after prolonged adhesion of endothelial cells to collagen

  5. Compartment syndrome without pain!

    LENUS (Irish Health Repository)

    O'Sullivan, M J

    2012-02-03

    We report the case of a young male patient who underwent intra-medullary nailing for a closed, displaced mid-shaft fracture of tibia and fibula. He was commenced on patient controlled analgesia post-operatively. A diagnosis of compartment syndrome in the patient\\'s leg was delayed because he did not exhibit a pain response. This ultimately resulted in a below-knee amputation of the patient\\'s leg. We caution against the use of patient controlled analgesia in any traumatised limb distal to the hip or the shoulder.

  6. Herpes simplex virus replication compartments can form by coalescence of smaller compartments

    International Nuclear Information System (INIS)

    Taylor, Travis J; McNamee, Elizabeth E.; Day, Cheryl; Knipe, David M.

    2003-01-01

    Herpes simplex virus (HSV) uses intranuclear compartmentalization to concentrate the viral and cellular factors required for the progression of the viral life cycle. Processes as varied as viral DNA replication, late gene expression, and capsid assembly take place within discrete structures within the nucleus called replication compartments. Replication compartments are hypothesized to mature from a few distinct structures, called prereplicative sites, that form adjacent to cellular nuclear matrix-associated ND10 sites. During productive infection, the HSV single-stranded DNA-binding protein ICP8 localizes to replication compartments. To further the understanding of replication compartment maturation, we have constructed and characterized a recombinant HSV-1 strain that expresses an ICP8 molecule with green fluorescent protein (GFP) fused to its C terminus. In transfected Vero cells that were infected with HSV, the ICP8-GFP protein localized to prereplicative sites in the presence of the viral DNA synthesis inhibitor phosphonoacetic acid (PAA) or to replication compartments in the absence of PAA. A recombinant HSV-1 strain expressing the ICP8-GFP virus replicated in Vero cells, but the yield was increased by 150-fold in an ICP8-complementing cell line. Using the ICP8-GFP protein as a marker for replication compartments, we show here that these structures start as punctate structures early in infection and grow into large, globular structures that eventually fill the nucleus. Large replication compartments were formed by small structures that either moved through the nucleus to merge with adjacent compartments or remained relatively stationary within the nucleus and grew by accretion and fused with neighboring structures

  7. Modeling of compartment fire

    International Nuclear Information System (INIS)

    Sathiah, P.; Siccama, A.; Visser, D.; Komen, E.

    2011-01-01

    Fire accident in a containment is a serious threat to nuclear reactors. Fire can cause substantial loss to life and property. The risk posed by fire can also exceed the risk from internal events within a nuclear reactor. Numerous research efforts have been performed to understand and analyze the phenomenon of fire in nuclear reactor and its consequences. Modeling of fire is an important subject in the field of fire safety engineering. Two approaches which are commonly used in fire modeling are zonal modeling and field modeling. The objective of this work is to compare zonal and field modeling approach against a pool fired experiment performed in a well-confined compartment. Numerical simulations were performed against experiments, which were conducted within PRISME program under the framework of OECD. In these experiments, effects of ventilation flow rate on heat release rate in a confined and mechanically ventilated compartment is investigated. Time dependent changes in gas temperature and oxygen mass fraction were measured. The trends obtained by numerical simulation performed using zonal model and field model compares well with experiments. Further validation is needed before this code can be used for fire safety analyses. (author)

  8. Myeloma cell-induced disruption of bone remodelling compartments leads to osteolytic lesions and generation of osteoclast-myeloma hybrid cells

    DEFF Research Database (Denmark)

    Andersen, Thomas L; Søe, Kent; Søndergaard, Teis Esben

    2010-01-01

    on the physical organisation of the myeloma cell microenvironment. The proximity between myeloma cells and osteoclasts or osteoblasts was shown to be conditioned by the recently discovered layer of flat cells that separates the osteoclasts and osteoblasts from the bone marrow, by forming a canopy over bone...

  9. Kidney compartment model

    International Nuclear Information System (INIS)

    Gullberg, G.T.

    1976-09-01

    A multiparameter kidney compartment model which quantitates the amount of iodohippurate concentration as a function of time in the blood, tissue, kidneys and bladder is developed from a system of differential equations which represent first order kinetics. The kinetic data are obtained using a gamma camera and an HP5407 computer system which allows one to delineate areas of interest for the blood and tissue, kidneys, and bladder thus separating the data into four data sets. The estimated tubular transit times have a high ratio of the signal to the variance whereas the estimates of the amount of iodohippurate in the blood, tissue and kidneys have a low ratio of the signal to the variance. Application of this model to patient data requires better statistics than available with conventional 131 I-hippurate doses; thus a true test of the efficacy awaits availability of 123 I-hippurate

  10. Xenopus egg cytoplasm with intact actin.

    Science.gov (United States)

    Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

    2014-01-01

    We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts. © 2014 Elsevier Inc. All rights reserved.

  11. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    International Nuclear Information System (INIS)

    Gou, Ke-Mian; Chang, Chia-Chun; Shen, Qing-Ji; Sung, Li-Ying; Liu, Ji-Long

    2014-01-01

    CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus

  12. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    Energy Technology Data Exchange (ETDEWEB)

    Gou, Ke-Mian [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193 (China); Chang, Chia-Chun [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Shen, Qing-Ji [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); Sung, Li-Ying, E-mail: liyingsung@ntu.edu.tw [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan, ROC (China); Liu, Ji-Long, E-mail: jilong.liu@dpag.ox.ac.uk [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom)

    2014-04-15

    CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus.

  13. Hydrogen Peroxide Probes Directed to Different Cellular Compartments

    Science.gov (United States)

    Malinouski, Mikalai; Zhou, You; Belousov, Vsevolod V.; Hatfield, Dolph L.; Gladyshev, Vadim N.

    2011-01-01

    Background Controlled generation and removal of hydrogen peroxide play important roles in cellular redox homeostasis and signaling. We used a hydrogen peroxide biosensor HyPer, targeted to different compartments, to examine these processes in mammalian cells. Principal Findings Reversible responses were observed to various redox perturbations and signaling events. HyPer expressed in HEK 293 cells was found to sense low micromolar levels of hydrogen peroxide. When targeted to various cellular compartments, HyPer occurred in the reduced state in the nucleus, cytosol, peroxisomes, mitochondrial intermembrane space and mitochondrial matrix, but low levels of the oxidized form of the biosensor were also observed in each of these compartments, consistent with a low peroxide tone in mammalian cells. In contrast, HyPer was mostly oxidized in the endoplasmic reticulum. Using this system, we characterized control of hydrogen peroxide in various cell systems, such as cells deficient in thioredoxin reductase, sulfhydryl oxidases or subjected to selenium deficiency. Generation of hydrogen peroxide could also be monitored in various compartments following signaling events. Conclusions We found that HyPer can be used as a valuable tool to monitor hydrogen peroxide generated in different cellular compartments. The data also show that hydrogen peroxide generated in one compartment could translocate to other compartments. Our data provide information on compartmentalization, dynamics and homeostatic control of hydrogen peroxide in mammalian cells. PMID:21283738

  14. Hydrogen peroxide probes directed to different cellular compartments.

    Directory of Open Access Journals (Sweden)

    Mikalai Malinouski

    2011-01-01

    Full Text Available Controlled generation and removal of hydrogen peroxide play important roles in cellular redox homeostasis and signaling. We used a hydrogen peroxide biosensor HyPer, targeted to different compartments, to examine these processes in mammalian cells.Reversible responses were observed to various redox perturbations and signaling events. HyPer expressed in HEK 293 cells was found to sense low micromolar levels of hydrogen peroxide. When targeted to various cellular compartments, HyPer occurred in the reduced state in the nucleus, cytosol, peroxisomes, mitochondrial intermembrane space and mitochondrial matrix, but low levels of the oxidized form of the biosensor were also observed in each of these compartments, consistent with a low peroxide tone in mammalian cells. In contrast, HyPer was mostly oxidized in the endoplasmic reticulum. Using this system, we characterized control of hydrogen peroxide in various cell systems, such as cells deficient in thioredoxin reductase, sulfhydryl oxidases or subjected to selenium deficiency. Generation of hydrogen peroxide could also be monitored in various compartments following signaling events.We found that HyPer can be used as a valuable tool to monitor hydrogen peroxide generated in different cellular compartments. The data also show that hydrogen peroxide generated in one compartment could translocate to other compartments. Our data provide information on compartmentalization, dynamics and homeostatic control of hydrogen peroxide in mammalian cells.

  15. Cytoplasmic lipid bodies of human neutrophilic leukocytes

    International Nuclear Information System (INIS)

    Weller, P.F.; Ackerman, S.J.; Nicholson-Weller, A.; Dvorak, A.M.

    1989-01-01

    The morphology and function of cytoplasmic lipid bodies in human neutrophils were evaluated. By transmission electron microscopy, neutrophil lipid bodies were cytoplasmic inclusions, usually several microns in diameter, that occasionally coalesced to attain a diameter up to 7 microM. Neutrophil lipid bodies were not enveloped by membrane but were often surrounded by a more electron-dense shell at their periphery. Normal peripheral blood neutrophils contained an average of approximately one lipid body per cell. Lipid bodies appeared in greater numbers in neutrophils from inflammatory lesions. Perturbation of neutrophils during conventional methods of cell isolation and purification modestly increased lipid body numbers in neutrophils, whereas incubation of neutrophils with 1 microM oleic acid rapidly induced lipid body formation over 30 to 60 minutes. After granulocytes were incubated for 2 hours with 3H-fatty acids, including arachidonic, oleic, and palmitic acids, electron microscopic autoradiography demonstrated that lipid bodies represented the predominant intracellular sites of localization of each of the three 3H-fatty acids. There was lesser labeling noted in the perinuclear cisterna, but not in cell membranes. Virtually all of each of the three 3H-fatty acids incorporated by the neutrophils were esterified into chromatographically resolved classes of neutral lipids or phospholipids. These findings indicate that cytoplasmic lipid bodies are more prominent in neutrophils in vivo engaged in inflammatory responses and that these organelles in human neutrophils function as sites of deposition of esterified, incorporated fatty acids

  16. The impact of pH inhomogeneities on CHO cell physiology and fed-batch process performance - two-compartment scale-down modelling and intracellular pH excursion.

    Science.gov (United States)

    Brunner, Matthias; Braun, Philipp; Doppler, Philipp; Posch, Christoph; Behrens, Dirk; Herwig, Christoph; Fricke, Jens

    2017-07-01

    Due to high mixing times and base addition from top of the vessel, pH inhomogeneities are most likely to occur during large-scale mammalian processes. The goal of this study was to set-up a scale-down model of a 10-12 m 3 stirred tank bioreactor and to investigate the effect of pH perturbations on CHO cell physiology and process performance. Short-term changes in extracellular pH are hypothesized to affect intracellular pH and thus cell physiology. Therefore, batch fermentations, including pH shifts to 9.0 and 7.8, in regular one-compartment systems are conducted. The short-term adaption of the cells intracellular pH are showed an immediate increase due to elevated extracellular pH. With this basis of fundamental knowledge, a two-compartment system is established which is capable of simulating defined pH inhomogeneities. In contrast to state-of-the-art literature, the scale-down model is included parameters (e.g. volume of the inhomogeneous zone) as they might occur during large-scale processes. pH inhomogeneity studies in the two-compartment system are performed with simulation of temporary pH zones of pH 9.0. The specific growth rate especially during the exponential growth phase is strongly affected resulting in a decreased maximum viable cell density and final product titer. The gathered results indicate that even short-term exposure of cells to elevated pH values during large-scale processes can affect cell physiology and overall process performance. In particular, it could be shown for the first time that pH perturbations, which might occur during the early process phase, have to be considered in scale-down models of mammalian processes. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. SCA-1 Labels a Subset of Estrogen-Responsive Bipotential Repopulating Cells within the CD24+ CD49fhi Mammary Stem Cell-Enriched Compartment

    Directory of Open Access Journals (Sweden)

    Genevieve V. Dall

    2017-02-01

    Full Text Available Estrogen stimulates breast development during puberty and mammary tumors in adulthood through estrogen receptor-α (ERα. These effects are proposed to occur via ERα+ luminal cells and not the mammary stem cells (MaSCs that are ERαneg. Since ERα+ luminal cells express stem cell antigen-1 (SCA-1, we sought to determine if SCA-1 could define an ERα+ subset of EpCAM+/CD24+/CD49fhi MaSCs. We show that the MaSC population has a distinct SCA-1+ population that is abundant in pre-pubertal mammary glands. The SCA-1+ MaSCs have less stem cell markers and less in vivo repopulating activity than their SCA-1neg counterparts. However, they express ERα and specifically enter the cell cycle at puberty. Using estrogen-deficient aromatase knockouts (ArKO, we showed that the SCA-1+ MaSC could be directly modulated by estrogen supplementation. Thus, SCA-1 enriches for an ERα+, estrogen-sensitive subpopulation within the CD24+/CD49fhi MaSC population that may be responsible for the hormonal sensitivity of the developing mammary gland.

  18. Maternal microchimerism: increased in the insulin positive compartment of type 1 diabetes pancreas but not in infiltrating immune cells or replicating islet cells.

    Directory of Open Access Journals (Sweden)

    Jody Ye

    Full Text Available Maternal microchimeric cells (MMc transfer across the placenta during pregnancy. Increased levels of MMc have been observed in several autoimmune diseases including type 1 diabetes but their role is unknown. It has been suggested that MMc are 1 effector cells of the immune response, 2 targets of the autoimmune response or 3 play a role in tissue repair. The aim of this study was to define the cellular phenotype of MMc in control (n = 14 and type 1 diabetes pancreas (n = 8.Using sex chromosome-based fluorescence in-situ hybridization, MMc were identified in male pancreas and their phenotype determined by concomitant immunofluorescence.In normal pancreas, MMc positive for endocrine, exocrine, duct and acinar markers were identified suggesting that these cells are derived from maternal progenitors. Increased frequencies of MMc were observed in type 1 diabetes pancreas (p = 0.03 with particular enrichment in the insulin positive fraction (p = 0.01. MMc did not contribute to infiltrating immune cells or Ki67+ islet cell populations in type 1 diabetes.These studies provide support for the hypothesis that MMc in human pancreas are derived from pancreatic precursors. Increased frequencies of MMc beta cells may contribute to the initiation of autoimmunity or to tissue repair but do not infiltrate islets in type 1 diabetes.

  19. A Novel View of the Adult Stem Cell Compartment From the Perspective of a Quiescent Population of Very Small Embryonic-Like Stem Cells.

    Science.gov (United States)

    Ratajczak, Mariusz Z; Ratajczak, Janina; Suszynska, Malwina; Miller, Donald M; Kucia, Magda; Shin, Dong-Myung

    2017-01-06

    Evidence has accumulated that adult hematopoietic tissues and other organs contain a population of dormant stem cells (SCs) that are more primitive than other, already restricted, monopotent tissue-committed SCs (TCSCs). These observations raise several questions, such as the developmental origin of these cells, their true pluripotent or multipotent nature, which surface markers they express, how they can be efficiently isolated from adult tissues, and what role they play in the adult organism. The phenotype of these cells and expression of some genes characteristic of embryonic SCs, epiblast SCs, and primordial germ cells suggests their early-embryonic deposition in developing tissues as precursors of adult SCs. In this review, we will critically discuss all these questions and the concept that small dormant SCs related to migratory primordial germ cells, described as very small embryonic-like SCs, are deposited during embryogenesis in bone marrow and other organs as a backup population for adult tissue-committed SCs and are involved in several processes related to tissue or organ rejuvenation, aging, and cancerogenesis. The most recent results on successful ex vivo expansion of human very small embryonic-like SC in chemically defined media free from feeder-layer cells open up new and exciting possibilities for their application in regenerative medicine. © 2017 American Heart Association, Inc.

  20. Differential dynamics of splicing factor SC35 during the cell cycle

    Indian Academy of Sciences (India)

    Fluorescence recovery after photobleaching (FRAP) experiments revealed that the mobility of GFP-SC35 was distinct in different mitotic compartments. Interestingly, the mobility of GFP-SC35 was 3-fold higher in the cytoplasm of metaphase cells compared with interphase speckles, the nucleoplasm or MIGs. Treatment of ...

  1. Reduced CD5(+) CD24(hi) CD38(hi) and interleukin-10(+) regulatory B cells in active anti-neutrophil cytoplasmic autoantibody-associated vasculitis permit increased circulating autoantibodies.

    Science.gov (United States)

    Aybar, L T; McGregor, J G; Hogan, S L; Hu, Y; Mendoza, C E; Brant, E J; Poulton, C J; Henderson, C D; Falk, R J; Bunch, D O

    2015-05-01

    Pathogenesis of anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis is B cell-dependent, although how particular B cell subsets modulate immunopathogenesis remains unknown. Although their phenotype remains controversial, regulatory B cells (Bregs ), play a role in immunological tolerance via interleukin (IL)-10. Putative CD19(+) CD24(hi) CD38(hi) and CD19(+) CD24(hi) CD27(+) Bregs were evaluated in addition to their CD5(+) subsets in 69 patients with ANCA-associated vasculitis (AAV). B cell IL-10 was verified by flow cytometry following culture with CD40 ligand and cytosine-phosphate-guanosine (CpG) DNA. Patients with active disease had decreased levels of CD5(+) CD24(hi) CD38(hi) B cells and IL-10(+) B cells compared to patients in remission and healthy controls (HCs). As IL-10(+) and CD5(+) CD24(hi) CD38(hi) B cells normalized in remission within an individual, ANCA titres decreased. The CD5(+) subset of CD24(hi) CD38(hi) B cells decreases in active disease and rebounds during remission similarly to IL-10-producing B cells. Moreover, CD5(+) B cells are enriched in the ability to produce IL-10 compared to CD5(neg) B cells. Together these results suggest that CD5 may identify functional IL-10-producing Bregs . The malfunction of Bregs during active disease due to reduced IL-10 expression may thus permit ANCA production. © 2014 British Society for Immunology.

  2. The subapical compartment : a traffic center in membrane polarity development

    NARCIS (Netherlands)

    Hoekstra, D; Tyteca, D; van IJzendoorn, SCD

    2004-01-01

    Spatially separated apical and basolateral plasma membrane domains that have distinct functions and molecular compositions are a characteristic feature of epithelial cell polarity. The subapical compartment (SAC), also known as the common endosome (CE), where endocytic pathways from both surfaces

  3. Dynamic and nucleolin-dependent localization of human cytomegalovirus UL84 to the periphery of viral replication compartments and nucleoli.

    Science.gov (United States)

    Bender, Brian J; Coen, Donald M; Strang, Blair L

    2014-10-01

    Protein-protein and protein-nucleic acid interactions within subcellular compartments are required for viral genome replication. To understand the localization of the human cytomegalovirus viral replication factor UL84 relative to other proteins involved in viral DNA synthesis and to replicating viral DNA in infected cells, we created a recombinant virus expressing a FLAG-tagged version of UL84 (UL84FLAG) and used this virus in immunofluorescence assays. UL84FLAG localization differed at early and late times of infection, transitioning from diffuse distribution throughout the nucleus to exclusion from the interior of replication compartments, with some concentration at the periphery of replication compartments with newly labeled DNA and the viral DNA polymerase subunit UL44. Early in infection, UL84FLAG colocalized with the viral single-stranded DNA binding protein UL57, but colocalization became less prominent as infection progressed. A portion of UL84FLAG also colocalized with the host nucleolar protein nucleolin at the peripheries of both replication compartments and nucleoli. Small interfering RNA (siRNA)-mediated knockdown of nucleolin resulted in a dramatic elimination of UL84FLAG from replication compartments and other parts of the nucleus and its accumulation in the cytoplasm. Reciprocal coimmunoprecipitation of viral proteins from infected cell lysates revealed association of UL84, UL44, and nucleolin. These results indicate that UL84 localization during infection is dynamic, which is likely relevant to its functions, and suggest that its nuclear and subnuclear localization is highly dependent on direct or indirect interactions with nucleolin. Importance: The protein-protein interactions among viral and cellular proteins required for replication of the human cytomegalovirus (HCMV) DNA genome are poorly understood. We sought to understand how an enigmatic HCMV protein critical for virus replication, UL84, localizes relative to other viral and cellular

  4. Stabilization and Degradation Mechanisms of Cytoplasmic Ataxin-1

    Directory of Open Access Journals (Sweden)

    Mayumi F. Kohiyama

    2015-01-01

    Full Text Available Aggregation-prone proteins in neurodegenerative disease disrupt cellular protein stabilization and degradation pathways. The neurodegenerative disease spinocerebellar ataxia type 1 (SCA1 is caused by a coding polyglutamine expansion in the Ataxin-1 gene ( ATXN1 , which gives rise to the aggregation-prone mutant form of ATXN1 protein. Cerebellar Purkinje neurons, preferentially vulnerable in SCA1, produce ATXN1 protein in both cytoplasmic and nuclear compartments. Cytoplasmic stabilization of ATXN1 by phosphorylation and 14-3-3-mediated mechanisms ultimately drive translocation of the protein to the nucleus where aggregation may occur. However, experimental inhibition of phosphorylation and 14-3-3 binding results in rapid degradation of ATXN1, thus preventing nuclear translocation and cellular toxicity. The exact mechanism of cytoplasmic ATXN1 degradation is currently unknown; further investigation of degradation may provide future therapeutic targets. This review examines the present understanding of cytoplasmic ATXN1 stabilization and potential degradation mechanisms during normal and pathogenic states.

  5. Cytoplasmic chromatin triggers inflammation in senescence and cancer.

    Science.gov (United States)

    Dou, Zhixun; Ghosh, Kanad; Vizioli, Maria Grazia; Zhu, Jiajun; Sen, Payel; Wangensteen, Kirk J; Simithy, Johayra; Lan, Yemin; Lin, Yanping; Zhou, Zhuo; Capell, Brian C; Xu, Caiyue; Xu, Mingang; Kieckhaefer, Julia E; Jiang, Tianying; Shoshkes-Carmel, Michal; Tanim, K M Ahasan Al; Barber, Glen N; Seykora, John T; Millar, Sarah E; Kaestner, Klaus H; Garcia, Benjamin A; Adams, Peter D; Berger, Shelley L

    2017-10-19

    Chromatin is traditionally viewed as a nuclear entity that regulates gene expression and silencing. However, we recently discovered the presence of cytoplasmic chromatin fragments that pinch off from intact nuclei of primary cells during senescence, a form of terminal cell-cycle arrest associated with pro-inflammatory responses. The functional significance of chromatin in the cytoplasm is unclear. Here we show that cytoplasmic chromatin activates the innate immunity cytosolic DNA-sensing cGAS-STING (cyclic GMP-AMP synthase linked to stimulator of interferon genes) pathway, leading both to short-term inflammation to restrain activated oncogenes and to chronic inflammation that associates with tissue destruction and cancer. The cytoplasmic chromatin-cGAS-STING pathway promotes the senescence-associated secretory phenotype in primary human cells and in mice. Mice deficient in STING show impaired immuno-surveillance of oncogenic RAS and reduced tissue inflammation upon ionizing radiation. Furthermore, this pathway is activated in cancer cells, and correlates with pro-inflammatory gene expression in human cancers. Overall, our findings indicate that genomic DNA serves as a reservoir to initiate a pro-inflammatory pathway in the cytoplasm in senescence and cancer. Targeting the cytoplasmic chromatin-mediated pathway may hold promise in treating inflammation-related disorders.

  6. Cloning of a novel cell type from human fetal liver expressing cytoplasmic CD3 delta and epsilon but not membrane CD3

    NARCIS (Netherlands)

    Hori, T.; de Waal Malefyt, R.; Duncan, B. W.; Harrison, M. R.; Roncarolo, M. G.; Spits, H.

    1991-01-01

    Seventeen-week human fetal liver cells cultured with a feeder cell mixture of irradiated PBL, irradiated JY cells (an EBV-transformed B cell line) and PHA contained a subpopulation of CD3- cells in addition to a major population of T cells with the mature phenotype. After 12 days in culture, CD3-

  7. Cytoplasmic transduction peptide (CTP): New approach for the delivery of biomolecules into cytoplasm in vitro and in vivo

    International Nuclear Information System (INIS)

    Kim, Daeyou; Jeon, Choonju; Kim, Jeong-Hwan; Kim, Mi-Seon; Yoon, Cheol-Hee; Choi, In-Soo; Kim, Sung-Hoon; Bae, Yong-Soo

    2006-01-01

    The protein transduction domain (PTD) of HIV-1 TAT has been extensively documented with regard to its membrane transduction potential, as well as its efficient delivery of biomolecules in vivo. However, the majority of PTD and PTD-conjugated molecules translocate to the nucleus rather than to the cytoplasm after transduction, due to the functional nuclear localization sequence (NLS). Here, we report a cytoplasmic transduction peptide (CTP), which was deliberately designed to ensure the efficient cytoplasmic delivery of the CTP-fused biomolecules. In comparison with PTD, CTP and its fusion partners exhibited a clear preference for cytoplasmic localization, and also markedly enhanced membrane transduction potential. Unlike the mechanism underlying PTD-mediated transduction, CTP-mediated transduction occurs independently of the lipid raft-dependent macropinocytosis pathway. The CTP-conjugated Smac/DIABLO peptide (Smac-CTP) was also shown to be much more efficient than Smac-PTD in the blockage of the antiapoptotic properties of XIAP, suggesting that cytoplasmic functional molecules can be more efficiently targeted by CTP-mediated delivery. In in vivo trafficking studies, CTP-fused β-gal exhibited unique organ tropisms to the liver and lymph nodes when systemically injected into mice, whereas PTD-β-gal exhibited no such tropisms. Taken together, our findings implicate CTP as a novel delivery peptide appropriate for (i) molecular targeting to cytoplasmic compartments in vitro, (ii) the development of class I-associated CTL vaccines, and (iii) special drug delivery in vivo, without causing any untoward effects on nuclear genetic material

  8. Double-compartment wrist arthrography

    International Nuclear Information System (INIS)

    Quinn, S.F.; Pittman, C.; Belsole, R.; Greene, T.L.; Rayhack, J.; Clark, R.A.; King, P.S.

    1987-01-01

    Seventy patients with clinical wrist problems were studied with double-compartment wrist arthrography. Midcarpal and radiocarpal compartment arthrograms were obtained in all patients. Digital subtraction technique was used to subtract out contrast from the first compartmental injection. Digital technique also allowed a dynamic record of each injection, which helped determine sites of intercompartmental communication. Postarthrography exercises recorded on videotape were performed after each injection. There were 34 normal studies. Abnormalities in the other 36 patients included: scapholunate communication (n = 9), lunatotriquetral communication (n = 6), communication with tendon sheaths (n = 4), communication with distal radioulnar compartment (n = 14), abnormal synovium process (n = 9), and communication through the radial or ulnar collateral ligament (n = 3). Double-compartment wrist arthrography may provide additional information for complex problems of the wrist

  9. The Capsicum annuum class IV chitinase ChitIV interacts with receptor-like cytoplasmic protein kinase PIK1 to accelerate PIK1-triggered cell death and defence responses

    Science.gov (United States)

    Kim, Dae Sung; Kim, Nak Hyun; Hwang, Byung Kook

    2015-01-01

    The pepper receptor-like cytoplasmic protein kinase, CaPIK1, which mediates signalling of plant cell death and defence responses was previously identified. Here, the identification of a class IV chitinase, CaChitIV, from pepper plants (Capsicum annuum), which interacts with CaPIK1 and promotes CaPIK1-triggered cell death and defence responses, is reported. CaChitIV contains a signal peptide, chitin-binding domain, and glycol hydrolase domain. CaChitIV expression was up-regulated by Xanthomonas campestris pv. vesicatoria (Xcv) infection. Notably, avirulent Xcv infection rapidly induced CaChitIV expression in pepper leaves. Bimolecular fluorescence complementation and co-immunoprecipitation revealed that CaPIK1 interacts with CaChitIV in planta, and that the CaPIK1–CaChitIV complex is localized mainly in the cytoplasm and plasma membrane. CaChitIV is also localized in the endoplasmic reticulum. Transient co-expression of CaChitIV with CaPIK1 enhanced CaPIK1-triggered cell death response and reactive oxygen species (ROS) and nitric oxide (NO) bursts. Co-silencing of both CaChitIV and CaPIK1 in pepper plants conferred enhanced susceptibility to Xcv infection, which was accompanied by a reduced induction of cell death response, ROS and NO bursts, and defence response genes. Ectopic expression of CaPIK1 in Arabidopsis enhanced basal resistance to Hyaloperonospora arabidopsidis infection. Together, the results suggest that CaChitIV positively regulates CaPIK1-triggered cell death and defence responses through its interaction with CaPIK1. PMID:25694549

  10. Uniparental Inheritance Promotes Adaptive Evolution in Cytoplasmic Genomes.

    Science.gov (United States)

    Christie, Joshua R; Beekman, Madeleine

    2017-03-01

    Eukaryotes carry numerous asexual cytoplasmic genomes (mitochondria and plastids). Lacking recombination, asexual genomes should theoretically suffer from impaired adaptive evolution. Yet, empirical evidence indicates that cytoplasmic genomes experience higher levels of adaptive evolution than predicted by theory. In this study, we use a computational model to show that the unique biology of cytoplasmic genomes-specifically their organization into host cells and their uniparental (maternal) inheritance-enable them to undergo effective adaptive evolution. Uniparental inheritance of cytoplasmic genomes decreases competition between different beneficial substitutions (clonal interference), promoting the accumulation of beneficial substitutions. Uniparental inheritance also facilitates selection against deleterious cytoplasmic substitutions, slowing Muller's ratchet. In addition, uniparental inheritance generally reduces genetic hitchhiking of deleterious substitutions during selective sweeps. Overall, uniparental inheritance promotes adaptive evolution by increasing the level of beneficial substitutions relative to deleterious substitutions. When we assume that cytoplasmic genome inheritance is biparental, decreasing the number of genomes transmitted during gametogenesis (bottleneck) aids adaptive evolution. Nevertheless, adaptive evolution is always more efficient when inheritance is uniparental. Our findings explain empirical observations that cytoplasmic genomes-despite their asexual mode of reproduction-can readily undergo adaptive evolution. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  11. Uniparental Inheritance Promotes Adaptive Evolution in Cytoplasmic Genomes

    Science.gov (United States)

    Christie, Joshua R.; Beekman, Madeleine

    2017-01-01

    Eukaryotes carry numerous asexual cytoplasmic genomes (mitochondria and plastids). Lacking recombination, asexual genomes should theoretically suffer from impaired adaptive evolution. Yet, empirical evidence indicates that cytoplasmic genomes experience higher levels of adaptive evolution than predicted by theory. In this study, we use a computational model to show that the unique biology of cytoplasmic genomes—specifically their organization into host cells and their uniparental (maternal) inheritance—enable them to undergo effective adaptive evolution. Uniparental inheritance of cytoplasmic genomes decreases competition between different beneficial substitutions (clonal interference), promoting the accumulation of beneficial substitutions. Uniparental inheritance also facilitates selection against deleterious cytoplasmic substitutions, slowing Muller’s ratchet. In addition, uniparental inheritance generally reduces genetic hitchhiking of deleterious substitutions during selective sweeps. Overall, uniparental inheritance promotes adaptive evolution by increasing the level of beneficial substitutions relative to deleterious substitutions. When we assume that cytoplasmic genome inheritance is biparental, decreasing the number of genomes transmitted during gametogenesis (bottleneck) aids adaptive evolution. Nevertheless, adaptive evolution is always more efficient when inheritance is uniparental. Our findings explain empirical observations that cytoplasmic genomes—despite their asexual mode of reproduction—can readily undergo adaptive evolution. PMID:28025277

  12. Dual-Compartment Inflatable Suitlock

    Science.gov (United States)

    Kennedy, Kriss J.; Guirgis, Peggy L.; Boyle, Robert M.

    2013-01-01

    There is a need for an improvement over current NASA Extravehicular Activity (EVA) technology. The technology must allow the capacity for quicker, more efficient egress/ingress, allow for shirtsleeve suit maintenance, be compact in transport, and be applicable to environments ranging from planetary surface (partial-g) to orbital or deep space zero-g environments. The technology must also be resistant to dust and other foreign contaminants that may be present on or around a planetary surface. The technology should be portable, and be capable of docking with a variety of habitats, ports, stations, vehicles, and other pressurized modules. The Dual-Compartment Inflatable Suitlock (DCIS) consists of three hard inline bulkheads, separating two cylindrical membrane-walled compartments. The Inner Bulkhead can be fitted with a variety of hatch types, docking flanges, and mating hardware, such as the Common Berthing Mechanism (CBM), for the purpose of mating with vehicles, habitats, and other pressurized modules. The Inner Bulkhead and Center Bulkhead function as the end walls of the Inner Compartment, which during operations, would stay pressurized, either matching the pressure of the habitat or acting as a lower-pressure transitional volume. The Inner Compartment contains donning/doffing fixtures and inner suit-port hatches. The Center Bulkhead has two integrated suit-ports along with a maintenance hatch. The Center Bulkhead and Outer Bulkhead function as the end walls of the Outer Compartment, which stays at vacuum during normal operations. This allows the crewmember to quickly don a suit, and egress the suitlock without waiting for the Outer Compartment to depressurize. The Outer Compartment can be pressurized infrequently for both nominal and off-nominal suit maintenance tasks, allowing shirtsleeve inspections and maintenance/repair of the environmental suits. The Outer Bulkhead has a pressure-assisted hatch door that stays open and stowed during EVA operations, but can

  13. Cytoplasmic Localization of HTLV-1 HBZ Protein: A Biomarker of HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP).

    Science.gov (United States)

    Baratella, Marco; Forlani, Greta; Raval, Goutham U; Tedeschi, Alessandra; Gout, Olivier; Gessain, Antoine; Tosi, Giovanna; Accolla, Roberto S

    2017-01-01

    HTLV-1 is the causative agent of a severe form of adult T cell leukemia/Lymphoma (ATL), and of a chronic progressive neuromyelopathy designated HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). Two important HTLV-1-encoded proteins, Tax-1 and HBZ, play crucial roles in the generation and maintenance of the oncogenic process. Less information is instead available on the molecular and cellular mechanisms leading to HAM/TSP. More importantly, no single specific biomarker has been described that unambiguously define the status of HAM/TSP. Here we report for the first time the finding that HBZ, described until now as an exclusive nuclear protein both in chronically infected and in ATL cells, is instead exclusively localized in the cytoplasm of peripheral blood mononuclear cells (PBMC) from patients suffering of HAM/TSP. Interestingly, at the single cell level, HBZ and Tax-1 proteins are never found co-expressed in the same cell, suggesting the existence of mechanisms of expression uncoupling of these two important HTLV-1 viral products in HAM/TSP patients. Cells expressing cytoplasmic HBZ were almost exclusively found in the CD4+ T cell compartment that was not, at least in a representative HAM/TSP patient, expressing the CD25 marker. Less than 1 percent CD8+ T cells were fond positive for HBZ, while B cells and NK cells were found negative for HBZ in HAM/TSP patients. Our results identify the cytoplasmic localization of HBZ in HAM/TSP patient as a possible biomarker of this rather neglected tropical disease, and raise important hypotheses on the role of HBZ in the pathogenesis of the neuromyelopathy associated to HTLV-1 infection.

  14. Cytoplasmic Localization of HTLV-1 HBZ Protein: A Biomarker of HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP.

    Directory of Open Access Journals (Sweden)

    Marco Baratella

    2017-01-01

    Full Text Available HTLV-1 is the causative agent of a severe form of adult T cell leukemia/Lymphoma (ATL, and of a chronic progressive neuromyelopathy designated HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP. Two important HTLV-1-encoded proteins, Tax-1 and HBZ, play crucial roles in the generation and maintenance of the oncogenic process. Less information is instead available on the molecular and cellular mechanisms leading to HAM/TSP. More importantly, no single specific biomarker has been described that unambiguously define the status of HAM/TSP. Here we report for the first time the finding that HBZ, described until now as an exclusive nuclear protein both in chronically infected and in ATL cells, is instead exclusively localized in the cytoplasm of peripheral blood mononuclear cells (PBMC from patients suffering of HAM/TSP. Interestingly, at the single cell level, HBZ and Tax-1 proteins are never found co-expressed in the same cell, suggesting the existence of mechanisms of expression uncoupling of these two important HTLV-1 viral products in HAM/TSP patients. Cells expressing cytoplasmic HBZ were almost exclusively found in the CD4+ T cell compartment that was not, at least in a representative HAM/TSP patient, expressing the CD25 marker. Less than 1 percent CD8+ T cells were fond positive for HBZ, while B cells and NK cells were found negative for HBZ in HAM/TSP patients. Our results identify the cytoplasmic localization of HBZ in HAM/TSP patient as a possible biomarker of this rather neglected tropical disease, and raise important hypotheses on the role of HBZ in the pathogenesis of the neuromyelopathy associated to HTLV-1 infection.

  15. Co-expression of nuclear and cytoplasmic HMGB1 is inversely associated with infiltration of CD45RO+ T cells and prognosis in patients with stage IIIB colon cancer

    International Nuclear Information System (INIS)

    Peng, Rui-Qing; Zeng, Yi-Xin; Zhang, Xiao-Shi; Wu, Xiao-Jun; Ding, Ya; Li, Chun-Yan; Yu, Xing-Juan; Zhang, Xing; Pan, Zhi-Zhong; Wan, De-Sen; Zheng, Li-Ming

    2010-01-01

    The intratumoral infiltration of T cells, especially memory T cells, is associated with a favorable prognosis in early colorectal cancers. However, the mechanism underlying this process remains elusive. This study examined whether high-mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) molecule, is involved in the infiltration of T cells and disease progression in locally advanced colon cancer. Seventy-two cases of pathologically-confirmed specimens were obtained from patients with stage IIIB (T3N1M0) colon cancer who underwent radical resection between January 1999 and May 2002 at the Cancer Center of Sun Yat-Sen University. The density of tumor-infiltrating lymphocytes (TILs) within the tumor tissue and the expression of HMGB1 in the cancer cells were examined via immunohistochemical analysis. The phenotype of CD45RO+ cells was confirmed using a flow cytometric assay. The association between HMGB1 expression, the density of TILs, and the 5-year survival rate were analyzed. The density of CD45RO+ T cells within the tumor was independently prognostic, although a higher density of CD3+ T cells was also associated with a favorable prognosis. More importantly, the expression of HMGB1 was observed in both the nucleus and the cytoplasm (co-expression pattern) in a subset of colon cancer tissues, whereas nuclear-only expression of HMGB1 (nuclear expression pattern) existed in most of the cancer tissues and normal mucosa. The co-expression pattern of HMGB1 in colon cancer cells was inversely associated with the infiltration of both CD3+ and CD45RO+ T cells and 5-year survival rates. This study revealed that the co-expression of HMGB1 is inversely associated with the infiltration of CD45RO+ T cells and prognosis in patients with stage IIIB colon cancer, indicating that the distribution patterns of HMGB1 might contribute to the progression of colon cancer via modulation of the local immune response

  16. Control of cytoplasmic and nuclear protein kinase A by phosphodiesterases and phosphatases in cardiac myocytes

    Science.gov (United States)

    Haj Slimane, Zeineb; Bedioune, Ibrahim; Lechêne, Patrick; Varin, Audrey; Lefebvre, Florence; Mateo, Philippe; Domergue-Dupont, Valérie; Dewenter, Matthias; Richter, Wito; Conti, Marco; El-Armouche, Ali; Zhang, Jin; Fischmeister, Rodolphe; Vandecasteele, Grégoire

    2014-01-01

    Aims The cAMP-dependent protein kinase (PKA) mediates β-adrenoceptor (β-AR) regulation of cardiac contraction and gene expression. Whereas PKA activity is well characterized in various subcellular compartments of adult cardiomyocytes, its regulation in the nucleus remains largely unknown. The aim of the present study was to compare the modalities of PKA regulation in the cytoplasm and nucleus of cardiomyocytes. Methods and results Cytoplasmic and nuclear cAMP and PKA activity were measured with targeted fluorescence resonance energy transfer probes in adult rat ventricular myocytes. β-AR stimulation with isoprenaline (Iso) led to fast cAMP elevation in both compartments, whereas PKA activity was fast in the cytoplasm but markedly slower in the nucleus. Iso was also more potent and efficient in activating cytoplasmic than nuclear PKA. Similar slow kinetics of nuclear PKA activation was observed upon adenylyl cyclase activation with L-858051 or phosphodiesterase (PDE) inhibition with 3-isobutyl-1-methylxantine. Consistently, pulse stimulation with Iso (15 s) maximally induced PKA and myosin-binding protein C phosphorylation in the cytoplasm, but marginally activated PKA and cAMP response element-binding protein phosphorylation in the nucleus. Inhibition of PDE4 or ablation of the Pde4d gene in mice prolonged cytoplasmic PKA activation and enhanced nuclear PKA responses. In the cytoplasm, phosphatase 1 (PP1) and 2A (PP2A) contributed to the termination of PKA responses, whereas only PP1 played a role in the nucleus. Conclusion Our study reveals a differential integration of cytoplasmic and nuclear PKA responses to β-AR stimulation in cardiac myocytes. This may have important implications in the physiological and pathological hypertrophic response to β-AR stimulation. PMID:24550350

  17. Upon Infection the Cellular WD Repeat-containing Protein 5 (WDR5) Localizes to Cytoplasmic Inclusion Bodies and Enhances Measles Virus Replication.

    Science.gov (United States)

    Ma, Dzwokai; George, Cyril X; Nomburg, Jason; Pfaller, Christian K; Cattaneo, Roberto; Samuel, Charles E

    2017-12-13

    Replication of negative-strand RNA viruses occurs in association with discrete cytoplasmic foci called inclusion bodies. Whereas inclusion bodies represent a prominent subcellular structure induced by viral infection, our knowledge of the cellular protein components involved in inclusion body formation and function is limited. Using measles virus-infected HeLa cells, we found that the WD repeat-containing protein 5 (WDR5), a subunit of histone H3 lysine 4 methyltransferases, was selectively recruited to virus-induced inclusion bodies. Furthermore, WDR5 was found in complexes containing viral proteins associated with RNA replication. WDR5 was not detected with mitochondria, stress granules, or other known secretory or endocytic compartments of infected cells. WDR5 deficiency decreased both viral protein production and infectious virus yields. Interferon production was modestly increased in WDR5 deficient cells. Thus, our study identifies WDR5 as a novel viral inclusion body-associated cellular protein and suggests a role for WDR5 in promoting viral replication. IMPORTANCE Measles virus is a human pathogen that remains a global concern with more than 100,000 measles-related deaths annually despite the availability of an effective vaccine. As measles continues to cause significant morbidity and mortality, understanding the virus-host interactions at the molecular level that affect virus replication efficiency is important for development and optimization of treatment procedures. Measles virus is an RNA virus that encodes six genes and replicates in the cytoplasm of infected cells in discrete cytoplasmic replication bodies, though little is known of the biochemical nature of these structures. Here we show that the cellular protein WDR5 is enriched in the cytoplasmic viral replication factories and enhances virus growth. WDR5-containing protein complex includes viral proteins responsible for viral RNA replication. Thus, we have identified WDR5 as a host factor that

  18. Tubular lysosome morphology and distribution within macrophages depend on the integrity of cytoplasmic microtubules

    International Nuclear Information System (INIS)

    Swanson, J.; Bushnell, A.; Silverstein, S.C.

    1987-01-01

    Pinocytosis of the fluorescent dye lucifer yellow labels elongated, membrane-bound tubular organelles in several cell types, including cultured human monocytes, thioglycolate-elicited mouse peritoneal macrophages, and the macrophage-like cell line J774.2. These tubular structures can be identified as lysosomes by acid phosphatase histochemistry and immunofluorescence localization of cathepsin L. The abundance of tubular lysosomes is markedly increased by treatment with phorbol 12-myristate 13-acetate. When labeled by pinocytosis of microperoxidase and examined by electron microscopic histochemistry, the tubular lysosomes have an outside diameter of ≅ 75 nm and a length of several micrometers; they radiate from the cell's centrosphere in alignment with cytoplasmic microtubules and intermediate filaments. Incubation of phorbol myristate acetate-treated macrophages at 4 0 C or in medium containing 5 μM colchicine or nocodazole at 37 0 C leads to disassembly of microtubules and fragmentation of the tubular lysosomes. Return of the cultures to 37 0 C or removal of nocodazole from the medium leads to reassembly of microtubules and the reappearance of tubular lysosomes within 10-20 min. The authors conclude that microtubules are essential for the maintenance of tubular lysosome morphology and that, in macrophages, a significant proportion of the lysosomal compartment is contained within these tubular structures

  19. Generation of H9 T-cells stably expressing a membrane-bound form of the cytoplasmic tail of the Env-glycoprotein: lack of transcomplementation of defective HIV-1 virions encoding C-terminally truncated Env

    Directory of Open Access Journals (Sweden)

    Bosch Valerie

    2006-05-01

    Full Text Available Abstract H9-T-cells do not support the replication of mutant HIV-1 encoding Env protein lacking its long cytoplasmic C-terminal domain (Env-CT. Here we describe the generation of a H9-T-cell population constitutively expressing the HIV-1 Env-CT protein domain anchored in the cellular membrane by it homologous membrane-spanning domain (TMD. We confirmed that the Env-TMD-CT protein was associated with cellular membranes, that its expression did not have any obvious cytotoxic effects on the cells and that it did not affect wild-type HIV-1 replication. However, as measured in both a single-round assay as well as in spreading infections, replication competence of mutant pNL-Tr712, lacking the Env-CT, was not restored in this H9 T-cell population. This means that the Env-CT per se cannot transcomplement the replication block of HIV-1 virions encoding C-terminally truncated Env proteins and suggests that the Env-CT likely exerts its function only in the context of the complete Env protein.

  20. Multi-compartment linear noise approximation

    International Nuclear Information System (INIS)

    Challenger, Joseph D; McKane, Alan J; Pahle, Jürgen

    2012-01-01

    The ability to quantify the stochastic fluctuations present in biochemical and other systems is becoming increasing important. Analytical descriptions of these fluctuations are attractive, as stochastic simulations are computationally expensive. Building on previous work, a linear noise approximation is developed for biochemical models with many compartments, for example cells. The procedure is then implemented in the software package COPASI. This technique is illustrated with two simple examples and is then applied to a more realistic biochemical model. Expressions for the noise, given in the form of covariance matrices, are presented. (paper)

  1. Protein synthesis and the recovery of both survival and cytoplasmic "petite" mutation in ultraviolet-treated yeast cells. I. Nuclear-directed protein synthesis.

    Science.gov (United States)

    Heude, M; Chanet, R; Moustacchi, E

    1975-04-01

    The contribution of nuclear-directed protein synthesis in the repair of lethal and mitochondrial genetic damage after UV-irradiation of exponential and stationary phage haploid yeast cells was examined. This was carried out using cycloheximide (CH), a specific inhibitor of nuclear protein synthesis. It appears that nuclear protein synthesis is required for the increase in survival seen after the liquid holding of cells at both stages, as well as for the "petite" recovery seen after the liquid holding of exponential phase cells. The characteristic negative liquid holding effect observed for the UV induction of "petites" in stationary phase cells (increase of the frequency of "petites" during storage) remained following all the treatments which inhibited nuclear protein synthesis. However, the application of photoreactivating light following dark holding with cycloheximide indicates that some steps of the repair of both nuclear and mitochondrial damage are performed in the absence of a synthesis of proteins.

  2. The vacuolar transport of aleurain-GFP and 2S albumin-GFP fusions is mediated by the same pre-vacuolar compartments in tobacco BY-2 and Arabidopsis suspension cultured cells.

    Science.gov (United States)

    Miao, Yansong; Li, Kwun Yee; Li, Hong-Ye; Yao, Xiaoqiang; Jiang, Liwen

    2008-12-01

    Soluble proteins reach vacuoles because they contain vacuolar sorting determinants (VSDs) that are recognized by vacuolar sorting receptor (VSR) proteins. Pre-vacuolar compartments (PVCs), defined by VSRs and GFP-VSR reporters in tobacco BY-2 cells, are membrane-bound intermediate organelles that mediate protein traffic from the Golgi apparatus to the vacuole in plant cells. Multiple pathways have been demonstrated to be responsible for vacuolar transport of lytic enzymes and storage proteins to the lytic vacuole (LV) and the protein storage vacuole (PSV), respectively. However, the nature of PVCs for LV and PSV pathways remains unclear. Here, we used two fluorescent reporters, aleurain-GFP and 2S albumin-GFP, that represent traffic of lytic enzymes and storage proteins to LV and PSV, respectively, to study the PVC-mediated transport pathways via transient expression in suspension cultured cells. We demonstrated that the vacuolar transport of aleurain-GFP and 2S albumin-GFP was mediated by the same PVC populations in both tobacco BY-2 and Arabidopsis suspension cultured cells. These PVCs were defined by the seven GFP-AtVSR reporters. In wortmannin-treated cells, the vacuolated PVCs contained the mRFP-AtVSR reporter in their limiting membranes, whereas the soluble aleurain-GFP or 2S albumin-GFP remained in the lumen of the PVCs, indicating a possible in vivo relationship between receptor and cargo within PVCs.

  3. On the relationship of cytoplasmic and nuclear 3H-estradiol bonding in human mammary carcinoma cells with special consideration of the sucrose density centrifugation in comparison to the charcoal method

    International Nuclear Information System (INIS)

    Semm, D.M.

    1982-01-01

    According to a hypothesis from Wittliff an estrogen-positive-receptor tumor would indicate the need for endrocrinological therapy if after sucrose density centrifugation on S-peak of 8-9 is found. In this work this hypothesis is studied, the sucrose density centrifugation is compared to the charcoal method, a normal value range is determined for the sucrose density centrifugation and the amount of cytoplasmic estrogen bonding is correlated with the estrogen chromatin bonding capacity. With malignantly degenerated breast cells the average 3 H-estradiol bonding capacities for the charcoal method were 74.09 fmol/mg protein and respectively 22.9 fmol/mg protein with incubation in 2.5x10 -8 M 3 H-estradiol. For the sucrose density centrifugation 42.81 fmol/mg protein was found as average value in the peak fraction (4-5 S-range). With the use of the charcoal method 78% of the carcinoma were estrogen-receptor positive compared to 57% using the sucrose density centrifugation (n=23). The average nucleus bonding amount in the case of 15 carcinomas which were studied for their estradiol chromatin bonding capacity was 588.1 dpm/1 μg DNA. Together with the estradiol receptor analysis the determination of nucleus bonding capacity appears to be a promising procedure especially for postclimacteric women for reducing the rate of non-responders. Beyond that this receptor combination could offer a contribution to the malignancy grading of nuclear mastopathy results procedure. Wittliff's hypothesis, a tumor is first estrogen sensitive when an 8-9 S-peak is present, could not be confirmed. Neither could the 8-9 S-peaks in the form given by Wittliff be reproduced nor could any positive correlation to cytoplasmic bonding or respectively to 3 H-estradiol chromatin bonding be found. (orig.) [de

  4. Dynamics of the association of heat shock protein HSPA6 (Hsp70B') and HSPA1A (Hsp70-1) with stress-sensitive cytoplasmic and nuclear structures in differentiated human neuronal cells.

    Science.gov (United States)

    Shorbagi, Sadek; Brown, Ian R

    2016-11-01

    Heat shock proteins (Hsps) are cellular repair agents that counter the effects of protein misfolding that is a characteristic feature of neurodegenerative diseases. HSPA1A (Hsp70-1) is a widely studied member of the HSPA (Hsp70) family. The little-studied HSPA6 (Hsp70B') is present in the human genome and absent in mouse and rat; hence, it is missing in current animal models of neurodegenerative diseases. Differentiated human neuronal SH-SY5Y cells were employed to compare the dynamics of the association of YFP-tagged HSPA6 and HSPA1A with stress-sensitive cytoplasmic and nuclear structures. Following thermal stress, live-imaging confocal microscopy and Fluorescence Recovery After Photobleaching (FRAP) demonstrated that HSPA6 displayed a prolonged and more dynamic association, compared to HSPA1A, with centrioles that play critical roles in neuronal polarity and migration. HSPA6 and HSPA1A also targeted nuclear speckles, rich in RNA splicing factors, and the granular component of the nucleolus that is involved in rRNA processing and ribosomal subunit assembly. HSPA6 and HSPA1A displayed similar FRAP kinetics in their interaction with nuclear speckles and the nucleolus. Subsequently, during the recovery from neuronal stress, HSPA6, but not HSPA1A, localized with the periphery of nuclear speckles (perispeckles) that have been characterized as transcription sites. The stress-induced association of HSPA6 with perispeckles displayed the greatest dynamism compared to the interaction of HSPA6 or HSPA1A with other stress-sensitive cytoplasmic and nuclear structures. This suggests involvement of HSPA6 in transcriptional recovery of human neurons from cellular stress that is not apparent for HSPA1A.

  5. Restoration of the CD4 T cell compartment after long-term highly active antiretroviral therapy without phenotypical signs of accelerated immunological aging

    NARCIS (Netherlands)

    Vrisekoop, Nienke; van Gent, Rogier; de Boer, Anne Bregje; Otto, Sigrid A.; Borleffs, Jan C. C.; Steingrover, Radjin; Prins, Jan M.; Kuijpers, Taco W.; Wolfs, Tom F. W.; Geelen, Sibyl P. M.; Vulto, Irma; Lansdorp, Peter; Tesselaar, Kiki; Borghans, José A. M.; Miedema, Frank

    2008-01-01

    It remains uncertain whether full T cell reconstitution can be established in HIV-infected children and adults with long-term sustained virological control by highly active antiretroviral therapy (HAART). In this study, we comprehensively analyzed various phenotypical markers of CD4 T cell recovery.

  6. Restoration of the CD4 T cell compartment after long-term highly active Antiretroviral therapy without phenotypical signs of accelerated immunological aging

    NARCIS (Netherlands)

    Vrisekoop, Nienke; van Gent, Rogier; de Boer, Anne Bregje; Otto, Sigrid A.; Borleffs, Jan C. C.; Stemgrover, Radjin; Prins, Jan M.; Kuijpers, Taco W.; Wolfs, Tom F. W.; Geelen, Sibyl P. M.; Vulto, Irma; Lansdorp, Peter; Tesselaar, Kiki; Borghans, Jose A. M.; Miedema, Frank

    2008-01-01

    It remains uncertain whether full T cell reconstitution can be established in HIV-infected children and adults with long-term sustained virological control by highly active antiretroviral therapy (HAART). In this study, we comprehensively analyzed various phenotypical markers of CD4 T cell recovery.

  7. Imaging of iliopsoas compartment disease

    International Nuclear Information System (INIS)

    Rocher, L.; Saint Maurice, J.P.; Le Quen, O.; Bazille, A.; Miquel, A.; Frouge, C.; Blery, M.

    1997-01-01

    Infection, neoplastic involvement, and hemorrhage, are the most frequent pathologies that involve the ilio-psoas compartment. The extension from contiguous pathological structures and particularly digestive and urological organs, are often the origin of abscess formation or malignant tumours. The radiological findings including ultrasonography, CT, and magnetic resonance imaging, show a low specificity, which improves if the clinical history is known. The final diagnosis is confirmed by puncture or biopsy. (author)

  8. Numerical treatment of compartment models

    International Nuclear Information System (INIS)

    Einarsson, B.

    1984-11-01

    This report describes and interactive program RADIO (Radioactive Decay Information Online) for studying the radioactive decay process, with applications to many ecological problems, but not necessarily involving radioactive processes. Starting with the compartment coefficients and initial values of the various compartments the problem is solved as a system of linear ordinary differential equations. The method of solution is the direct use of matrix exponentials or the backward differences method. A program INVERS is also available for the solution of the inverse problem, that is parameter estimation in a system of linear ordinary differential equations when the solution is available pointwise. The output can be printed on a line printer either from a result file or from the plot file, which of course also can be used to produce graphic output. The plot file is processed by the plotting program VISION or by the auxiliary printing program RADAR. Another file can be used for a later restart from the point of time where the previous computation was aborted or from an arbitrary point of time if the relevant starting information is available. This is useful in order to avoid the manual input of a compartment matrix if it is similar to one used before. When the program RADIO is run the user answers to the question asked by the program. The programs are written in Fortran 77 for the Digital Equipment VAX 11 with graphical presentation on a Tektronix 4010, and are available from the author. (Author)

  9. Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E. coli

    Directory of Open Access Journals (Sweden)

    Hatahet Feras

    2010-09-01

    Full Text Available Abstract Background The formation of native disulfide bonds is a complex and essential post-translational modification for many proteins. The large scale production of these proteins can be difficult and depends on targeting the protein to a compartment in which disulfide bond formation naturally occurs, usually the endoplasmic reticulum of eukaryotes or the periplasm of prokaryotes. It is currently thought to be impossible to produce large amounts of disulfide bond containing protein in the cytoplasm of wild-type bacteria such as E. coli due to the presence of multiple pathways for their reduction. Results Here we show that the introduction of Erv1p, a sulfhydryl oxidase and FAD-dependent catalyst of disulfide bond formation found in the inter membrane space of mitochondria, allows the efficient formation of native disulfide bonds in heterologously expressed proteins in the cytoplasm of E. coli even without the disruption of genes involved in disulfide bond reduction, for example trxB and/or gor. Indeed yields of active disulfide bonded proteins were higher in BL21 (DE3 pLysSRARE, an E. coli strain with the reducing pathways intact, than in the commercial Δgor ΔtrxB strain rosetta-gami upon co-expression of Erv1p. Conclusions Our results refute the current paradigm in the field that disruption of at least one of the reducing pathways is essential for the efficient production of disulfide bond containing proteins in the cytoplasm of E. coli and open up new possibilities for the use of E. coli as a microbial cell factory.

  10. Regulation of autophagy by cytoplasmic p53.

    Science.gov (United States)

    Tasdemir, Ezgi; Maiuri, M Chiara; Galluzzi, Lorenzo; Vitale, Ilio; Djavaheri-Mergny, Mojgan; D'Amelio, Marcello; Criollo, Alfredo; Morselli, Eugenia; Zhu, Changlian; Harper, Francis; Nannmark, Ulf; Samara, Chrysanthi; Pinton, Paolo; Vicencio, José Miguel; Carnuccio, Rosa; Moll, Ute M; Madeo, Frank; Paterlini-Brechot, Patrizia; Rizzuto, Rosario; Szabadkai, Gyorgy; Pierron, Gérard; Blomgren, Klas; Tavernarakis, Nektarios; Codogno, Patrice; Cecconi, Francesco; Kroemer, Guido

    2008-06-01

    Multiple cellular stressors, including activation of the tumour suppressor p53, can stimulate autophagy. Here we show that deletion, depletion or inhibition of p53 can induce autophagy in human, mouse and nematode cells subjected to knockout, knockdown or pharmacological inhibition of p53. Enhanced autophagy improved the survival of p53-deficient cancer cells under conditions of hypoxia and nutrient depletion, allowing them to maintain high ATP levels. Inhibition of p53 led to autophagy in enucleated cells, and cytoplasmic, not nuclear, p53 was able to repress the enhanced autophagy of p53(-/-) cells. Many different inducers of autophagy (for example, starvation, rapamycin and toxins affecting the endoplasmic reticulum) stimulated proteasome-mediated degradation of p53 through a pathway relying on the E3 ubiquitin ligase HDM2. Inhibition of p53 degradation prevented the activation of autophagy in several cell lines, in response to several distinct stimuli. These results provide evidence of a key signalling pathway that links autophagy to the cancer-associated dysregulation of p53.

  11. A physical perspective on cytoplasmic streaming.

    Science.gov (United States)

    Goldstein, Raymond E; van de Meent, Jan-Willem

    2015-08-06

    Organisms show a remarkable range of sizes, yet the dimensions of a single cell rarely exceed 100 µm. While the physical and biological origins of this constraint remain poorly understood, exceptions to this rule give valuable insights. A well-known counterexample is the aquatic plant Chara, whose cells can exceed 10 cm in length and 1 mm in diameter. Two spiralling bands of molecular motors at the cell periphery drive the cellular fluid up and down at speeds up to 100 µm s(-1), motion that has been hypothesized to mitigate the slowness of metabolite transport on these scales and to aid in homeostasis. This is the most organized instance of a broad class of continuous motions known as 'cytoplasmic streaming', found in a wide range of eukaryotic organisms-algae, plants, amoebae, nematodes and flies-often in unusually large cells. In this overview of the physics of this phenomenon, we examine the interplay between streaming, transport and cell size and discuss the possible role of self-organization phenomena in establishing the observed patterns of streaming.

  12. Genetic expression of induced rice sterility under alien-cytoplasm

    International Nuclear Information System (INIS)

    Wang Naiyuan; Cai Zhijun; Liang Kangjing; Li Yu

    2005-01-01

    Rice restorer lines were treated with 60 Co γ-ray and 4 male sterile mutants obtained with the fertility of controlled by 4 non-allelic recessive genes, respectively. Sixty combinations were made by using male sterile plants/fertile plants as male parents, and 15 different cytoplasmic substitution lines of the same cell nucleus as female parents. The result showed that F 1 spikelets were normal and fertile, and different numbers of male sterile plants were segregated in F 2 . Complete fertility genotype was not found among interactions between induced male sterile genes and alien-cytoplasms. (authors)

  13. Forearm Compartment Syndrome Caused by Reperfusion Injury

    Directory of Open Access Journals (Sweden)

    Ufuk Sayar

    2014-01-01

    Full Text Available Compartment syndrome is commonly seen following lower extremity ischemia. However, upper extremities’ compartment syndrome, especially after any vascular surgical procedures, is infrequent. Herein we report a case of an acute forearm compartment syndrome that was developed after delayed brachial artery embolectomy.

  14. Acute compartment syndrome after medial gastrocnemius tear.

    Science.gov (United States)

    Sit, Yan Kit; Lui, Tun Hing

    2015-02-01

    Acute compartment syndrome after medial gastrocnemius tear is very rare. It can involve the superficial posterior compartment alone or progress to involve all the 4 compartments of the lower legs. Those patients with high pain tolerance and minor trauma can lead to delayed presentation. Immediate fasciotomy is the treatment of choice. Therapeutic Level IV, Case Study. © 2014 The Author(s).

  15. 14 CFR 25.787 - Stowage compartments.

    Science.gov (United States)

    2010-01-01

    ... STANDARDS: TRANSPORT CATEGORY AIRPLANES Design and Construction Personnel and Cargo Accommodations § 25.787 Stowage compartments. (a) Each compartment for the stowage of cargo, baggage, carry-on articles, and... to compartments located below, or forward, of all occupants in the airplane. If the airplane has a...

  16. The Cytoplasmic Tail Domain of Epstein-Barr Virus gH Regulates Membrane Fusion Activity through Altering gH Binding to gp42 and Epithelial Cell Attachment

    Directory of Open Access Journals (Sweden)

    Jia Chen

    2016-11-01

    Full Text Available Epstein-Barr virus (EBV is associated with infectious mononucleosis and a variety of cancers as well as lymphoproliferative disorders in immunocompromised patients. EBV mediates viral entry into epithelial and B cells using fusion machinery composed of four glycoproteins: gB, the gH/gL complex, and gp42. gB and gH/gL are required for both epithelial and B cell fusion. The specific role of gH/gL in fusion has been the most elusive among the required herpesvirus entry glycoproteins. Previous mutational studies have focused on the ectodomain of EBV gH and not on the gH cytoplasmic tail domain (CTD. In this study, we chose to examine the function of the gH CTD by making serial gH truncation mutants as well as amino acid substitution mutants to determine the importance of the gH CTD in epithelial and B cell fusion. Truncation of 8 amino acids (aa 698 to 706 of the gH CTD resulted in diminished fusion activity using a virus-free syncytium formation assay and fusion assay. The importance of the amino acid composition of the gH CTD was also investigated by amino acid substitutions that altered the hydrophobicity or hydrophilicity of the CTD. These mutations also resulted in diminished fusion activity. Interestingly, some of the gH CTD truncation mutants and hydrophilic tail substitution mutants lost the ability to bind to gp42 and epithelial cells. In summary, our studies indicate that the gH CTD is an important functional domain.

  17. Cytoplasmic streaming velocity as a plant size determinant.

    Science.gov (United States)

    Tominaga, Motoki; Kimura, Atsushi; Yokota, Etsuo; Haraguchi, Takeshi; Shimmen, Teruo; Yamamoto, Keiichi; Nakano, Akihiko; Ito, Kohji

    2013-11-11

    Cytoplasmic streaming is active transport widely occurring in plant cells ranging from algae to angiosperms. Although it has been revealed that cytoplasmic streaming is generated by organelle-associated myosin XI moving along actin bundles, the fundamental function in plants remains unclear. We generated high- and low-speed chimeric myosin XI by replacing the motor domains of Arabidopsis thaliana myosin XI-2 with those of Chara corallina myosin XI and Homo sapiens myosin Vb, respectively. Surprisingly, the plant sizes of the transgenic Arabidopsis expressing high- and low-speed chimeric myosin XI-2 were larger and smaller, respectively, than that of the wild-type plant. This size change correlated with acceleration and deceleration, respectively, of cytoplasmic streaming. Our results strongly suggest that cytoplasmic streaming is a key determinant of plant size. Furthermore, because cytoplasmic streaming is a common system for intracellular transport in plants, our system could have applications in artificial size control in plants. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Membrane order in the plasma membrane and endocytic recycling compartment.

    Science.gov (United States)

    Iaea, David B; Maxfield, Frederick R

    2017-01-01

    The cholesterol content of membranes plays an important role in organizing membranes for signal transduction and protein trafficking as well as in modulating the biophysical properties of membranes. While the properties of model or isolated membranes have been extensively studied, there has been little evaluation of internal membranes in living cells. Here, we use a Nile Red based probe, NR12S, and ratiometric live cell imaging, to analyze the membrane order of the plasma membrane and endocytic recycling compartment. We find that after a brief incubation to allow endocytosis, NR12S is distributed between the plasma membrane and the endocytic recycling compartment. The NR12S reports that the endocytic recycling compartment is more highly ordered than the plasma membrane. We also find that the plasma membrane and the endocytic recycling compartment are differentially affected by altering cellular cholesterol levels. The membrane order of the plasma membrane, but not the endocytic recycling compartment, is altered significantly when cellular cholesterol content is increased or decreased by 20%. These results demonstrate that changes in cellular cholesterol differentially alter membrane order within different organelles.

  19. Magnetite nanoparticles as reporters for microcarrier processing in cytoplasm

    Energy Technology Data Exchange (ETDEWEB)

    Reibetanz, Uta, E-mail: uta.reibetanz@medizin.uni-leipzig.de [Translational Centre for Regenerative Medicine (TRM) Leipzig, Universitaet Leipzig, Philipp-Rosenthal-Strasse 55, 04103 Leipzig (Germany); Institute for Medical Physics and Biophysics, Medical Faculty, Universitaet Leipzig, Haertelstrasse 16-18, 04107 Leipzig (Germany); Jankuhn, Steffen, E-mail: jankuhn@uni-leipzig.de [Division of Nuclear Solid State Physics, Faculty of Physics and Geosciences, Universitaet Leipzig, Linnestrasse 5, 04103 Leipzig (Germany); Office for Environmental Protection and Occupational Safety, Universitaet Leipzig, Ritterstrasse 24, 04109 Leipzig (Germany)

    2011-10-15

    The development and therapeutic application of drug delivery systems based on colloidal microcarriers layer-by-layer coated with biopolyelectrolytes requires the investigation of their processing inside the cell for the successful and efficient transport and release of the active agents. The present study is focused on the time-dependent multilayer decomposition and the subsequent release of active agents to the cytoplasm. Magnetite nanoparticles (MNP) were used as reporter agents integrated into the protamine sulfate/dextran sulfate basis multilayer on colloidal SiO{sub 2} cores. This functionalization allows the monitoring of the multilayer decomposition due to the detection of the MNP release, visualized by means of proton-induced X-ray emission (PIXE) by elemental distribution of Si and Fe. The direct correlation between the microcarrier localization in endolysosomes and cytoplasm of HEK293T/17 cells via confocal laser scanning microscopy (CLSM) and the elemental distribution (PIXE) allows tracing the fate of the MNP-coated microcarriers in cytoplasm, and thus the processing of the multilayer. Microcarrier/cell co-incubation experiments of 6 h, 24 h, 48 h, and 72 h show that a MNP release and a slight expansion into the cytoplasm occurs after a longer co-incubation of 72 h.

  20. The antimicrobial peptides lactoferricin B and magainin 2 cross over the bacterial cytoplasmic membrane and reside in the cytoplasm.

    Science.gov (United States)

    Haukland, H H; Ulvatne, H; Sandvik, K; Vorland, L H

    2001-11-23

    The localization of immunolabelled antimicrobial peptides was studied using transmission electron microscopy. Staphylococcus aureus and Escherichia coli were exposed to lactoferricin B (17-41), lactoferricin B (17-31) and D-lactoferricin B (17-31). E. coli was also exposed to cecropin P1 and magainin 2. The lactoferricins were found in the cytoplasm of both bacteria. In S. aureus the amount of cytoplasmic lactoferricin B (17-41) was time- and concentration-dependent, reaching a maximum within 30 min. Cecropin P1 was confined to the cell wall, while magainin 2 was found in the cytoplasm of E. coli. The finding of intracellularly localized magainin is not reported previously.

  1. Extrasynaptic N-methyl-D-aspartate (NMDA) receptor stimulation induces cytoplasmic translocation of the CDKL5 kinase and its proteasomal degradation.

    Science.gov (United States)

    Rusconi, Laura; Kilstrup-Nielsen, Charlotte; Landsberger, Nicoletta

    2011-10-21

    Mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) have been found in patients with epileptic encephalopathy characterized by early onset intractable epilepsy, including infantile spasms and other types of seizures, severe developmental delay, and often the development of Rett syndrome-like features. Despite its clear involvement in proper brain development, CDKL5 functions are still far from being understood. In this study, we analyzed the subcellular localization of the endogenous kinase in primary murine hippocampal neurons. CDKL5 was localized both in nucleus and cytoplasm and, conversely to proliferating cells, did not undergo constitutive shuttling between these compartments. Nevertheless, glutamate stimulation was able to induce the exit of the kinase from the nucleus and its subsequent accumulation in the perinuclear cytoplasm. Moreover, we found that sustained glutamate stimulation promoted CDKL5 proteasomal degradation. Both events were mediated by the specific activation of extrasynaptic pool of N-methyl-d-aspartate receptors. Proteasomal degradation was also induced by withdrawal of neurotrophic factors and hydrogen peroxide treatment, two different paradigms of cell death. Altogether, our results indicate that both subcellular localization and expression of CDKL5 are modulated by the activation of extrasynaptic N-methyl-D-aspartate receptors and suggest regulation of CDKL5 by cell death pathways.

  2. Extrasynaptic N-Methyl-d-aspartate (NMDA) Receptor Stimulation Induces Cytoplasmic Translocation of the CDKL5 Kinase and Its Proteasomal Degradation*

    Science.gov (United States)

    Rusconi, Laura; Kilstrup-Nielsen, Charlotte; Landsberger, Nicoletta

    2011-01-01

    Mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) have been found in patients with epileptic encephalopathy characterized by early onset intractable epilepsy, including infantile spasms and other types of seizures, severe developmental delay, and often the development of Rett syndrome-like features. Despite its clear involvement in proper brain development, CDKL5 functions are still far from being understood. In this study, we analyzed the subcellular localization of the endogenous kinase in primary murine hippocampal neurons. CDKL5 was localized both in nucleus and cytoplasm and, conversely to proliferating cells, did not undergo constitutive shuttling between these compartments. Nevertheless, glutamate stimulation was able to induce the exit of the kinase from the nucleus and its subsequent accumulation in the perinuclear cytoplasm. Moreover, we found that sustained glutamate stimulation promoted CDKL5 proteasomal degradation. Both events were mediated by the specific activation of extrasynaptic pool of N-methyl-d-aspartate receptors. Proteasomal degradation was also induced by withdrawal of neurotrophic factors and hydrogen peroxide treatment, two different paradigms of cell death. Altogether, our results indicate that both subcellular localization and expression of CDKL5 are modulated by the activation of extrasynaptic N-methyl-d-aspartate receptors and suggest regulation of CDKL5 by cell death pathways. PMID:21832092

  3. Members of the HCMV US12 family of predicted heptaspanning membrane proteins have unique intracellular distributions, including association with the cytoplasmic virion assembly complex

    International Nuclear Information System (INIS)

    Das, Subhendu; Pellett, Philip E.

    2007-01-01

    The human cytomegalovirus (HCMV) US12 gene family is a group of 10 predicted seven-transmembrane domain proteins that have some features in common with G-protein-coupled receptors. Little is known of their patterns of expression, localization, or functional interactions. Here, we studied the intracellular localization of three US12 family members, US14, US17, and US18, with respect to various intracellular markers and the cytoplasmic virion assembly compartment (AC). The three proteins have distinct patterns of expression, which include associations with the AC. US14 is often distributed in a uniform granular manner throughout the cytoplasm, concentrating in the AC in some cells. US17 is expressed in a segmented manner, with its N-terminal domain localizing to the periphery of what we show here to be the AC and the C-terminal domain localizing to nuclei and the cytoplasm [Das, S., Skomorovska-Prokvolit, Y., Wang, F. Z., Pellett, P.E., 2006. Infection-dependent nuclear localization of US17, a member of the US12 family of human cytomegalovirus-encoded seven-transmembrane proteins. J. Virol. 80, 1191-1203]. Here, we show that the C-terminal domain is present at the center of the AC, in close association with markers of early endosomes; the N-terminal staining corresponds to an area stained by markers for the Golgi and trans-Golgi. US18 is distributed throughout the cytoplasm, concentrating in the AC at later stages of infection; it is localized more to the periphery of the AC than are US14 and US17C, in association with markers of the trans-Golgi. Although not detected in virions, their structures and localization in various zones within the AC suggest possible roles for these proteins in the process of virion maturation and egress

  4. Cytoplasmic Dynein Promotes HIV-1 Uncoating

    Directory of Open Access Journals (Sweden)

    Paulina Pawlica

    2014-11-01

    Full Text Available Retroviral capsid (CA cores undergo uncoating during their retrograde transport (toward the nucleus, and/or after reaching the nuclear membrane. However, whether HIV-1 CA core uncoating is dependent upon its transport is not understood. There is some evidence that HIV-1 cores retrograde transport involves cytoplasmic dynein complexes translocating on microtubules. Here we investigate the role of dynein-dependent transport in HIV-1 uncoating. To interfere with dynein function, we depleted dynein heavy chain (DHC using RNA interference, and we over-expressed p50/dynamitin. In immunofluorescence microscopy experiments, DHC depletion caused an accumulation of CA foci in HIV-1 infected cells. Using a biochemical assay to monitor HIV-1 CA core disassembly in infected cells, we observed an increase in amounts of intact (pelletable CA cores upon DHC depletion or p50 over-expression. Results from these two complementary assays suggest that inhibiting dynein-mediated transport interferes with HIV-1 uncoating in infected cells, indicating the existence of a functional link between HIV-1 transport and uncoating.

  5. Cytoplasmic Domains and Voltage-Dependent Potassium Channel Gating

    Science.gov (United States)

    Barros, Francisco; Domínguez, Pedro; de la Peña, Pilar

    2012-01-01

    The basic architecture of the voltage-dependent K+ channels (Kv channels) corresponds to a transmembrane protein core in which the permeation pore, the voltage-sensing components and the gating machinery (cytoplasmic facing gate and sensor–gate coupler) reside. Usually, large protein tails are attached to this core, hanging toward the inside of the cell. These cytoplasmic regions are essential for normal channel function and, due to their accessibility to the cytoplasmic environment, constitute obvious targets for cell-physiological control of channel behavior. Here we review the present knowledge about the molecular organization of these intracellular channel regions and their role in both setting and controlling Kv voltage-dependent gating properties. This includes the influence that they exert on Kv rapid/N-type inactivation and on activation/deactivation gating of Shaker-like and eag-type Kv channels. Some illustrative examples about the relevance of these cytoplasmic domains determining the possibilities for modulation of Kv channel gating by cellular components are also considered. PMID:22470342

  6. Actin polymerisation at the cytoplasmic face of eukaryotic nuclei

    Directory of Open Access Journals (Sweden)

    David-Watine Brigitte

    2006-05-01

    Full Text Available Abstract Background There exists abundant molecular and ultra-structural evidence to suggest that cytoplasmic actin can physically interact with the nuclear envelope (NE membrane system. However, this interaction has yet to be characterised in living interphase cells. Results Using a fluorescent conjugate of the actin binding drug cytochalasin D (CD-BODIPY we provide evidence that polymerising actin accumulates in vicinity to the NE. In addition, both transiently expressed fluorescent actin and cytoplasmic micro-injection of fluorescent actin resulted in accumulation of actin at the NE-membrane. Consistent with the idea that the cytoplasmic phase of NE-membranes can support this novel pool of perinuclear actin polymerisation we show that isolated, intact, differentiated primary hepatocyte nuclei support actin polymerisation in vitro. Further this phenomenon was inhibited by treatments hindering steric access to outer-nuclear-membrane proteins (e.g. wheat germ agglutinin, anti-nesprin and anti-nucleoporin antibodies. Conclusion We conclude that actin polymerisation occurs around interphase nuclei of living cells at the cytoplasmic phase of NE-membranes.

  7. Endoplasmic-reticulum-mediated microtubule alignment governs cytoplasmic streaming.

    Science.gov (United States)

    Kimura, Kenji; Mamane, Alexandre; Sasaki, Tohru; Sato, Kohta; Takagi, Jun; Niwayama, Ritsuya; Hufnagel, Lars; Shimamoto, Yuta; Joanny, Jean-François; Uchida, Seiichi; Kimura, Akatsuki

    2017-04-01

    Cytoplasmic streaming refers to a collective movement of cytoplasm observed in many cell types. The mechanism of meiotic cytoplasmic streaming (MeiCS) in Caenorhabditis elegans zygotes is puzzling as the direction of the flow is not predefined by cell polarity and occasionally reverses. Here, we demonstrate that the endoplasmic reticulum (ER) network structure is required for the collective flow. Using a combination of RNAi, microscopy and image processing of C. elegans zygotes, we devise a theoretical model, which reproduces and predicts the emergence and reversal of the flow. We propose a positive-feedback mechanism, where a local flow generated along a microtubule is transmitted to neighbouring regions through the ER. This, in turn, aligns microtubules over a broader area to self-organize the collective flow. The proposed model could be applicable to various cytoplasmic streaming phenomena in the absence of predefined polarity. The increased mobility of cortical granules by MeiCS correlates with the efficient exocytosis of the granules to protect the zygotes from osmotic and mechanical stresses.

  8. Estudio longitudinal de anticuerpos anticitoplasma de neutrófilos en pacientes con anemia drepanocítica Longitudinal study of antineutrophil cytoplasmic antibodies in patients with sickle cell anemia

    Directory of Open Access Journals (Sweden)

    Ana María Guerreiro Hernández

    2000-08-01

    Full Text Available Se realizó un estudio longitudinal para detectar anticuerpos anticitoplasma de neutrófilos (ANCA en 13 pacientes con anemia drepanocítica en crisis vasooclusiva y en estado basal, mediante un método de inmunofluorescencia indirecta. Del total de 34 muestras de suero obtenidas, 16 fueron en crisis vasooclusiva y en 12 de ellas, correspondientes a 10 pacientes, se demostró la presencia de p-ANCA. En el resto de las muestras en crisis vasooclusiva y en estado basal no se observó la presencia de p-ANCA o c-ANCA. Los resultados obtenidos sugieren la posible participación de los p-ANCA en el daño isquémico, así como la importancia de su medición en el diagnóstico de las crisis vasooclusivas (CVO en los pacientes con anemia drepanocítica (ADA longitudinal study was made to detect antineutrophil cytoplasmic antibodies (ANCA in 13 patients with sickle cell anemia in vasocclusive crisis and basal state by using an indirect immunofluorescence method. Of 34 serum samples, 16 were in vasocclusive crisis and 12 of them corresponding to 10 patients revealed the presence of p-ANCA. Neither p-ANCA nor c-ANCA was observed in the rest of the samples taken in vasoclussive crisis and in basal state. The results achieved signaled a possible involvement of p-ANCA in ischemic damage as well as the importance of their measurement in the diagnosis of vasocclusive crisis in patients with sickle cell anemia

  9. Cytoplasmic p21 is a potential predictor for cisplatin sensitivity in ovarian cancer

    International Nuclear Information System (INIS)

    Xia, Xi; Weng, Yanjie; Liao, Shujie; Han, Zhiqiang; Liu, Ronghua; Zhu, Tao; Wang, Shixuan; Xu, Gang; Meng, Li; Zhou, Jianfeng; Ma, Ding; Ma, Quanfu; Li, Xiao; Ji, Teng; Chen, Pingbo; Xu, Hongbin; Li, Kezhen; Fang, Yong; Weng, Danhui

    2011-01-01

    P21 (WAF1/Cip1) binds to cyclin-dependent kinase complexes and inhibits their activities. It was originally described as an inhibitor of cancer cell proliferation. However, many recent studies have shown that p21 promotes tumor progression when accumulated in the cell cytoplasm. So far, little is known about the correlation between cytoplasmic p21 and drug resistance. This study was aimed to investigate the role of p21 in the cisplatin resistance of ovarian cancer. RT-PCR, western blot and immunofluorescence were used to detect p21 expression and location in cisplatin-resistant ovarian cancer cell line C13* and its parental line OV2008. Regulation of cytoplasmic p21 was performed through transfection of p21 siRNA, Akt2 shRNA and Akt2 constitutively active vector in the two cell lines; their effects on cisplatin-induced apoptosis were evaluated by flow cytometry. Tumor tissue sections of clinical samples were analyzed by immunohistochemistry. p21 predominantly localizes to the cytoplasm in C13* compared to OV2008. Persistent exposure to low dose cisplatin in OV2008 leads to p21 translocation from nuclear to cytoplasm, while it had not impact on p21 localization in C13*. Knockdown of cytoplasmic p21 by p21 siRNA transfection in C13* notably increased cisplatin-induced apoptosis through activation of caspase 3. Inhibition of p21 translocation into the cytoplasm by transfection of Akt2 shRNA into C13* cells significantly increased cisplatin-induced apoptosis, while induction of p21 translocation into the cytoplasm by transfection of constitutively active Akt2 in OV2008 enhanced the resistance to cisplatin. Immunohistochemical analysis of clinical ovarian tumor tissues demonstrated that cytoplasmic p21 was negatively correlated with the response to cisplatin based treatment. Cytoplasmic p21 is a novel biomarker of cisplatin resistance and it may represent a potential therapeutic target for ovarian tumors that are refractory to conventional treatment

  10. Characterization of cytoplasmic male sterility of rice with Lead Rice cytoplasm in comparison with that with Chinsurah Boro II cytoplasm.

    Science.gov (United States)

    Itabashi, Etsuko; Kazama, Tomohiko; Toriyama, Kinya

    2009-02-01

    Rice with LD-type cytoplasmic male sterility (CMS) possesses the cytoplasm of 'Lead Rice' and its fertility is recovered by a nuclear fertility restorer gene Rf1. Rf1 promotes processing of a CMS-associated mitochondrial RNA of atp6-orf79, which consists of atp6 and orf79, in BT-CMS with the cytoplasm of 'Chinsurah Boro II'. In this study, we found that LD-cytoplasm contained a sequence variant of orf79 downstream of atp6. Northern blot analysis showed that atp6-orf79 RNA of LD-cytoplasm was co-transcribed and was processed in the presence of Rf1 in the same manner as in BT-cytoplasm. Western blot analysis showed that the ORF79 peptide did not accumulate in an LD-CMS line, while ORF79 accumulated in a BT-CMS line and was diminished by Rf1. These results suggest that accumulation of ORF79 is not the cause of CMS in LD-cytoplasm and the mechanism of male-sterility induction/fertility restoration in LD-CMS is different from that in BT-CMS.

  11. Acute compartment syndrome caused by uncontrolled hypothyroidism.

    Science.gov (United States)

    Modi, Anar; Amin, Hari; Salzman, Matthew; Morgan, Farah

    2017-06-01

    Acute compartment syndrome is increased tissue pressure exceeding perfusion pressure in a closed compartment resulting in nerve and muscle ischemia. Common precipitating causes are crush injuries, burns, substance abuse, osseous or vascular limb trauma. This is a case of 42year old female with history of hypothyroidism who presented to emergency room with acute onset of severe pain and swelling in right lower extremity. Physical examination was concerning for acute compartment syndrome of right leg which was confirmed by demonstration of elevated compartmental pressures. No precipitating causes were readily identified. Further laboratory testing revealed uncontrolled hypothyroidism. Management included emergent fasciotomy and initiating thyroid hormone replacement. This case represents a rare association between acute compartment syndrome and uncontrolled hypothyroidism. We also discuss the pathogenesis of compartment syndrome in hypothyroid patients and emphasize the importance of evaluating for less common causes, particularly in setting of non-traumatic compartment syndrome. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Forearm Compartment Syndrome: Evaluation and Management.

    Science.gov (United States)

    Kistler, Justin M; Ilyas, Asif M; Thoder, Joseph J

    2018-02-01

    Compartment syndrome of the forearm is uncommon but can have devastating consequences. Compartment syndrome is a result of osseofascial swelling leading to decreased tissue perfusion and tissue necrosis. There are numerous causes of forearm compartment syndrome and high clinical suspicion must be maintained to avoid permanent disability. The most widely recognized symptoms include pain out of proportion and pain with passive stretch of the wrist and digits. Early diagnosis and decompressive fasciotomy are essential in the treatment of forearm compartment syndrome. Closure of fasciotomy wounds can often be accomplished by primary closure but many patients require additional forms of soft tissue coverage procedures. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Hydrogen peroxide probes directed to different cellular compartments.

    OpenAIRE

    Mikalai Malinouski; You Zhou; Vsevolod V Belousov; Dolph L Hatfield; Vadim N Gladyshev

    2011-01-01

    Background Controlled generation and removal of hydrogen peroxide play important roles in cellular redox homeostasis and signaling. We used a hydrogen peroxide biosensor HyPer, targeted to different compartments, to examine these processes in mammalian cells. Principal Findings Reversible responses were observed to various redox perturbations and signaling events. HyPer expressed in HEK 293 cells was found to sense low micromolar levels of hydrogen peroxide. When targeted to various cellular ...

  14. Proteomic response of Bacillus subtilis to lantibiotics reflects differences in interaction with the cytoplasmic membrane

    NARCIS (Netherlands)

    Wenzel, M.; Kohl, B.; Münch, D.; Raatschen, N.; Albada, H.B.; Hamoen, L.; Metzler-Nolte, N.; Sahl, H.G.; Bandow, J.E.

    2012-01-01

    Mersacidin, gallidermin, and nisin are lantibiotics, antimicrobial peptides containing lanthionine. They show potent antibacterial activity. All three interfere with cell wall biosynthesis by binding lipid II, but they display different levels of interaction with the cytoplasmic membrane. On one end

  15. Nucleoporin Nup98 mediates galectin-3 nuclear-cytoplasmic trafficking

    Energy Technology Data Exchange (ETDEWEB)

    Funasaka, Tatsuyoshi, E-mail: funasaka@staff.kanazawa-u.ac.jp [Laboratory of Molecular and Cellular Biology, Department of Biology, Faculty of Natural Systems, Institute of Science and Engineering, Kanazawa University, Ishikawa (Japan); Balan, Vitaly; Raz, Avraham [Department of Oncology and Pathology, Karmanos Cancer Institute, Wayne State University, School of Medicine, Detroit, MI (United States); Wong, Richard W., E-mail: rwong@staff.kanazawa-u.ac.jp [Laboratory of Molecular and Cellular Biology, Department of Biology, Faculty of Natural Systems, Institute of Science and Engineering, Kanazawa University, Ishikawa (Japan); Bio-AFM Frontier Research Center, Kanazawa Kanazawa University, Ishikawa (Japan)

    2013-04-26

    Highlights: •Nuclear pore protein Nup98 is a novel binding partner of galectin-3. •Nup98 transports galectin-3 into cytoplasm. •Nup98 depletion leads to galectin-3 nuclear transport and induces growth retardation. •Nup98 may involve in ß-catenin pathway through interaction with galectin-3. -- Abstract: Nucleoporin Nup98 is a component of the nuclear pore complex, and is important in transport across the nuclear pore. Many studies implicate nucleoporin in cancer progression, but no direct mechanistic studies of its effect in cancer have been reported. We show here that Nup98 specifically regulates nucleus–cytoplasm transport of galectin-3, which is a ß-galactoside-binding protein that affects adhesion, migration, and cancer progression, and controls cell growth through the ß-catenin signaling pathway in cancer cells. Nup98 interacted with galectin-3 on the nuclear membrane, and promoted galectin-3 cytoplasmic translocation whereas other nucleoporins did not show these functions. Inversely, silencing of Nup98 expression by siRNA technique localized galectin-3 to the nucleus and retarded cell growth, which was rescued by Nup98 transfection. In addition, Nup98 RNA interference significantly suppressed downstream mRNA expression in the ß-catenin pathway, such as cyclin D1 and FRA-1, while nuclear galectin-3 binds to ß-catenin to inhibit transcriptional activity. Reduced expression of ß-catenin target genes is consistent with the Nup98 reduction and the galectin-3–nucleus translocation rate. Overall, the results show Nup98’s involvement in nuclear–cytoplasm translocation of galectin-3 and ß-catenin signaling pathway in regulating cell proliferation, and the results depicted here suggest a novel therapeutic target/modality for cancers.

  16. Subcellular localization, mobility, and kinetic activity of glucokinase in glucose-responsive insulin-secreting cells.

    Science.gov (United States)

    Stubbs, M; Aiston, S; Agius, L

    2000-12-01

    We investigated the subcellular localization, mobility, and activity of glucokinase in MIN6 cells, a glucose-responsive insulin-secreting beta-cell line. Glucokinase is present in the cytoplasm and a vesicular/granule compartment that is partially colocalized with insulin granules. The granular staining of glucokinase is preserved after permeabilization of the cells with digitonin. There was no evidence for changes in distribution of glucokinase between the cytoplasm and the granule compartment during incubation of the cells with glucose. The rate of release of glucokinase and of phosphoglucoisomerase from digitonin-permeabilized cells was slower when cells were incubated at an elevated glucose concentration (S0.5 approximately 15 mmol/l). This effect of glucose was counteracted by competitive inhibitors of glucokinase (5-thioglucose and mannoheptulose) but was unaffected by fructose analogs and may be due to changes in cell shape or conformation of the cytoskeleton that are secondary to glucose metabolism. Based on the similar release of glucokinase and phosphoglucoisomerase, we found no evidence for specific binding of cytoplasmic digitonin-extractable glucokinase. The affinity of beta-cells for glucose is slightly lower than that in cell extracts and, unlike that in hepatocytes, is unaffected by fructose, tagatose, or a high-K+ medium, which is consistent with the lack of change in glucokinase distribution or release. We conclude that glucokinase is present in two locations, cytoplasm and the granular compartment, and that it does not translocate between them. This conclusion is consistent with the lack of adaptive changes in the glucose phosphorylation affinity. The glucokinase activity associated with the insulin granules may have a role in either direct or indirect coupling between glucose phosphorylation and insulin secretion.

  17. Cytoplasmic transfer of heritable elements other than mtDNA from SAMP1 mice into mouse tumor cells suppresses their ability to form tumors in C57BL6 mice.

    Science.gov (United States)

    Shimizu, Akinori; Tani, Haruna; Takibuchi, Gaku; Ishikawa, Kaori; Sakurazawa, Ryota; Inoue, Takafumi; Hashimoto, Tetsuo; Nakada, Kazuto; Takenaga, Keizo; Hayashi, Jun-Ichi

    2017-11-04

    In a previous study, we generated transmitochondrial P29mtSAMP1 cybrids, which had nuclear DNA from the C57BL6 (referred to as B6) mouse strain-derived P29 tumor cells and mitochondrial DNA (mtDNA) exogenously-transferred from the allogeneic strain SAMP1. Because P29mtSAMP1 cybrids did not form tumors in syngeneic B6 mice, we proposed that allogeneic SAMP1 mtDNA suppressed tumor formation of P29mtSAMP1 cybrids. To test this hypothesis, current study generated P29mt(sp)B6 cybrids carrying all genomes (nuclear DNA and mtDNA) from syngeneic B6 mice by eliminating SAMP1 mtDNA from P29mtSAMP1 cybrids and reintroducing B6 mtDNA. However, the P29mt(sp)B6 cybrids did not form tumors in B6 mice, even though they had no SAMP1 mtDNA, suggesting that SAMP1 mtDNA is not involved in tumor suppression. Then, we examined another possibility of whether SAMP1 mtDNA fragments potentially integrated into the nuclear DNA of P29mtSAMP1 cybrids are responsible for tumor suppression. We generated P29 H (sp)B6 cybrids by eliminating nuclear DNA from P29mt(sp)B6 cybrids and reintroducing nuclear DNA with no integrated SAMP1 mtDNA fragment from mtDNA-less P29 cells resistant to hygromycin in selection medium containing hygromycin. However, the P29 H (sp)B6 cybrids did not form tumors in B6 mice, even though they carried neither SAMP1 mtDNA nor nuclear DNA with integrated SAMP1 mtDNA fragments. Moreover, overproduction of reactive oxygen species (ROS) and bacterial infection were not involved in tumor suppression. These observations suggest that tumor suppression was caused not by mtDNA with polymorphic mutations or infection of cytozoic bacteria but by hypothetical heritable cytoplasmic elements other than mtDNA from SAMP1 mice. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  18. High performance glucose/O2 compartment-less biofuel cell using DNA/CNTs as platform for immobilizing bilirubin oxidase as novel biocathode and integrated NH2-CNTs/dendrimer/glucose dehydrogenase/nile blue as bioanode

    International Nuclear Information System (INIS)

    Korani, Aazam; Salimi, Abdollah

    2015-01-01

    Highlights: • A biocathode based on immobilization of bilirubin oxidase onto MWCNTs/DNA is designed. • The performance of MWCNTs/DNA/BOD biocathode for O 2 reduction reaction is improved. • Compared to MWCNTs/BOD,at present biocathode current density to ORR increased 3 folds. • The onset potential for ORR is 0.57 V and its current density increased to 270 μA cm −2 . • A glucose/O 2 BFC with voltage of 0.66 V, J = 172 μAcm −2 and power of 45 μW cm −2 fabricated. - Abstract: Herein, deoxyribonucleic acid (DNA)/multi-walled carbon nanotube (MWCNTs) with enhanced negative charged density was used as a novel electrochemical platform for oriented immobilization of bilirubin oxidase. The proposed support improved the direct electron transfer kinetics of BOD and its catalytic activity toward oxygen reduction reaction (ORR). In comparison to BOD enzyme which immobilized directly onto MWCNTs the current density increased three folds and reached to 270 μA cm −2 at 0.405 V with an onset potential of 0.57 V (vs. Ag/AgCl). The ability of this modified electrode as a biocathode is investigated after assembling with bioanode. The bioanode prepared with covalent attachment of glucose dehydrogenase enzyme (GDH) and nile blue (NB) as an efficient mediator for coenzyme regeneration onto glassy carbon electrode modified with amino-carbon nanotubes(MWCNTs-NH 2 ) and carboxyl terminated polyamidoamin dendrimer (PAMAM-Den) as a multifunctional linker. Finally, the performance of one-compartment glucose/O 2 biofuel cell without separators is also investigated. The open circuit voltage of the cell and maximum current density are obtained 660 mV and 172 μA cm −2 , respectively, while the maximum power density of 45 μW cm −2 is achieved at 428 mV of the cell voltage in buffer solution saturated with O 2 and containing 50 mM of glucose. The stability of the constructed EBFC is investigated under continuous operation at maximum power. It is observed that the biofuel

  19. Clinical aspects of lower leg compartment syndrome

    NARCIS (Netherlands)

    Brand, Johan Gerard Henric van den

    2004-01-01

    A compartment syndrome is a condition in which increased pressure within a limited space compromises the circulation and function of tissues within that space. Although pathofysiology is roughly similar in chronic exertional and acute compartment syndrome of the lower leg, the clinical

  20. Curious Sex Ratios and Cytoplasmic Genes

    Indian Academy of Sciences (India)

    instances of curious sex ratios exemplify an important principle: the fitness ..... markable transition - the whole means of sex determination has changed. No longer ... to the cytoplasmic symbiont is self-evident; the symbionts simply increase the.

  1. TRIM5α association with cytoplasmic bodies is not required for antiretroviral activity

    International Nuclear Information System (INIS)

    Song, Byeongwoon; Diaz-Griffero, Felipe; Park, Do Hyun; Rogers, Thomas; Stremlau, Matthew; Sodroski, Joseph

    2005-01-01

    The tripartite motif (TRIM) protein, TRIM5α, restricts infection by particular retroviruses. Many TRIM proteins form cytoplasmic bodies of unknown function. We investigated the relationship between cytoplasmic body formation and the structure and antiretroviral activity of TRIM5α. In addition to diffuse cytoplasmic staining, the TRIM5α proteins from several primate species were located in cytoplasmic bodies of different sizes; by contrast, TRIM5α from spider monkeys did not form cytoplasmic bodies. Despite these differences, all of the TRIM5α proteins exhibited the ability to restrict infection by particular retroviruses. Treatment of cells with geldanamycin, an Hsp90 inhibitor, resulted in disappearance or reduction of the TRIM5α-associated cytoplasmic bodies, yet exerted little effect on the restriction of retroviral infection. Studies of green fluorescent protein-TRIM5α fusion proteins indicated that no TRIM5α domain is specifically required for association with cytoplasmic bodies. Apparently, the formation of cytoplasmic bodies is not required for the antiretroviral activity of TRIM5α

  2. Characterization of depolarization-coupled release of glutamate from cultured mouse cerebellar granule cells using DL-threo-beta-benzyloxyaspartate (DL-TBOA) to distinguish between the vesicular and cytoplasmic pools

    DEFF Research Database (Denmark)

    Bak, Lasse K; Schousboe, Arne; Waagepetersen, Helle S

    2003-01-01

    Release of preloaded [3H]D-aspartate in response to depolarization induced by N-methyl-D-aspartate (NMDA) or the endogenous agonist glutamate was characterized using cultured glutamatergic cerebellar granule neurons. Release from the vesicular and the cytoplasmic glutamate pools, respectively, wa...

  3. Nuclear translocation and regulation of intranuclear distribution of cytoplasmic poly(A-binding protein are distinct processes mediated by two Epstein Barr virus proteins.

    Directory of Open Access Journals (Sweden)

    Richard Park

    Full Text Available Many viruses target cytoplasmic polyA binding protein (PABPC to effect widespread inhibition of host gene expression, a process termed viral host-shutoff (vhs. During lytic replication of Epstein Barr Virus (EBV we observed that PABPC was efficiently translocated from the cytoplasm to the nucleus. Translocated PABPC was diffusely distributed but was excluded from viral replication compartments. Vhs during EBV infection is regulated by the viral alkaline nuclease, BGLF5. Transfection of BGLF5 alone into BGLF5-KO cells or uninfected 293 cells promoted translocation of PAPBC that was distributed in clumps in the nucleus. ZEBRA, a viral bZIP protein, performs essential functions in the lytic program of EBV, including activation or repression of downstream viral genes. ZEBRA is also an essential replication protein that binds to viral oriLyt and interacts with other viral replication proteins. We report that ZEBRA also functions as a regulator of vhs. ZEBRA translocated PABPC to the nucleus, controlled the intranuclear distribution of PABPC, and caused global shutoff of host gene expression. Transfection of ZEBRA alone into 293 cells caused nuclear translocation of PABPC in the majority of cells in which ZEBRA was expressed. Co-transfection of ZEBRA with BGLF5 into BGLF5-KO cells or uninfected 293 cells rescued the diffuse intranuclear pattern of PABPC seen during lytic replication. ZEBRA mutants defective for DNA-binding were capable of regulating the intranuclear distribution of PABPC, and caused PABPC to co-localize with ZEBRA. One ZEBRA mutant, Z(S186E, was deficient in translocation yet was capable of altering the intranuclear distribution of PABPC. Therefore ZEBRA-mediated nuclear translocation of PABPC and regulation of intranuclear PABPC distribution are distinct events. Using a click chemistry-based assay for new protein synthesis, we show that ZEBRA and BGLF5 each function as viral host shutoff factors.

  4. Cytoplasmic TRADD Confers a Worse Prognosis in Glioblastoma

    Directory of Open Access Journals (Sweden)

    Sharmistha Chakraborty

    2013-08-01

    Full Text Available Tumor necrosis factor receptor 1 (TNFR1-associated death domain protein (TRADD is an important adaptor in TNFR1 signaling and has an essential role in nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB activation and survival signaling. Increased expression of TRADD is sufficient to activate NF-κB. Recent studies have highlighted the importance of NF-κB activation as a key pathogenic mechanism in glioblastoma multiforme (GBM, the most common primary malignant brain tumor in adults.We examined the expression of TRADD by immunohistochemistry (IHC and find that TRADD is commonly expressed at high levels in GBM and is detected in both cytoplasmic and nuclear distribution. Cytoplasmic IHC TRADD scoring is significantly associated with worse progression-free survival (PFS both in univariate and multivariate analysis but is not associated with overall survival (n = 43 GBMs. PFS is a marker for responsiveness to treatment. We propose that TRADD-mediated NF-κB activation confers chemoresistance and thus a worse PFS in GBM. Consistent with the effect on PFS, silencing TRADD in glioma cells results in decreased NF-κB activity, decreased proliferation of cells, and increased sensitivity to temozolomide. TRADD expression is common in glioma-initiating cells. Importantly, silencing TRADD in GBM-initiating stem cell cultures results in decreased viability of stem cells, suggesting that TRADD may be required for maintenance of GBM stem cell populations. Thus, our study suggests that increased expression of cytoplasmic TRADD is both an important biomarker and a key driver of NF-κB activation in GBM and supports an oncogenic role for TRADD in GBM.

  5. Mutant p53 protein localized in the cytoplasm inhibits autophagy.

    Science.gov (United States)

    Morselli, Eugenia; Tasdemir, Ezgi; Maiuri, Maria Chiara; Galluzzi, Lorenzo; Kepp, Oliver; Criollo, Alfredo; Vicencio, José Miguel; Soussi, Thierry; Kroemer, Guido

    2008-10-01

    The knockout, knockdown or chemical inhibition of p53 stimulates autophagy. Moreover, autophagy-inducing stimuli such as nutrient depletion, rapamycin or lithium cause the depletion of cytoplasmic p53, which in turn is required for the induction of autophagy. Here, we show that retransfection of p53(-/-) HCT 116 colon carcinoma cells with wild type p53 decreases autophagy down to baseline levels. Surprisingly, one third among a panel of 22 cancer-associated p53 single amino acid mutants also inhibited autophagy when transfected into p53(-/-) cells. Those variants of p53 that preferentially localize to the cytoplasm effectively repressed autophagy, whereas p53 mutants that display a prominently nuclear distribution failed to inhibit autophagy. The investigation of a series of deletion mutants revealed that removal of the DNA-binding domain from p53 fails to interfere with its role in the regulation of autophagy. Altogether, these results identify the cytoplasmic localization of p53 as the most important feature for p53-mediated autophagy inhibition. Moreover, the structural requirements for the two biological activities of extranuclear p53, namely induction of apoptosis and inhibition of autophagy, are manifestly different.

  6. Expression of cytoplasmic CD3 epsilon proteins in activated human adult natural killer (NK) cells and CD3 gamma, delta, epsilon complexes in fetal NK cells. Implications for the relationship of NK and T lymphocytes

    NARCIS (Netherlands)

    Lanier, L. L.; Chang, C.; Spits, H.; Phillips, J. H.

    1992-01-01

    NK cells have been defined as CD3-, CD16+, and/or CD56+ lymphocytes that mediate MHC-unrestricted cytotoxicity against certain tumors and virus-infected cells. Although CD3 epsilon transcripts have been detected in some NK clones, it has generally been thought that NK cells do not express CD3

  7. Immunomodulatory and antitumor effects in vivo by the cytoplasmic fraction of Lactobacillus casei and Bifidobacterium longum.

    Science.gov (United States)

    Lee, Jung-Woo; Shin, Jung-Gul; Kim, Eun Hee; Kang, Hae Eun; Yim, In Been; Kim, Ji Yeon; Joo, Hong-Gu; Woo, Hee Jong

    2004-03-01

    The immunomodulatory and antitumor effects of lactic acid bacteria (LABs) were investigated. Cytoplasmic fraction of Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium longum were tested for the antiproliferative activity in vitro to SNUC2A, SNU1, NIH/3T3 and Jurkat cell lines by crystal violet assay. All cytoplasmic fraction suppressed proliferation of tumor cells, though L. casei and B. longum were more effective. From these results, cytoplasmic fraction of L. casei and B. longum with Y400 as a control were administered as dietary supplements to Balb/c mice for 2, and 4 consecutive wks. Administration for 4 wks enhanced the number of total T cells, NK cells and MHC class II+ cells, and CD4-CD8+ T cells in flow cytometry analysis. To determine of antitumor activity of LABs preparation in vivo, F9 teratocarcinoma cells were inoculated on mice at 14th day. Body weight was decreased with increased survival rate in all groups with the cytoplasm of LABs. Our results showed that cytoplasmic fraction of LABs had direct antiproliferative effects on tumor cell lines in vitro, effects on immune cells in vivo, and antitumor effects on tumor-bearing mice with prolonged survival periods.

  8. Genetic analysis of the cytoplasmic dynein subunit families.

    Science.gov (United States)

    Pfister, K Kevin; Shah, Paresh R; Hummerich, Holger; Russ, Andreas; Cotton, James; Annuar, Azlina Ahmad; King, Stephen M; Fisher, Elizabeth M C

    2006-01-01

    Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles.

  9. Genetic analysis of the cytoplasmic dynein subunit families.

    Directory of Open Access Journals (Sweden)

    K Kevin Pfister

    2006-01-01

    Full Text Available Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles.

  10. Abdominal compartment syndrome with acute reperfusion syndrome

    International Nuclear Information System (INIS)

    Maleeva, A.

    2017-01-01

    Abdominal compartment syndrome was recognized clinically in the 19th century when Marey and Burt observed its association with declines in respiratory function. Abdominal compartment syndrome is first used as a medical terminology from Fietsman in a case of ruptured abdominal aortic aneurysm. A condition caused by abnormally increased pressure within the abdomen. Causes of abdominal compartment syndrome include trauma, surgery, or infection. Common symptoms: abdominal distension, fast heart rate, insufficient urine production, or low blood pressure Medical procedure: nasogastric intubation Surgery: laparotomy Specialists: radiologist, primary care provider (PCP), surgeon, and emergency medicine doctor [6, 10]. Keywords: Stomach. Gastroparesis . Diabetes Mellitus [bg

  11. Venture from the Interior-Herpesvirus pUL31 Escorts Capsids from Nucleoplasmic Replication Compartments to Sites of Primary Envelopment at the Inner Nuclear Membrane.

    Science.gov (United States)

    Bailer, Susanne M.

    2017-11-25

    Herpesviral capsid assembly is initiated in the nucleoplasm of the infected cell. Size constraints require that newly formed viral nucleocapsids leave the nucleus by an evolutionarily conserved vescular transport mechanism called nuclear egress. Mature capsids released from the nucleoplasm are engaged in a membrane-mediated budding process, composed of primary envelopment at the inner nuclear membrane and de-envelopment at the outer nuclear membrane. Once in the cytoplasm, the capsids receive their secondary envelope for maturation into infectious virions. Two viral proteins conserved throughout the herpesvirus family, the integral membrane protein pUL34 and the phosphoprotein pUL31, form the nuclear egress complex required for capsid transport from the infected nucleus to the cytoplasm. Formation of the nuclear egress complex results in budding of membrane vesicles revealing its function as minimal virus-encoded membrane budding and scission machinery. The recent structural analysis unraveled details of the heterodimeric nuclear egress complex and the hexagonal coat it forms at the inside of budding vesicles to drive primary envelopment. With this review, I would like to present the capsid-escort-model where pUL31 associates with capsids in nucleoplasmic replication compartments for escort to sites of primary envelopment thereby coupling capsid maturation and nuclear egress.

  12. The components of the unique Zur regulon of Cupriavidus metallidurans mediate cytoplasmic zinc handling.

    Science.gov (United States)

    Bütof, Lucy; Schmidt-Vogler, Christopher; Herzberg, Martin; Große, Cornelia; Nies, Dietrich H

    2017-08-14

    Zinc is an essential trace element and at the same time it is toxic at high concentrations. In the beta-proteobacterium Cupriavidus metallidurans the highly efficient removal of surplus zinc from the periplasm is responsible for its outstanding metal resistance. Rather than having a typical Zur-dependent, high-affinity ATP-binding cassette transporter of the ABC protein superfamily for zinc uptake at low concentrations, C. metallidurans instead has the secondary zinc importer ZupT of the ZRT/IRT (ZIP) family. It is important to understand, therefore, how this zinc-resistant bacterium copes when it is exposed to low zinc concentrations. Members of the Zur regulon in C. metallidurans were identified by comparing the transcriptomes of a Δ zur mutant and its parent strain. The consensus sequence of the Zur-binding box was derived for the zupTp promoter-regulatory region using a truncation assay. The motif was used to predict possible Zur-boxes upstream of Zur regulon members. Binding of Zur to these boxes was confirmed. Two Zur-boxes upstream of the cobW 1 gene, encoding a putative zinc chaperone, proved to be required for complete repression of cobW 1 and its downstream genes in cells cultivated in mineral salts medium. A Zur box upstream of each of zur-cobW 2 , cobW 3 and zupT permitted low-expression level of these genes plus their up-regulation under zinc starvation conditions. This demonstrates a compartmentalization of zinc homeostasis in C. metallidurans with the periplasm being responsible for removal of surplus zinc and cytoplasmic components for management of zinc as an essential co-factor, with both compartments connected by ZupT. Importance Elucidating zinc homeostasis is necessary to understand both host-pathogen interactions and performance of free-living bacteria in their natural environment. Escherichia coli acquires zinc under low zinc concentrations by the Zur-controlled ZnuABC importer of the ABC superfamily, and this was also the paradigm for other

  13. The statolith compartment in Chara rhizoids contains carbohydrate and protein

    Science.gov (United States)

    Wang-Cahill, F.; Kiss, J. Z.

    1995-01-01

    In contrast to higher plants, the alga Chara has rhizoids with single membrane-bound compartments that function as statoliths in gravity perception. Previous work has demonstrated that these statoliths contain barium sulfate crystals. In this study, we show that statoliths in Chara rhizoids react with a Coomassie Brilliant Blue cytochemical stain for proteins. While statoliths did not react with silver methenamine carbohydrate cytochemistry, the monoclonal antibody CCRC-M2, which is against a carbohydrate (sycamore-maple rhamnogalacturonan I), labeled the statolith compartment. These results demonstrate that in addition to barium sulfate, statoliths in Chara rhizoids have an organic matrix that consists of protein and carbohydrate moieties. Since the statoliths were silver methenamine negative, the carbohydrate in this compartment could be a 3-linked polysaccharide. CCRC-M2 also labeled Golgi cisternae, Golgi-associated vesicles, apical vesicles, and cell walls in the rhizoids. The specificity of CCRC-M2 immunolabeling was verified by several control experiments, including the demonstration that labeling was abolished when the antibody was preabsorbed with its antigen. Since in this and a previous study (John Z. Kiss and L. Andrew Staehelin, American Journal of Botany 80: 273-282, 1993) antibodies against higher plant carbohydrates crossreacted with cell walls of Chara in a specific manner, Characean algae may be a useful model system in biochemical and molecular studies of cell walls.

  14. Cytoplasmic Dynein Regulation by Subunit Heterogeneity and Its Role in Apical Transport

    Science.gov (United States)

    Tai, Andrew W.; Chuang, Jen-Zen; Sung, Ching-Hwa

    2001-01-01

    Despite the existence of multiple subunit isoforms for the microtubule motor cytoplasmic dynein, it has not yet been directly shown that dynein complexes with different compositions exhibit different properties. The 14-kD dynein light chain Tctex-1, but not its homologue RP3, binds directly to rhodopsin's cytoplasmic COOH-terminal tail, which encodes an apical targeting determinant in polarized epithelial Madin-Darby canine kidney (MDCK) cells. We demonstrate that Tctex-1 and RP3 compete for binding to dynein intermediate chain and that overexpressed RP3 displaces endogenous Tctex-1 from dynein complexes in MDCK cells. Furthermore, replacement of Tctex-1 by RP3 selectively disrupts the translocation of rhodopsin to the MDCK apical surface. These results directly show that cytoplasmic dynein function can be regulated by its subunit composition and that cytoplasmic dynein is essential for at least one mode of apical transport in polarized epithelia. PMID:11425878

  15. In Vivo Imaging of Diacylglycerol at the Cytoplasmic Leaflet of Plant Membranes.

    Science.gov (United States)

    Vermeer, Joop E M; van Wijk, Ringo; Goedhart, Joachim; Geldner, Niko; Chory, Joanne; Gadella, Theodorus W J; Munnik, Teun

    2017-07-01

    Diacylglycerol (DAG) is an important intermediate in lipid biosynthesis and plays key roles in cell signaling, either as a second messenger itself or as a precursor of phosphatidic acid. Methods to identify distinct DAG pools have proven difficult because biochemical fractionation affects the pools, and concentrations are limiting. Here, we validate the use of a genetically encoded DAG biosensor in living plant cells. The sensor is composed of a fusion between yellow fluorescent protein and the C1a domain of protein kinase C (YFP-C1aPKC) that specifically binds DAG, and was stably expressed in suspension-cultured tobacco BY-2 cells and whole Arabidopsis thaliana plants. Confocal imaging revealed that the majority of the YFP-C1aPKC fluorescence did not locate to membranes but was present in the cytosol and nucleus. Treatment with short-chain DAG or PMA (phorbol-12-myristate-13-acetate), a phorbol ester that binds the C1a domain of PKC, caused the recruitment of the biosensor to the plasma membrane. These results indicate that the biosensor works and that the basal DAG concentration in the cytoplasmic leaflet of membranes (i.e. accessible to the biosensor) is in general too low, and confirms that the known pools in plastids, the endoplasmic reticulum and mitochondria are located at the luminal face of these compartments (i.e. inaccessible to the biosensor). Nevertheless, detailed further analysis of different cells and tissues discovered four novel DAG pools, namely at: (i) the trans-Golgi network; (ii) the cell plate during cytokinesis; (iii) the plasma membrane of root epidermal cells in the transition zone, and (iv) the apex of growing root hairs. The results provide new insights into the spatiotemporal dynamics of DAG in plants and offer a new tool to monitor this in vivo. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  16. Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 3: developmental changes in spermatid flagellum and cytoplasmic droplet and interaction of sperm with the zona pellucida and egg plasma membrane.

    Science.gov (United States)

    Hermo, Louis; Pelletier, R-Marc; Cyr, Daniel G; Smith, Charles E

    2010-04-01

    Spermiogenesis constitutes the steps involved in the metamorphosis of spermatids into spermatozoa. It involves modification of several organelles in addition to the formation of several structures including the flagellum and cytoplasmic droplet. The flagellum is composed of a neck region and middle, principal, and end pieces. The axoneme composed of nine outer microtubular doublets circularly arranged to form a cylinder around a central pair of microtubules is present throughout the flagellum. The middle and principal pieces each contain specific components such as the mitochondrial sheath and fibrous sheath, respectively, while outer dense fibers are common to both. A plethora of proteins are constituents of each of these structures, with each playing key roles in functions related to the fertility of spermatozoa. At the end of spermiogenesis, a portion of spermatid cytoplasm remains associated with the released spermatozoa, referred to as the cytoplasmic droplet. The latter has as its main feature Golgi saccules, which appear to modify the plasma membrane of spermatozoa as they move down the epididymal duct and hence may be partly involved in male gamete maturation. The end product of spermatogenesis is highly streamlined and motile spermatozoa having a condensed nucleus equipped with an acrosome. Spermatozoa move through the female reproductive tract and eventually penetrate the zona pellucida and bind to the egg plasma membrane. Many proteins have been implicated in the process of fertilization as well as a plethora of proteins involved in the development of spermatids and sperm, and these are high lighted in this review. Copyright 2009 Wiley-Liss, Inc.

  17. Abdominal Compartment Syndrome in Surgical Patients

    African Journals Online (AJOL)

    abdominal hypertension and abdominal compartment syndrome, affect ... timely surgical intervention is crucial. Key words: .... On the second postoperative day, he was noted to be restless ... Although surgery is very effective in managing ACS.

  18. Compartment syndrome in a labrador retriever

    International Nuclear Information System (INIS)

    Williams, J.; Bailey, M.Q.; Schertel, E.R.; Valentine, A.

    1994-01-01

    Compartment syndrome is an elevation of interstitial pressure in a closed osseofascial compartment that results in microvascular compromise. This report documents a clinical syndrome in the crus of a fourteen-month-old intact male Labrador Retriever which was consistent with trauma-induced compartment syndrome. A six month history of recurring trauma or complications resulted in the need for referral. Survey radiography and ultrasonography aided in the diagnosis, but the definitive answer was provided by femoral angiography. The patient was successfully treated and was discharged with normal limb function. One year later, there were no complications observed. Compartment syndrome is not uncommon in humans, and is routinely considered in certain blunt and most penetrating traumas. However, few reports of this complication in animals are found

  19. Multi-compartment microscopic diffusion imaging

    OpenAIRE

    Kaden, Enrico; Kelm, Nathaniel D.; Carson, Robert P.; Does, Mark D.; Alexander, Daniel C.

    2016-01-01

    This paper introduces a multi-compartment model for microscopic diffusion anisotropy imaging. The aim is to estimate microscopic features specific to the intra- and extra-neurite compartments in nervous tissue unconfounded by the effects of fibre crossings and orientation dispersion, which are ubiquitous in the brain. The proposed MRI method is based on the Spherical Mean Technique (SMT), which factors out the neurite orientation distribution and thus provides direct estimates of the microsco...

  20. Emergence of nuclear heparanase induces differentiation of human mammary cancer cells

    International Nuclear Information System (INIS)

    Nobuhisa, Tetsuji; Naomoto, Yoshio; Takaoka, Munenori; Tabuchi, Yoko; Ookawa, Keizou; Kitamoto, Dai; Gunduz, Esra; Gunduz, Mehmet; Nagatsuka, Hitoshi; Haisa, Minoru; Matsuoka, Junji; Nakajima, Motowo; Tanaka, Noriaki

    2005-01-01

    The study of epithelial differentiation touches upon many modern aspects of biology. The epithelium is in constant dialogue with the underlying mesenchyme to control stem cell activity, proliferation in transit-amplifying compartments, lineage commitment, terminal differentiation and, ultimately, cell death. There are spatially distinct compartments dedicated to each of these events. Recently we reported that heparanase is expressed in nucleus as well as in the cytoplasm and that nuclear heparanase seems to be related to cell differentiation. In this study, we investigated the role of nuclear heparanase in differentiation by transducing human mammary epithelial cancer cells with heparanase which was delivered specifically into nucleus. We observed that expression of nuclear heparanase allowed the cells to differentiate with the appearance of lipid droplets. This finding supports the idea that heparanase plays a novel role in epithelial cell differentiation apart from its known enzymatic function

  1. Cytoplasmic Control of Sense-Antisense mRNA Pairs

    Directory of Open Access Journals (Sweden)

    Flore Sinturel

    2015-09-01

    Full Text Available Transcriptome analyses have revealed that convergent gene transcription can produce many 3′-overlapping mRNAs in diverse organisms. Few studies have examined the fate of 3′-complementary mRNAs in double-stranded RNA-dependent nuclear phenomena, and nothing is known about the cytoplasmic destiny of 3′-overlapping messengers or their impact on gene expression. Here, we demonstrate that the complementary tails of 3′-overlapping mRNAs can interact in the cytoplasm and promote post-transcriptional regulatory events including no-go decay (NGD in Saccharomyces cerevisiae. Genome-wide experiments confirm that these messenger-interacting mRNAs (mimRNAs form RNA duplexes in wild-type cells and thus have potential roles in modulating the mRNA levels of their convergent gene pattern under different growth conditions. We show that the post-transcriptional fate of hundreds of mimRNAs is controlled by Xrn1, revealing the extent to which this conserved 5′-3′ cytoplasmic exoribonuclease plays an unexpected but key role in the post-transcriptional control of convergent gene expression.

  2. Cytoplasmic Control of Sense-Antisense mRNA Pairs.

    Science.gov (United States)

    Sinturel, Flore; Navickas, Albertas; Wery, Maxime; Descrimes, Marc; Morillon, Antonin; Torchet, Claire; Benard, Lionel

    2015-09-22

    Transcriptome analyses have revealed that convergent gene transcription can produce many 3'-overlapping mRNAs in diverse organisms. Few studies have examined the fate of 3'-complementary mRNAs in double-stranded RNA-dependent nuclear phenomena, and nothing is known about the cytoplasmic destiny of 3'-overlapping messengers or their impact on gene expression. Here, we demonstrate that the complementary tails of 3'-overlapping mRNAs can interact in the cytoplasm and promote post-transcriptional regulatory events including no-go decay (NGD) in Saccharomyces cerevisiae. Genome-wide experiments confirm that these messenger-interacting mRNAs (mimRNAs) form RNA duplexes in wild-type cells and thus have potential roles in modulating the mRNA levels of their convergent gene pattern under different growth conditions. We show that the post-transcriptional fate of hundreds of mimRNAs is controlled by Xrn1, revealing the extent to which this conserved 5'-3' cytoplasmic exoribonuclease plays an unexpected but key role in the post-transcriptional control of convergent gene expression. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  3. Titration of a cytoplasmic polyhedrosis virus by a tissue microculture assay: some applications.

    Science.gov (United States)

    Belloncik, S; Chagnon, A

    1980-01-01

    A simple tissue microculture technique was developed for the titration of a cytoplasmic polyhedrosis virus (CPV) from Euxoa scandens. The procedure was similar to the 50% tissue culture infectious dose assay, but a single infected cell, detected by the presence of cytoplasmic polyhedra, was scored rather than the degeneration of cell monolayers. The filtration of CPV suspensions resulted in decreased virus titers under certain conditions. This microculture assay was used to determine the effect of cell disruption methods on virus yields. Sonication of infected cells was more efficient than freeze-thawing for the recovery of nonoccluded virus.

  4. Expression of Anion Exchanger 1 Sequestrates p16 in the Cytoplasm in Gastric, Colonic Adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Wei-Wei Shen

    2007-10-01

    Full Text Available p16INK4A (p16 binds to cyclin-dependent kinase 4/6, negatively regulates cell growth. Recent studies have led to an understanding of additional biologic functions for p16; however, the detailed mechanisms involved are still elusive. In this article, we show an unexpected expression of anion exchanger 1 (AEi in the cytoplasm in poorly, moderately differentiated gastric, colonic adenocarcinoma cells, in its interaction with p16, thereby sequestrating the protein in the cytoplasm. Genetic alterations of p16, AEi were not detectable. Forced expression of AEi in these cells sequestrated more p16 in the cytoplasm, whereas small interfering RNA-mediated silencing of AEi in the cells induced the release of p16 from the cytoplasm to the nucleus, leading to cell death, growth inhibition of tumor cells. By analyzing tissue samples obtained from patients with gastric, colonic cancers, we found that 83.33% of gastric cancers, 56.52% of colonic cancers coexpressed AEi, p16 in the cytoplasm. We conclude that AEi plays a crucial role in the pathogenesis of gastric, colonic adenocarcinoma, that p16 dysfunction is a novel pathway of carcinogenesis.

  5. Differential CARM1 Isoform Expression in Subcellular Compartments and among Malignant and Benign Breast Tumors.

    Directory of Open Access Journals (Sweden)

    David Shlensky

    Full Text Available Coactivator-associated arginine methyltransferase 1 (CARM1 is a coactivator for ERα and cancer-relevant transcription factors, and can methylate diverse cellular targets including histones. CARM1 is expressed in one of two alternative splice isoforms, full-length CARM1 (CARM1FL and truncated CARM1 (CARM1ΔE15. CARM1FL and CARM1ΔE15 function differently in transcriptional regulation, protein methylation, and mediation of pre-mRNA splicing in cellular models.To investigate the functional roles and the prognosis potential of CARM1 alternative spliced isoforms in breast cancer, we used recently developed antibodies to detect differential CARM1 isoform expression in subcellular compartments and among malignant and benign breast tumors.Immunofluorescence in MDA-MB-231 and BG-1 cell lines demonstrated that CARM1ΔE15 is the dominant isoform expressed in the cytoplasm, and CARM1FL is more nuclear localized. CARM1ΔE15 was found to be more sensitive to Hsp90 inhibition than CARM1FL, indicating that the truncated isoform may be the oncogenic form. Clinical cancer samples did not have significantly higher expression of CARM1FL or CARM1ΔE15 than benign breast samples at the level of mRNA or histology. Furthermore neither CARM1FL nor CARM1ΔE15 expression correlated with breast cancer molecular subtypes, tumor size, or lymph node involvement.The analysis presented here lends new insights into the possible oncogenic role of CARM1ΔE15. This study also demonstrates no obvious association of CARM1 isoform expression and clinical correlates in breast cancer. Recent studies, however, have shown that CARM1 expression correlates with poor prognosis, indicating a need for further studies of both CARM1 isoforms in a large cohort of breast cancer specimens.

  6. Involvement of the mitochondrial compartment in human NCL fibroblasts

    International Nuclear Information System (INIS)

    Pezzini, Francesco; Gismondi, Floriana; Tessa, Alessandra; Tonin, Paola; Carrozzo, Rosalba; Mole, Sara E.; Santorelli, Filippo M.; Simonati, Alessandro

    2011-01-01

    Highlights: ► Mitochondrial reticulum fragmentation occurs in human CLN1 and CLN6 fibroblasts. ► Likewise mitochondrial shift-to periphery and decreased mitochondrial density are seen. ► Enhanced caspase-mediated apoptosis occurs following STS treatment in CLN1 fibroblasts. -- Abstract: Neuronal ceroid lipofuscinosis (NCL) are a group of progressive neurodegenerative disorders of childhood, characterized by the endo-lysosomal storage of autofluorescent material. Impaired mitochondrial function is often associated with neurodegeneration, possibly related to the apoptotic cascade. In this study we investigated the possible effects of lysosomal accumulation on the mitochondrial compartment in the fibroblasts of two NCL forms, CLN1 and CLN6. Fragmented mitochondrial reticulum was observed in all cells by using the intravital fluorescent marker Mitotracker, mainly in the perinuclear region. This was also associated with intense signal from the lysosomal markers Lysotracker and LAMP2. Likewise, mitochondria appeared to be reduced in number and shifted to the cell periphery by electron microscopy; moreover the mitochondrial markers VDCA and COX IV were reduced following quantitative Western blot analysis. Whilst there was no evidence of increased cell death under basal condition, we observed a significant increase in apoptotic nuclei following Staurosporine treatment in CLN1 cells only. In conclusion, the mitochondrial compartment is affected in NCL fibroblasts invitro, and CLN1 cells seem to be more vulnerable to the negative effects of stressed mitochondrial membrane than CLN6 cells.

  7. Detection of antineutrophil cytoplasmic antibodies (ANCAs)

    DEFF Research Database (Denmark)

    Damoiseaux, Jan; Csernok, Elena; Rasmussen, Niels

    2017-01-01

    of diagnosis) from 251 patients with ANCA-associated vasculitis (AAV), including granulomatosis with polyangiitis and microscopic polyangiitis, and from 924 disease controls were tested for the presence of cytoplasmic pattern/perinuclear pattern and atypical ANCA (A-ANCA) by indirect immunofluorescence (IIF...

  8. Curious Sex Ratios and Cytoplasmic Genes

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 2; Issue 6. Curious Sex Ratios and Cytoplasmic Genes Microbes Can Distort the Sex Ratio of Populations. Stephen J Freeland Laurence D Hurst. General Article Volume 2 Issue 6 June 1997 pp 68-78 ...

  9. Investigation of radiation enhanced reactivation of cytoplasmic replicating human virus

    International Nuclear Information System (INIS)

    Bockstahler, L.E.; Haynes, K.F.; Stafford, J.E.

    1976-01-01

    When monolayers of CV-1 monkey kidney cells were exposed to ultraviolet (uv) radiation (0 to 200 erg/nm 2 ) or x rays (0 to 10 krads) before infection with uv-irradiated herpes simplex virus, an increase in the infectivity of this nuclear replicating virus occurred as measured by plaque formation. These phenomena are known as uv (Weigle) reactivation (WR) and x-ray reactivation (x-ray R). In this study the presence of WR and x-ray R was examined in CV-1 cells infected with uv-irradiated vaccinia virus or poliovirus, both cytoplasmic replicating viruses. Little or no WR or x-ray R was observed for either of these viruses. These results suggest that WR and x-ray R in mammalian cells may be restricted to viruses which are synthesized in the cell nucleus

  10. Birbeck granule-like "organized smooth endoplasmic reticulum" resulting from the expression of a cytoplasmic YFP-tagged langerin.

    Directory of Open Access Journals (Sweden)

    Cédric Lenormand

    Full Text Available Langerin is required for the biogenesis of Birbeck granules (BGs, the characteristic organelles of Langerhans cells. We previously used a Langerin-YFP fusion protein having a C-terminal luminal YFP tag to dynamically decipher the molecular and cellular processes which accompany the traffic of Langerin. In order to elucidate the interactions of Langerin with its trafficking effectors and their structural impact on the biogenesis of BGs, we generated a YFP-Langerin chimera with an N-terminal, cytosolic YFP tag. This latter fusion protein induced the formation of YFP-positive large puncta. Live cell imaging coupled to a fluorescence recovery after photobleaching approach showed that this coalescence of proteins in newly formed compartments was static. In contrast, the YFP-positive structures present in the pericentriolar region of cells expressing Langerin-YFP chimera, displayed fluorescent recovery characteristics compatible with active membrane exchanges. Using correlative light-electron microscopy we showed that the coalescent structures represented highly organized stacks of membranes with a pentalaminar architecture typical of BGs. Continuities between these organelles and the rough endoplasmic reticulum allowed us to identify the stacks of membranes as a form of "Organized Smooth Endoplasmic Reticulum" (OSER, with distinct molecular and physiological properties. The involvement of homotypic interactions between cytoplasmic YFP molecules was demonstrated using an A206K variant of YFP, which restored most of the Langerin traffic and BG characteristics observed in Langerhans cells. Mutation of the carbohydrate recognition domain also blocked the formation of OSER. Hence, a "double-lock" mechanism governs the behavior of YFP-Langerin, where asymmetric homodimerization of the YFP tag and homotypic interactions between the lectin domains of Langerin molecules participate in its retention and the subsequent formation of BG-like OSER. These

  11. Tumor-associated Tn-MUC1 glycoform is internalized through the macrophage galactose-type C-type lectin and delivered to the HLA class I and II compartments in dendritic cells

    DEFF Research Database (Denmark)

    Napoletano, Chiara; Rughetti, Aurelia; Agervig Tarp, Mads P

    2007-01-01

    . This results in the expression of tumor-associated glycoforms and in MUC1 carrying the tumor-specific glycan Tn (GalNAcalpha1-O-Ser/Thr). Glycopeptides corresponding to three tandem repeats of MUC1, enzymatically glycosylated with 9 or 15 mol of GalNAc, were shown to specifically bind and to be internalized...... and ELISA done on subcellular fractions of iDCs showed that the Tn-MUC1 glycopeptides colocalized with HLA class I and II compartments after internalization. Importantly, although Tn-MUC1 recombinant protein was bound and internalized by MGL, the glycoprotein entered the HLA class II compartment......, but not the HLA class I pathway. These data indicate that MGL expressed on iDCs is an optimal receptor for the internalization of short GalNAcs carrying immunogens to be delivered into HLA class I and II compartments. Such glycopeptides therefore represent a new way of targeting the HLA class I and II pathways...

  12. Activation of chromatin degradation by a protein factor of thymocyte cytoplasm of irradiated mice

    International Nuclear Information System (INIS)

    Soldatenkov, V.A.; Filippovich, I.V.

    1986-01-01

    A cytoplasmic thymocyte fraction isolated 1 h after irradiation of mice accelerates chromatin degradation in isolated nuclei. Treatment of the cytoplasmic fraction by heat and injection of cycloheximide to mice prevent the acceleration of DNA degradation. The analysis of the chromatin degradation products and the kinetics of this process at acid and alkaline pH shows that activation of DNA degradation in thymocytes by a factor obtained from the irradiated cell cytoplasm is specific for a Ca 2+ , Mg 2+ -dependent enzyme. The time- and dose-dependent parameters of the appearance in the thymocyte cytoplasm of the factor influencing degradation of chromatin are in a good agreement with both the time of the onset of its postirradiation degradation and the dose dependence of this process

  13. Iliopsoas compartment lesions: a radiologic evaluation

    International Nuclear Information System (INIS)

    Leao, Alberto Ribeiro de Souza; Amaral, Raquel Portugal Guimaraes; Abud, Thiago Giansante; Demarchi, Guilherme Tadeu Sauaia; Freire Filho, Edison de Oliveira; Novack, Paulo Rogerio; Campos, Flavio do Amaral; Shigueoka, David Carlos; Fernandes, Artur da Rocha Correa; Szejnfeld, Jacob; D'Ippolito, Giuseppe

    2007-01-01

    The iliopsoas compartment, a posterior boundary of the retroperitoneum, is comprised of the psoas major, psoas minor and iliac muscles. The symptoms picture in patients presenting with pathological involvement of this compartment may show a wide range of nonspecific clinical presentations that may lead to delayed diagnosis. However, in the search of an etiological diagnosis, it is already known that inflammation, tumors, and hemorrhages account for almost all the lesions affecting the iliopsoas compartment. By means of a retrospective analysis of radiological studies in patients with iliopsoas compartment lesions whose diagnosis was confirmed by anatomopathological evaluation or clinical follow-up, we have reviewed its anatomy as well as the main forms of involvement, with the purpose of identifying radiological signs that may help to narrow down the potential differential diagnoses. As each lesion is approached we will discuss the main radiological findings such as presence of gas in pyogenic abscesses, bone destruction and other bone changes of vertebral bodies in lesions secondary to tuberculosis, involvement of fascial planes in cases of neoplasms, and differences in signal density and intensity of hematomas secondary to hemoglobin degradation, among others. So, we have tried to present cases depicting the most frequent lesions involving the iliopsoas compartment, with emphasis on those signs that can lead us to a more specific etiological diagnosis. (author)

  14. Iliopsoas compartment lesions: a radiologic evaluation

    Energy Technology Data Exchange (ETDEWEB)

    Leao, Alberto Ribeiro de Souza; Amaral, Raquel Portugal Guimaraes; Abud, Thiago Giansante; Demarchi, Guilherme Tadeu Sauaia; Freire Filho, Edison de Oliveira; Novack, Paulo Rogerio; Campos, Flavio do Amaral; Shigueoka, David Carlos; Fernandes, Artur da Rocha Correa; Szejnfeld, Jacob; D' Ippolito, Giuseppe [Universidade Federal de Sao Paulo (UNIFESP-EPM), Sao Paulo, SP (Brazil). Dept. de Diagnostico por Imagem]. E-mail: ar.leao@uol.com.br; Santos, Jose Eduardo Mourao [Universidade Federal de Sao Paulo (UNIFESP-EPM), Sao Paulo, SP (Brazil)

    2007-07-15

    The iliopsoas compartment, a posterior boundary of the retroperitoneum, is comprised of the psoas major, psoas minor and iliac muscles. The symptoms picture in patients presenting with pathological involvement of this compartment may show a wide range of nonspecific clinical presentations that may lead to delayed diagnosis. However, in the search of an etiological diagnosis, it is already known that inflammation, tumors, and hemorrhages account for almost all the lesions affecting the iliopsoas compartment. By means of a retrospective analysis of radiological studies in patients with iliopsoas compartment lesions whose diagnosis was confirmed by anatomopathological evaluation or clinical follow-up, we have reviewed its anatomy as well as the main forms of involvement, with the purpose of identifying radiological signs that may help to narrow down the potential differential diagnoses. As each lesion is approached we will discuss the main radiological findings such as presence of gas in pyogenic abscesses, bone destruction and other bone changes of vertebral bodies in lesions secondary to tuberculosis, involvement of fascial planes in cases of neoplasms, and differences in signal density and intensity of hematomas secondary to hemoglobin degradation, among others. So, we have tried to present cases depicting the most frequent lesions involving the iliopsoas compartment, with emphasis on those signs that can lead us to a more specific etiological diagnosis. (author)

  15. Adenovirus or HA-2 fusogenic peptide-assisted lipofection increases cytoplasmic levels of plasmid in nondividing endothelium with little enhancement of transgene expression.

    Science.gov (United States)

    Subramanian, Ajit; Ma, Haiching; Dahl, Kris N; Zhu, Jingya; Diamond, Scott L

    2002-01-01

    Adenovirus-assisted lipofection has been reported to increase transfection efficiency through mechanisms potentially involving endosome escape and/or nuclear targeting activity. Similarly, transfection with the viral fusogenic peptide HA-2 of the influenza virus hemagglutinin can increase transfection efficiency. However, there are few studies examining the mechanism and intracellular trafficking of these viral and/or viral fusogenic peptide-assisted lipofections. Endosome escape was directly assayed with T7 RNA polymerase bound to plasmid (pTM beta gal) expressing beta-galactosidase under a T7 promoter to detect transcribable plasmid that escapes the endosomal compartment. Lipofection of pTM beta gal with replication-deficient adenovirus (Ad5-null) at a multiplicity of infection (MOI) of 100 and 1000 increased cytoplasmic levels of transcribable plasmid by 24- and 117-fold, respectively, over lipofection alone, without an effect on total plasmid uptake. However, lipofection of pCMV beta gal with Ad5-null at a MOI of 100 and 1000 increased transgene expression only seven- and eight-fold, respectively, over lipofection alone. Thus, a 24-fold increase in endosome escape saturated expression from pCMV beta gal and provided only a seven-fold benefit in nondividing cells, which was not significantly increased with further increases in endosome escape. A cationic form of HA-2 (HA-K(4)) also caused significant enhancements in endosome escape, as detected with the cytoplasmic transcription assay. However, HA-K(4) enhancement of endosome escape did not correlate with transgene expression from pCMV beta gal, consistent with the detection of HA-K(4)-mediated partitioning of plasmid to the insoluble fraction of the cell lysate. These results indicate that enhancement of endosome escape in nondividing cells does not fully alleviate rate limits related to nuclear import of the plasmid. Copyright 2001 John Wiley & Sons, Ltd.

  16. Microtubule–microtubule sliding by kinesin-1 is essential for normal cytoplasmic streaming in Drosophila oocytes

    Science.gov (United States)

    Lu, Wen; Winding, Michael; Lakonishok, Margot; Wildonger, Jill

    2016-01-01

    Cytoplasmic streaming in Drosophila oocytes is a microtubule-based bulk cytoplasmic movement. Streaming efficiently circulates and localizes mRNAs and proteins deposited by the nurse cells across the oocyte. This movement is driven by kinesin-1, a major microtubule motor. Recently, we have shown that kinesin-1 heavy chain (KHC) can transport one microtubule on another microtubule, thus driving microtubule–microtubule sliding in multiple cell types. To study the role of microtubule sliding in oocyte cytoplasmic streaming, we used a Khc mutant that is deficient in microtubule sliding but able to transport a majority of cargoes. We demonstrated that streaming is reduced by genomic replacement of wild-type Khc with this sliding-deficient mutant. Streaming can be fully rescued by wild-type KHC and partially rescued by a chimeric motor that cannot move organelles but is active in microtubule sliding. Consistent with these data, we identified two populations of microtubules in fast-streaming oocytes: a network of stable microtubules anchored to the actin cortex and free cytoplasmic microtubules that moved in the ooplasm. We further demonstrated that the reduced streaming in sliding-deficient oocytes resulted in posterior determination defects. Together, we propose that kinesin-1 slides free cytoplasmic microtubules against cortically immobilized microtubules, generating forces that contribute to cytoplasmic streaming and are essential for the refinement of posterior determinants. PMID:27512034

  17. The Cellular Distribution of RanGAP1 Is Regulated by CRM1-Mediated Nuclear Export in Mammalian Cells.

    Directory of Open Access Journals (Sweden)

    Keith Cha

    Full Text Available The Ran GTPase activating protein RanGAP1 plays an essential role in nuclear transport by stimulating RanGTP hydrolysis in the cytoplasmic compartment. In mammalian cells, unmodified RanGAP1 is predominantly cytoplasmic, whereas modification by small ubiquitin-related modifier protein (SUMO targets RanGAP1 to the cytoplasmic filaments of nuclear pore complex (NPC. Although RanGAP1 contains nine putative nuclear export signals and a nuclear localization signal, little is known if RanGAP1 shuttles between the nuclear and cytoplasmic compartments and how its primary localization in the cytoplasm and at the NPC is regulated. Here we show that inhibition of CRM1-mediated nuclear export using RNAi-knockdown of CRM1 and inactivation of CRM1 by leptomycin B (LMB results in nuclear accumulation of RanGAP1. LMB treatment induced a more robust redistribution of RanGAP1 from the cytoplasm to the nucleoplasm compared to CRM1 RNAi and also uniquely triggered a decrease or loss of RanGAP1 localization at the NPC, suggesting that LMB treatment is more effective in inhibiting CRM1-mediated nuclear export of RanGAP1. Our time-course analysis of LMB treatment reveals that the NPC-associated RanGAP1 is much more slowly redistributed to the nucleoplasm than the cytoplasmic RanGAP1. Furthermore, LMB-induced nuclear accumulation of RanGAP1 is positively correlated with an increase in levels of SUMO-modified RanGAP1, suggesting that SUMOylation of RanGAP1 may mainly take place in the nucleoplasm. Lastly, we demonstrate that the nuclear localization signal at the C-terminus of RanGAP1 is required for its nuclear accumulation in cells treated with LMB. Taken together, our results elucidate that RanGAP1 is actively transported between the nuclear and cytoplasmic compartments, and that the cytoplasmic and NPC localization of RanGAP1 is dependent on CRM1-mediated nuclear export.

  18. Compartmented pyruvate in perfused working heart

    International Nuclear Information System (INIS)

    Buenger, R.

    1985-01-01

    Pyruvate compartmentation and lactate dehydrogenase (LDH) were studied in isolated perfused working guinea pig hearts. The mean intracellular pyruvate (Pyr) contents increased with perfusate Pyr (0-2 mM) but varied only slightly with glucose (0-10 mM) and additional insulin (0.04-5 U/l), respectively. With 5-10 mM glucose plus 5 U/l insulin, but not with Pyr or lactate (Lac) as substrates, a near equilibrium between the LDH and the glycerol-3-phosphate dehydrogenase seemed to exist. Evidence for an inhibitory effect of Pyr on the activity of the LDH system of the perfused hearts was not obtained. With [U- 14 C]glucose as sole substrate, the specific activity of coronary venous Lac was near half that of precursor glucose. 14 CO 2 production was thus in quantitative agreement with rates of pyruvate oxidation that were determined as glucose uptake minus (Pyr + Lac) release. In contrast, with 0.2 mM [1- 14 C]Pyr plus 5 mM glucose, the ratio of 14 CO 2 production to specific activity of Lac overestimated Pyr oxidation judged from myocardial substrate balances and O 2 uptake, respectively; here, at least three pools of [ 14 C]HCO-3 and [ 14 C]lac, respectively, were kinetically demonstrable during washout of trace amounts of 14 C-labeled Pyr. Evidently, the specific activity of Lac was equivalent to that of mitochondrial oxidized Pyr provided [ 14 C]glucose was the sole or major precursor of cellular pyruvate. However, exogenously applied [1- 14 C]Pyr of high specific activity seemed to induce intracellular formation of both a highly and lowly labeled Pyr; the latter Pyr compartment did not seem in ready equilibrium with the cell physiologically prevailing highly labeled Pyr pool

  19. Compartment analysis of 125I-labelled albumin washout from coronary vessels of isolated perfused hearts

    International Nuclear Information System (INIS)

    Cheng Eap Ng; Seh-Hoon Song

    1978-01-01

    Albumin labelled with 125 I was used as a tracer to investigate the washout kinetics of plasma from the coronary circulation of isolated perfused feline hearts. Compartmentalization with experimental results showed at least two compartments. The model was compared with a three-compartment model found previously for red blood cells. The results indicate that there is a separation of plasma and RBC in the coronary microcirculation. (author)

  20. Promising SINEs for embargoing nuclear-cytoplasmic export as an anticancer strategy.

    Science.gov (United States)

    Tan, David S P; Bedard, Philippe L; Kuruvilla, John; Siu, Lillian L; Razak, Albiruni R Abdul

    2014-05-01

    In cancer cells, the nuclear-cytoplasmic transport machinery is frequently disrupted, resulting in mislocalization and loss of function for many key regulatory proteins. In this review, the mechanisms by which tumor cells co-opt the nuclear transport machinery to facilitate carcinogenesis, cell survival, drug resistance, and tumor progression will be elucidated, with a particular focus on the role of the nuclear-cytoplasmic export protein. The recent development of a new generation of selective inhibitors of nuclear export (XPO1 antagonists) and how these novel anticancer drugs may bring us closer to the implementation of this therapeutic strategy in the clinic will be discussed.

  1. Post-dialysis urea concentration: comparison between one- compartment model and two-compartment model

    International Nuclear Information System (INIS)

    Tamrin, N S Ahmad; Ibrahim, N

    2014-01-01

    The reduction of the urea concentration in blood can be numerically projected by using one-compartment model and two-compartment model with no variation in body fluid. This study aims to compare the simulated values of post-dialysis urea concentration for both models with the clinical data obtained from the hospital. The clinical assessment of adequacy of a treatment is based on the value of Kt/V. Further, direct calculation using clinical data and one-compartment model are presented in the form of ratio. It is found that the ratios of postdialysis urea concentration simulated using two-compartment model are higher compared to the ratios of post-dialysis urea concentration using one-compartment model. In addition, most values of post-dialysis urea concentration simulated using two-compartment model are much closer to the clinical data compared to values simulated using one-compartment model. Kt/V values calculated directly using clinical data are found to be higher than Kt/V values derived from one-compartment model

  2. Intracellular compartimentation of abscisic acid (ABA) in guard cells and mesophyll cells under exposure to SO sub 2. Kompartimentierung von Abscisinsaeure (ABA) in Schliess- und Mesophyllzellen unter SO sub 2 -Belastung

    Energy Technology Data Exchange (ETDEWEB)

    Baier, M.; Daeter, W.; Hartung, W. (Wuerzburg Univ. (Germany, F.R.). Lehrstuhl fuer Botanik 1)

    1989-07-01

    The effect of SO{sub 2} on the intracellular compartimentation of ABA in guard cells and mesophyll cells of Valerianella locusta was investigated, using the efflux compartmental analysis, as described by Behl and Hartung (1986). The cytoplasmic ABA content of the guard cells was reduced drastically by 6 {mu}molxm{sup -3} SO{sub 2} (20% of the controls). The vacuolar content was decreased less dramatically (70% of the controls). The ABA distribution of mesophyll cells remained uneffected by 6 {mu}molxm{sup -3} SO{sub 2}. The SO{sub 2} effects are explained by an acidification of the compartments. (orig.).

  3. Effects of combustible stacking in large compartments

    DEFF Research Database (Denmark)

    Gentili, Filippo; Giuliani, Luisa; Bontempi, Franco

    2013-01-01

    This paper focuses on the modelling of fire in case of various distributions of combustible materials in a large compartment. Large compartments often represent a challenge for structural fire safety, because of lack of prescriptive rules to follow and difficulties of taking into account the effect...... of non uniform distribution of the combustible materials and fire propagation. These aspects are discussed in this paper with reference to an industrial steel building, taken as case study. Fires triggered by the burning of wooden pallets stored in the premises have been investigated with respect...

  4. Activation of 5-[125I]iodonaphthyl-1-azide via excitation of fluorescent (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)) lipid analogs in living cells. A potential tool for identification of compartment-specific proteins and proteins involved in intracellular transport and metabolism of lipids

    International Nuclear Information System (INIS)

    Rosenwald, A.G.; Pagano, R.E.; Raviv, Y.

    1991-01-01

    We describe a new technique for analysis of proteins located near fluorescent lipid analogs in intact living cells using the membrane-permeant, photoactivatable probe, 5-[ 125 I]iodonaphthyl-1-azide ([ 125 I]INA). [ 125 I] INA can be activated directly with UV light or indirectly through excitation of adjacent fluorophores (photosensitizers) with visible light to modify nearby proteins covalently with 125 I. In this report we demonstrate that fluorescent phospholipids and sphingolipids containing N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-6-aminocaproic acid serve as appropriate photosensitizers for [ 125 I]INA. Using Chinese hamster ovary fibroblasts, we optimized the labeling conditions with respect to lipid concentration and time of irradiation and then examined the profiles of cellular proteins that were labeled when fluorescent analogs of ceramide, sphingomyelin, and phosphatidic acid were used as photosensitizers in living cells. The use of different fluorescent lipids, which label different subcellular compartments of cells as determined by fluorescence microscopy, derivatized different sets of cellular proteins with 125 I. The labeled proteins were subsets of the total set of proteins available for derivatization as determined by direct activation of [ 125 I]INA. Most proteins labeled by this procedure were pelleted by centrifugation of cell lysates at high speed (260,000 x g), but several soluble proteins were also labeled under these conditions. The implications of using this technique for identification of compartment-specific proteins and proteins involved in lipid metabolism and transport are discussed

  5. CONTINUOUS MEASUREMENT OF THE CYTOPLASMIC PH IN LACTOCOCCUS-LACTIS WITH A FLUORESCENT PH INDICATOR

    NARCIS (Netherlands)

    MOLENAAR, D; ABEE, T; KONINGS, WN

    1991-01-01

    The cytoplasmic pH of Lactococcus lactis was studied with the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5 (and-6)-carboxyfluorescein (BCECF). A novel method was applied for loading bacterial cells with BCECF, which consists of briefly treating a dense cell suspension with acid in the

  6. Simultaneous determination of reactive oxygen and nitrogen species in mitochondrial compartments of apoptotic HepG2 cells and PC12 cells based on microchip electrophoresis-laser-induced fluorescence.

    Science.gov (United States)

    Chen, Zhenzhen; Li, Qingling; Sun, Qianqian; Chen, Hao; Wang, Xu; Li, Na; Yin, Miao; Xie, Yanxia; Li, Hongmin; Tang, Bo

    2012-06-05

    Determination of intracellular bioactive species will afford beneficial information related to cell metabolism, signal transduction, cell function, and disease treatment. In this study, the first application of a microchip electrophoresis-laser-induced fluorescence (MCE-LIF) method for concurrent determination of reactive oxygen species (ROS) and reactive nitrogen species (RNS), i.e., superoxide (O(2)(-•)) and nitric oxide (NO) in mitochondria, was developed using fluorescent probes 2-chloro-1,3-dibenzothiazolinecyclohexene (DBZTC) and 3-amino,4-aminomethyl-2',7'-difluorescein (DAF-FM), respectively. Potential interference of intracellular dehydroascorbic acid (DHA) and ascorbic acid (AA) for NO detection with DAF-FM was eliminated through oxidation of AA with the addition of ascorbate oxidase, followed by subsequent MCE separation. Fluorescent products of O(2)(-•) and NO, DBZTC oxide (DBO), and DAF-FM triazole (DAF-FMT) showed excellent baseline separation within 1 min with a running buffer of 40 mM Tris solution (pH 7.4) and a separating electric field of 500 V/cm. The levels of DBO and DAF-FMT in mitochondria isolated from normal HepG2 cells and PC12 cells were evaluated using this method. Furthermore, the changes of DBO and DAF-FMT levels in mitochondria isolated from apoptotic HepG2 cells and PC12 cells could also be detected. The current approach was proved to be simple, fast, reproducible, and efficient. Measurement of the two species with the method will be beneficial to understand ROS/RNS distinctive functions. In addition, it will provide new insights into the role that both species play in biological systems.

  7. Transport and phosphorylation of choline in higher plant cells. Phosphorus-31 nuclear magnetic resonance studies

    Energy Technology Data Exchange (ETDEWEB)

    Bligny, R.; Foray, M.F.; Roby, C.; Douce, R.

    1989-03-25

    When sycamore cells were suspended in basal medium containing choline, the latter was taken up by the cells very rapidly. A facilitated diffusion system appertained at low concentrations of choline and exhibited Michaelis-Menten kinetics. At higher choline concentrations simple diffusion appeared to be the principal mode of uptake. Addition of choline to the perfusate of compressed sycamore cells monitored by /sup 31/P NMR spectroscopy resulted in a dramatic accumulation of P-choline in the cytoplasmic compartment containing choline kinase and not in the vacuole. The total accumulation of P-choline over a 10-h period exhibited Michaelis-Menten kinetics. During this period, in the absence of Pi in the perfusion medium there was a marked depletion of glucose-6-P, and the cytoplasmic Pi resonance disappeared almost completely. When a threshold of cytoplasmic Pi was attained, the phosphorylation of choline was sustained by the continuous release of Pi from the vacuole although at a much lower rate. However, when 100 microM inorganic phosphate was present in the perfusion medium, externally added Pi was preferentially used to sustain P-choline synthesis. It is clear, therefore, that cytosolic choline kinase associated with a carrier-mediated transport system for choline uptake appeared as effective systems for continuously trapping cytoplasmic Pi including vacuolar Pi entering the cytoplasm.

  8. Bilateral post-traumatic gluteal compartment syndrome: A case report and review of literature

    Directory of Open Access Journals (Sweden)

    Devashis Barick

    2015-01-01

    Full Text Available Gluteal compartment is a rare site for compartment syndrome. Gluteal compartment syndrome has most commonly been described in the literature as occurring after prolonged immobility associated with substance abuse, improper operative positioning, sickle cell-induced infarct, post-traumatic and spontaneous superior gluteal artery rupture, exercise, and post-arterial embolization of the internal iliac artery prior to abdominal aortic aneurysm repair. Trauma is rarely associated with this syndrome. Gluteal compartment syndrome occurs in approximately 0.9% of trauma patients. Posttraumatic gluteal compartment syndrome develops because of edema with traumatic contusions, crush injuries and hematoma formation due to blunt superior or inferior gluteal artery injuries in all compartments of the gluteal region Only 6 previous cases have been reported in the literature. Two previous cases involved positioning for urological procedures, while the other cited causes of bilateral gluteal compartment syndrome include exercise-induced, trauma, and prolonged immobilization from substance abuse. One of the most immediately devastating results of a missed compartment syndrome is the risk of the development of rhabdomyolysis with the resulting squeal of myoglobinuria, hyperkalemia, and acidosis resulting in renal failure, shock, multiple organ failure, disseminated intravascular coagulation, and possibly death. Here we report a case of posttraumatic bilateral compartment syndrome which developed secondary to pressure due to patient being trapped under a vehicle following a vehicular accident. He was operated upon and a bilateral fasciotomy was done. Although he did not develop any renal complications, the sciatic nerve palsy on the left side did not recover. The patient is still under follow up.

  9. The DEAD box helicase RDE-12 promotes amplification of RNAi in cytoplasmic foci in C. elegans.

    Science.gov (United States)

    Yang, Huan; Vallandingham, Jim; Shiu, Philip; Li, Hua; Hunter, Craig P; Mak, Ho Yi

    2014-04-14

    RNAi is a potent mechanism for downregulating gene expression. Conserved RNAi pathway components are found in animals, plants, fungi, and other eukaryotes. In C. elegans, the RNAi response is greatly amplified by the synthesis of abundant secondary small interfering RNAs (siRNAs). Exogenous double-stranded RNA is processed by Dicer and RDE-1/Argonaute into primary siRNA that guides target mRNA recognition. The RDE-10/RDE-11 complex and the RNA-dependent RNA polymerase RRF-1 then engage the target mRNA for secondary siRNA synthesis. However, the molecular link between primary siRNA production and secondary siRNA synthesis remains largely unknown. Furthermore, it is unclear whether the subcellular sites for target mRNA recognition and degradation coincide with sites where siRNA synthesis and amplification occur. In the C. elegans germline, cytoplasmic P granules at the nuclear pores and perinuclear Mutator foci contribute to target mRNA surveillance and siRNA amplification, respectively. We report that RDE-12, a conserved phenylalanine-glycine (FG) domain-containing DEAD box helicase, localizes in P granules and cytoplasmic foci that are enriched in RSD-6 but are excluded from the Mutator foci. Our results suggest that RDE-12 promotes secondary siRNA synthesis by orchestrating the recruitment of RDE-10 and RRF-1 to primary siRNA-targeted mRNA in distinct cytoplasmic compartments. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Endogenous Mouse Dicer Is an Exclusively Cytoplasmic Protein.

    Directory of Open Access Journals (Sweden)

    Christian Much

    2016-06-01

    Full Text Available Dicer is a large multi-domain protein responsible for the ultimate step of microRNA and short-interfering RNA biogenesis. In human and mouse cell lines, Dicer has been shown to be important in the nuclear clearance of dsRNA as well as the establishment of chromatin modifications. Here we set out to unambiguously define the cellular localization of Dicer in mice to understand if this is a conserved feature of mammalian Dicer in vivo. To this end, we utilized an endogenously epitope tagged Dicer knock-in mouse allele. From primary mouse cell lines and adult tissues, we determined with certainty by biochemical fractionation and confocal immunofluorescence microscopy that endogenous Dicer is exclusively cytoplasmic. We ruled out the possibility that a fraction of Dicer shuttles to and from the nucleus as well as that FGF or DNA damage signaling induce Dicer nuclear translocation. We also explored Dicer localization during the dynamic and developmental context of embryogenesis, where Dicer is ubiquitously expressed and strictly cytoplasmic in all three germ layers as well as extraembryonic tissues. Our data exclude a direct role for Dicer in the nuclear RNA processing in the mouse.

  11. Endogenous Mouse Dicer Is an Exclusively Cytoplasmic Protein.

    Science.gov (United States)

    Much, Christian; Auchynnikava, Tania; Pavlinic, Dinko; Buness, Andreas; Rappsilber, Juri; Benes, Vladimir; Allshire, Robin; O'Carroll, Dónal

    2016-06-01

    Dicer is a large multi-domain protein responsible for the ultimate step of microRNA and short-interfering RNA biogenesis. In human and mouse cell lines, Dicer has been shown to be important in the nuclear clearance of dsRNA as well as the establishment of chromatin modifications. Here we set out to unambiguously define the cellular localization of Dicer in mice to understand if this is a conserved feature of mammalian Dicer in vivo. To this end, we utilized an endogenously epitope tagged Dicer knock-in mouse allele. From primary mouse cell lines and adult tissues, we determined with certainty by biochemical fractionation and confocal immunofluorescence microscopy that endogenous Dicer is exclusively cytoplasmic. We ruled out the possibility that a fraction of Dicer shuttles to and from the nucleus as well as that FGF or DNA damage signaling induce Dicer nuclear translocation. We also explored Dicer localization during the dynamic and developmental context of embryogenesis, where Dicer is ubiquitously expressed and strictly cytoplasmic in all three germ layers as well as extraembryonic tissues. Our data exclude a direct role for Dicer in the nuclear RNA processing in the mouse.

  12. Production of ABA responses requires both the nuclear and cytoplasmic functional involvement of PYR1

    International Nuclear Information System (INIS)

    Park, EunJoo; Kim, Tae-Houn

    2017-01-01

    Abscisic acid (ABA) enhances stress tolerant responses in plants against unfavorable environmental conditions. In Arabidopsis, ABA promotes interactions between PYR/PYL/RCARs and PP2C, thereby allowing SnRK2s to phosphorylate downstream components required for the regulation of gene expression or for gating ion channels. Because PYR1 is known to localize to nucleus and cytoplasm it is a question whether nuclear or cytoplasmic PYR1 confer different functions to the ABA signaling pathway, as has been previously shown for regulatory proteins. In order to answer this question, transgenic lines expressing nuclear PYR1 were generated in an ABA insensitive mutant background. Enforced nuclear expression of PYR1 was examined by confocal microscopy and western blot analysis. Physiological analyses of the transgenic lines demonstrated that nuclear PYR1 is sufficient to generate ABA responses, such as, the inhibition of seed germination, root growth inhibition, the induction of gene expression, and stomatal closing movement. However, for the full recovery of ABA responses in the mutant background cytoplasmic PYR1 was required. The study suggests both nuclear and cytoplasmic PYR1 participate in the control of ABA signal transduction. - Highlights: • Nuclear and cytoplasmic functions of PYR1 were studied in the mutant which lacked majority of ABA responses. • Nuclear PYR1 reconstituted partially the ABA responses during seed germination, root growth, and guard cell movement. • Both the nuclear and cytoplasmic functions of PYR1 were required for the full generation of ABA responses.

  13. Raman microspectroscopy of nucleus and cytoplasm for human colon cancer diagnosis.

    Science.gov (United States)

    Liu, Wenjing; Wang, Hongbo; Du, Jingjing; Jing, Chuanyong

    2017-11-15

    Subcellular Raman analysis is a promising clinic tool for cancer diagnosis, but constrained by the difficulty of deciphering subcellular spectra in actual human tissues. We report a label-free subcellular Raman analysis for use in cancer diagnosis that integrates subcellular signature spectra by subtracting cytoplasm from nucleus spectra (Nuc.-Cyt.) with a partial least squares-discriminant analysis (PLS-DA) model. Raman mapping with the classical least-squares (CLS) model allowed direct visualization of the distribution of the cytoplasm and nucleus. The PLS-DA model was employed to evaluate the diagnostic performance of five types of spectral datasets, including non-selective, nucleus, cytoplasm, ratio of nucleus to cytoplasm (Nuc./Cyt.), and nucleus minus cytoplasm (Nuc.-Cyt.), resulting in diagnostic sensitivity of 88.3%, 84.0%, 98.4%, 84.5%, and 98.9%, respectively. Discriminating between normal and cancerous cells of actual human tissues through subcellular Raman markers is feasible, especially when using the nucleus-cytoplasm difference spectra. The subcellular Raman approach had good stability, and had excellent diagnostic performance for rectal as well as colon tissues. The insights gained from this study shed new light on the general applicability of subcellular Raman analysis in clinical trials. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Antineutrophil cytoplasmic autoantibody-associated small-vessel vasculitis

    NARCIS (Netherlands)

    Kallenberg, Cees G. M.

    Purpose of reviews This review focuses on recent advance in the diagnosis pathogenesis and treatment of antineutrophil cytoplasmic autoantibody-associated small-vessel vasculitis. Recent findings Antineutrophil cytoplasmic autoantibodies are closely associated with Wegener's granulomatosis and

  15. Bayesian Inference of Forces Causing Cytoplasmic Streaming in Caenorhabditis elegans Embryos and Mouse Oocytes.

    Science.gov (United States)

    Niwayama, Ritsuya; Nagao, Hiromichi; Kitajima, Tomoya S; Hufnagel, Lars; Shinohara, Kyosuke; Higuchi, Tomoyuki; Ishikawa, Takuji; Kimura, Akatsuki

    2016-01-01

    Cellular structures are hydrodynamically interconnected, such that force generation in one location can move distal structures. One example of this phenomenon is cytoplasmic streaming, whereby active forces at the cell cortex induce streaming of the entire cytoplasm. However, it is not known how the spatial distribution and magnitude of these forces move distant objects within the cell. To address this issue, we developed a computational method that used cytoplasm hydrodynamics to infer the spatial distribution of shear stress at the cell cortex induced by active force generators from experimentally obtained flow field of cytoplasmic streaming. By applying this method, we determined the shear-stress distribution that quantitatively reproduces in vivo flow fields in Caenorhabditis elegans embryos and mouse oocytes during meiosis II. Shear stress in mouse oocytes were predicted to localize to a narrower cortical region than that with a high cortical flow velocity and corresponded with the localization of the cortical actin cap. The predicted patterns of pressure gradient in both species were consistent with species-specific cytoplasmic streaming functions. The shear-stress distribution inferred by our method can contribute to the characterization of active force generation driving biological streaming.

  16. Systemic Administration of Carbon Monoxide-Releasing Molecule-3 Protects the Skeletal Muscle in Porcine Model of Compartment Syndrome.

    Science.gov (United States)

    Bihari, Aurelia; Cepinskas, Gediminas; Sanders, David; Lawendy, Abdel-Rahman

    2018-05-01

    Acute limb compartment syndrome, a complication of musculoskeletal trauma, results in muscle necrosis and cell death. Carbon monoxide, liberated from the carbon monoxide-releasing molecule-3, has been shown protective in a rat model of compartment syndrome. The purpose of this study was to test the effect of carbon monoxide-releasing molecule-3 in a preclinical large animal model of compartment syndrome, with the ultimate goal of developing a pharmacologic adjunct treatment for compartment syndrome. Animal research study. Basic research laboratory in a hospital setting. Male Yorkshire-Landrace pigs (50-60 kg). Pigs underwent 6 hours of intracompartmental pressure elevation by infusing fluid into the anterior compartment of the right hind limb. Carbon monoxide-releasing molecule-3 was administered systemically (2 mg/kg, IV) at fasciotomy, followed by 3-hour reperfusion. Muscle perfusion, inflammation, injury, and apoptosis were assessed in the skeletal muscle. Systemic leukocyte activation was assessed during compartment syndrome and reperfusion. Elevation of hind limb intracompartmental pressure resulted in significant microvascular perfusion deficits (44% ± 1% continuously perfused capillaries in compartment syndrome vs 76% ± 4% in sham; p molecule-3 at fasciotomy increased the number of continuously perfused capillaries (68% ± 3%; p molecule-3 at fasciotomy offered protection against compartment syndrome-induced microvascular perfusion deficit, tissue injury, and systemic leukocyte activation. The data suggest the potential therapeutic application of carbon monoxide-releasing molecule-3 to patients at risk of developing compartment syndrome.

  17. Development of a compartment model based on CFD simulations for description of mixing in bioreactors

    Directory of Open Access Journals (Sweden)

    Crine, M.

    2010-01-01

    Full Text Available Understanding and modeling the complex interactions between biological reaction and hydrodynamics are a key problem when dealing with bioprocesses. It is fundamental to be able to accurately predict the hydrodynamics behavior of bioreactors of different size and its interaction with the biological reaction. CFD can provide detailed modeling about hydrodynamics and mixing. However, it is computationally intensive, especially when reactions are taken into account. Another way to predict hydrodynamics is the use of "Compartment" or "Multi-zone" models which are much less demanding in computation time than CFD. However, compartments and fluxes between them are often defined by considering global quantities not representative of the flow. To overcome the limitations of these two methods, a solution is to combine compartment modeling and CFD simulations. Therefore, the aim of this study is to develop a methodology in order to propose a compartment model based on CFD simulations of a bioreactor. The flow rate between two compartments can be easily computed from the velocity fields obtained by CFD. The difficulty lies in the definition of the zones in such a way they can be considered as perfectly mixed. The creation of the model compartments from CFD cells can be achieved manually or automatically. The manual zoning consists in aggregating CFD cells according to the user's wish. The automatic zoning defines compartments as regions within which the value of one or several properties are uniform with respect to a given tolerance. Both manual and automatic zoning methods have been developed and compared by simulating the mixing of an inert scalar. For the automatic zoning, several algorithms and different flow properties have been tested as criteria for the compartment creation.

  18. Leg 201Tl-SPECT in chronic exertional compartment syndrome

    International Nuclear Information System (INIS)

    Elkadri, N.; Slim, I.; Blondet, C.; Choquet, Ph.; Constantinesco, A.; Lecocq, J.

    2004-01-01

    Leg 201 Tl-SPECT in chronic exertional compartment syndrome Background: The chronic exertional compartment syndrome is one of the most frequent origins regarding leg pain due to sport training. The diagnosis can be established by invasive compartment pressure measurement. The aim of this study is to evaluate the role that could have 201 Tl-SPECT for patients with suspicion of compartment syndrome. Patients and methods: 51 leg 201 Tl-SPECT exams were performed (exercise - and rest without reinjection) in 49 patients; 28 had compartment syndrome confirmed by pressure measurement. About 100 MBq of 201 Tl were injected during exercise, when pain appeared or at least after 25 minutes exercise. We studied mean percentages of level uptake for each compartment, referred to the maximal uptake of both legs. Results: 47 compartments were concerned by compartment syndrome and 361 compartments were not. Scintigraphic patterns in compartments are reversible ischaemia (45%), uptake stability (36%) or reverse redistribution (19%); these patterns are not linked to compartment syndrome. However, there is a significant difference of rest 201 Tl level uptake between compartments with and without compartment syndrome and a significant correlation between muscular pressure measurement and rest level uptake. Conclusion: 201 Tl-SPECT shows that only ischaemia does not explain compartment syndrome. Moreover, it allows to predict pressure variation during exercise but it does not offer any interest in order to select patients for muscular invasive pressure measurement. (author)

  19. Phase separation between nucleoid and cytoplasm in Escherichia coli as defined by immersive refractometry.

    Science.gov (United States)

    Valkenburg, J A; Woldringh, C L

    1984-01-01

    The refractive indices of nucleoid and cytoplasm in Escherichia coli were derived theoretically and experimentally. For the theoretical estimates, we made use of the known macromolecular composition of E. coli B/r (G. Churchward and H. Bremer, J. Theor. Biol. 94:651-670, 1982) and of estimates of cell and nucleoid volumes. These were obtained from micrographs of living bacteria made with a confocal scanning light microscope. The theoretical values were calculated, assuming that all DNA occurred in the nucleoid and that all protein and RNA occurred in the cytoplasm. Comparison with experimental refractive index values directly obtained by immersive refractometry showed that, besides its DNA, the nucleoid must contain an additional amount of solids equivalent to 8.6% (wt/vol) protein. With the nucleoid containing 6.8% (wt/vol) DNA and 8.6% (wt/vol) protein and the cytoplasm containing 21% (wt/vol) protein and 4% (wt/vol) RNA, a mass difference is obtained, which accounts for the phase separation observed between the nucleoid and cytoplasm in living cells by phase-contrast microscopy. The decrease in the refractive index of the nucleoid relative to that of the cytoplasm observed upon, for instance, OsO4 fixation was interpreted as being indicative of the loss of protein content in the nucleoid. Images PMID:6389508

  20. Elucidating the role of select cytoplasmic proteins in altering diffusion of integrin receptors.

    Science.gov (United States)

    Sander, Suzanne; Arora, Neha; Smith, Emily A

    2012-06-01

    Cytoplasmic proteins that affect integrin diffusion in the cell membrane are identified using a combination of fluorescence recovery after photobleaching (FRAP) and RNA interference. Integrin receptors are essential for many cellular events, and alterations in lateral diffusion are one mechanism for modulating their function. In cells expressing native cytoplasmic protein concentrations and spread on a slide containing integrin extracellular ligand, 45 ± 2% of the integrin is mobile with a time-dependent 5.2 ± 0.9 × 10(-9) cm(2)/s diffusion coefficient at 1 s. The time exponent is 0.90 ± 0.07, indicating integrin diffusion moderately slows at longer times. The role of a specific cytoplasmic protein in altering integrin diffusion is revealed through changes in the FRAP curve after reducing the cytoplasmic protein's expression. Decreased expression of cytoplasmic proteins rhea, focal adhesion kinase (FAK), or steamer duck decreases the integrin mobile fraction. For rhea and FAK, there is a concomitant shift to Brownian (i.e., time-independent) diffusion at reduced concentrations of these proteins. In contrast, when the expression of actin 42A, dreadlocks, paxillin, integrin-linked kinase (ILK), or vinculin is reduced, integrin diffusion generally becomes more constrained with an increase in the integrin mobile fraction. This same change in integrin diffusion is measured in the absence of integrin extracellular ligand. The results indicate breaking the extracellular ligand-integrin-cytoskeletal linkage alters integrin diffusion properties, and, in most cases, there is no correlation between integrin and lipid diffusion properties.

  1. Labelling of human resting lymphocytes by continuous infusion of (/sup 3/H)thymidine. 1. Characterization of cytoplasmic label

    Energy Technology Data Exchange (ETDEWEB)

    Schick, P; Trepel, F; Maisel, K H; Past, W; Reisert, I; Begemann, H; Pilgrim, C [Ulm Univ. (Germany, F.R.)

    1978-01-01

    After continuous /sup 3/H-TdR infusion in vivo or incubation with /sup 3/H-TdR in vitro human blood lymphocytes were examined by light-microscopic and electron-microscopic autoradiography. Using relatively long autoradiographic exposure times (50-300 days) not only nuclear but also cytoplasmic labelling was visualized, the cytoplasmic label being present in up to 96% of the cells. The cytoplasmic label was predominantly associated with the mitochondria and was removed from the cells nearly completely by treatment with DNase but not with RNase or cold perchloric acid. It is concluded that this cytoplasmic label mainly represents /sup 3/H-TdR incorporated into mitochondrial DNA which is continuously renewed in an average turnover time of 14 days or less. This value is compatible with a turnover time of 11 days for mitochondrial DNA in mammalian cells reported in the literature.

  2. Multi-compartment Aerosol Transport Model

    Energy Technology Data Exchange (ETDEWEB)

    Hubbard, Joshua Allen; Santarpia, Joshua; Brotherton, Christopher M.; Omana, Michael Alexis; Rivera, Danielle; Lucero, Gabriel Anthony

    2017-06-01

    A simple aerosol transport model was developed for a multi-compartmented cleanroom. Each compartment was treated as a well-mixed volume with ventilating supply and return air. Gravitational settling, intercompartment transport, and leakage of exterior air into the system were included in the model. A set of first order, coupled, ordinary differential equations was derived from the conservation equations of aerosol mass and air mass. The system of ODEs was then solved in MATLAB using pre-existing numerical methods. The model was verified against cases of (1) constant inlet-duct concentration, and (2) exponentially decaying inlet-duct concentration. Numerical methods resulted in normalized error of less than 10 -9 when model solutions were compared to analytical solutions. The model was validated against experimental measurements from a single field test and showed good agreement in the shape and magnitude of the aerosol concentration profile with time.

  3. A modern definition of mediastinal compartments.

    Science.gov (United States)

    Carter, Brett W; Tomiyama, Noriyuki; Bhora, Faiz Y; Rosado de Christenson, Melissa L; Nakajima, Jun; Boiselle, Phillip M; Detterbeck, Frank C; Marom, Edith M

    2014-09-01

    Division of the mediastinum into compartments is used to help narrow the differential diagnosis of newly detected mediastinal masses, to assist in planning biopsy and surgical procedures, and to facilitate communication among clinicians of multiple disciplines. Several traditional mediastinal division schemes exist based upon arbitrary landmarks on the lateral chest radiograph. We describe a modern, computed tomography-based mediastinal division scheme, which has been accepted by the International Thymic Malignancy Interest Group as a new standard. This clinical classification defines a prevascular (anterior), a visceral (middle), and a paravertebral (posterior) compartment, with anatomic boundaries defined clearly by computed tomography. It is our intention that this definition be used in the reporting of clinical cases and the design of prospective clinical trials.

  4. Hermetic compartments leak-tightness enhancement

    International Nuclear Information System (INIS)

    Murani, J.

    2000-01-01

    In connection with the enhancement of the nuclear safety of the Jaslovske Bohunice V-1 NPP actions for the increase of the leak tightness are performed. The reconstruction has been done in the following directions: hermetic compartments leak tightness enhancement; air lock installation; installation of air lock in SP 4 vent system; integrated leakage rate test to hermetic compartments with leak detection. After 'major' leaks on the hermetic boundary components had been eliminated, since 1994 works on a higher qualitative level began. The essence of the works consists in the detection and identification of leaks in the structural component of the hermetic boundary during the planned refueling outages. The results of the Small Reconstruction and gradual enhancement of leak tightness are presented

  5. New quantitative approaches reveal the spatial preference of nuclear compartments in mammalian fibroblasts.

    Science.gov (United States)

    Weston, David J; Russell, Richard A; Batty, Elizabeth; Jensen, Kirsten; Stephens, David A; Adams, Niall M; Freemont, Paul S

    2015-03-06

    The nuclei of higher eukaryotic cells display compartmentalization and certain nuclear compartments have been shown to follow a degree of spatial organization. To date, the study of nuclear organization has often involved simple quantitative procedures that struggle with both the irregularity of the nuclear boundary and the problem of handling replicate images. Such studies typically focus on inter-object distance, rather than spatial location within the nucleus. The concern of this paper is the spatial preference of nuclear compartments, for which we have developed statistical tools to quantitatively study and explore nuclear organization. These tools combine replicate images to generate 'aggregate maps' which represent the spatial preferences of nuclear compartments. We present two examples of different compartments in mammalian fibroblasts (WI-38 and MRC-5) that demonstrate new knowledge of spatial preference within the cell nucleus. Specifically, the spatial preference of RNA polymerase II is preserved across normal and immortalized cells, whereas PML nuclear bodies exhibit a change in spatial preference from avoiding the centre in normal cells to exhibiting a preference for the centre in immortalized cells. In addition, we show that SC35 splicing speckles are excluded from the nuclear boundary and localize throughout the nucleoplasm and in the interchromatin space in non-transformed WI-38 cells. This new methodology is thus able to reveal the effect of large-scale perturbation on spatial architecture and preferences that would not be obvious from single cell imaging.

  6. Monoterpene biosynthesis potential of plant subcellular compartments.

    Science.gov (United States)

    Dong, Lemeng; Jongedijk, Esmer; Bouwmeester, Harro; Van Der Krol, Alexander

    2016-01-01

    Subcellular monoterpene biosynthesis capacity based on local geranyl diphosphate (GDP) availability or locally boosted GDP production was determined for plastids, cytosol and mitochondria. A geraniol synthase (GES) was targeted to plastids, cytosol, or mitochondria. Transient expression in Nicotiana benthamiana indicated local GDP availability for each compartment but resulted in different product levels. A GDP synthase from Picea abies (PaGDPS1) was shown to boost GDP production. PaGDPS1 was also targeted to plastids, cytosol or mitochondria and PaGDPS1 and GES were coexpressed in all possible combinations. Geraniol and geraniol-derived products were analyzed by GC-MS and LC-MS, respectively. GES product levels were highest for plastid-targeted GES, followed by mitochondrial- and then cytosolic-targeted GES. For each compartment local boosting of GDP biosynthesis increased GES product levels. GDP exchange between compartments is not equal: while no GDP is exchanged from the cytosol to the plastids, 100% of GDP in mitochondria can be exchanged to plastids, while only 7% of GDP from plastids is available for mitochondria. This suggests a direct exchange mechanism for GDP between plastids and mitochondria. Cytosolic PaGDPS1 competes with plastidial GES activity, suggesting an effective drain of isopentenyl diphosphate from the plastids to the cytosol. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  7. Perfluoroalkyl acid distribution in various plant compartments ...

    Science.gov (United States)

    Crop uptake of perfluoroalkyl acids (PFAAs) from biosolids-amended soil has been identified as a potential pathway for PFAA entry into the terrestrial food chain. This study compared the uptake of PFAAs in greenhouse-grown radish (Raphanus sativus), celery (Apium graveolens var.dulce), tomato (Lycopersicon lycopersicum), and sugar snap pea (Pisum sativum var. macrocarpon) from an industrially impacted biosolids-amended soil, a municipal biosolids­ amended soil, and a control soil. Individual concentrations of PFAAs, on a dry weight basis, in mature, edible portions of crops grown in soil amended with PFAA industrially impacted biosolids were highest for perfluorooctanoate (PFOA; 67 ng/g) in radish root, perfluorobutanoate (PFBA;232 ng/g) in celery shoot, and PFBA (150 ng/g) in pea fruit. Comparatively, PFAA concentrations in edible compartments of crops grown in the municipal biosolids-amended soil and in the control soil were less than 25 ng/g. Bioaccumulation factors (BAFs) were calculated for the root, shoot, and fruit compartments (as applicable) of all crops grown in the industrially impacted soil. BAFs were highest for PFBA in the shoots of all crops, as well as in the fruit compartment of pea. Root­ soil concentration factors (RCFs) for tomato and pea were independent of PFAA chain length, while radish and celery RCFs showed a slight decrease with increasing chain length. Shoot-soil concentration factors (SCFs) for all crops showed a decrease with incre

  8. Salus: Kernel Support for Secure Process Compartments

    Directory of Open Access Journals (Sweden)

    Raoul Strackx

    2015-01-01

    Full Text Available Consumer devices are increasingly being used to perform security and privacy critical tasks. The software used to perform these tasks is often vulnerable to attacks, due to bugs in the application itself or in included software libraries. Recent work proposes the isolation of security-sensitive parts of applications into protected modules, each of which can be accessed only through a predefined public interface. But most parts of an application can be considered security-sensitive at some level, and an attacker who is able to gain inapplication level access may be able to abuse services from protected modules. We propose Salus, a Linux kernel modification that provides a novel approach for partitioning processes into isolated compartments sharing the same address space. Salus significantly reduces the impact of insecure interfaces and vulnerable compartments by enabling compartments (1 to restrict the system calls they are allowed to perform, (2 to authenticate their callers and callees and (3 to enforce that they can only be accessed via unforgeable references. We describe the design of Salus, report on a prototype implementation and evaluate it in terms of security and performance. We show that Salus provides a significant security improvement with a low performance overhead, without relying on any non-standard hardware support.

  9. Evaluation of acute compartment syndrome of extremities in ...

    African Journals Online (AJOL)

    Arun Kumar Agnihotri

    compartment syndrome in children; Acute compartment syndrome and fasciotomy. INTRODUCTIONᴪ .... these patients were manipulated under general anaesthesia ... of these children. The clinical diagnosis of increased ICP is not easy.

  10. Compartment syndrome can also be seen in the forearm

    DEFF Research Database (Denmark)

    Asmar, Ali; Broholm, Rikke; Bülow, Jens

    2014-01-01

    Chronic compartment syndrome is a challenge for the clinician and symptomatic similar to neuropathies, tenosynovitis, stress fractures and referred pain from lumbar cervicalis. Thus, chronic compartment syndrome of the upper extremities is probably an underdiagnosed condition. In patients...

  11. 14 CFR 23.853 - Passenger and crew compartment interiors.

    Science.gov (United States)

    2010-01-01

    ... Photographic Film PH1.25 (available from the American National Standards Institute, 1430 Broadway, New York, N... stowage compartments and compartments for stowing small items such as magazines and maps) must be self...

  12. Pleckstrin Homology Domain Diffusion in Dictyostelium Cytoplasm Studied Using Fluorescence Correlation Spectroscopy

    NARCIS (Netherlands)

    Engel, Ruchira; Hink, Mark A.; Bosgraaf, Leonard; Haastert, Peter J.M. van; Visser, Antonie J.W.G.

    2004-01-01

    The translocation of pleckstrin homology (PH) domain-containing proteins from the cytoplasm to the plasma membrane plays an important role in the chemotaxis mechanism of Dictyostelium cells. The diffusion of three PH domain-green fluorescent protein (GFP) fusions (PH2-GFP, PH10-GFP, and PH-CRAC

  13. Pleckstrin homology domain diffusion in Dictyostelium cytoplasm studied using fluorescence correlation spectroscopy

    NARCIS (Netherlands)

    Ruchira, A.; Hink, M.A.; Bosgraaf, L.; Haastert, van P.J.M.; Visser, A.J.W.G.

    2004-01-01

    The translocation of pleckstrin homology (PH) domain-containing proteins from the cytoplasm to the plasma membrane plays an important role in the chemotaxis mechanism of Dictyostelium cells. The diffusion of three PH domain-green fluorescent protein (GFP) fusions (PH2-GFP, PH10-GFP, and PH-CRAC

  14. Electron tomography of fusiform vesicles and their organization in urothelial cells.

    Directory of Open Access Journals (Sweden)

    Samo Hudoklin

    Full Text Available The formation of fusiform vesicles (FVs is one of the most distinctive features in the urothelium of the urinary bladder. FVs represent compartments for intracellular transport of urothelial plaques, which modulate the surface area of the superficial urothelial (umbrella cells during the distension-contraction cycle. We have analysed the three-dimensional (3D structure of FVs and their organization in umbrella cells of mouse urinary bladders. Compared to chemical fixation, high pressure freezing gave a new insight into the ultrastructure of urothelial cells. Electron tomography on serial sections revealed that mature FVs had a shape of flattened discs, with a diameter of up to 1.2 µm. The lumen between the two opposing asymmetrically thickened membranes was very narrow, ranging from 5 nm to 10 nm. Freeze-fracturing and immunolabelling confirmed that FVs contain two opposing urothelial plaques connected by a hinge region that made an omega shaped curvature. In the central cytoplasm, 4-15 FVs were often organized into stacks. In the subapical cytoplasm, FVs were mainly organized as individual vesicles. Distension-contraction cycles did not affect the shape of mature FVs; however, their orientation changed from parallel in distended to perpendicular in contracted bladder with respect to the apical plasma membrane. In the intermediate cells, shorter and more dilated immature FVs were present. The salient outcome from this research is the first comprehensive, high resolution 3D view of the ultrastructure of FVs and how they are organized differently depending on their location in the cytoplasm of umbrella cells. The shape of mature FVs and their organization into tightly packed stacks makes them a perfect storage compartment, which transports large amounts of urothelial plaques while occupying a small volume of umbrella cell cytoplasm.

  15. 46 CFR 169.627 - Compartments containing diesel fuel tanks.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Compartments containing diesel fuel tanks. 169.627... SCHOOL VESSELS Machinery and Electrical Ventilation § 169.627 Compartments containing diesel fuel tanks. Unless they are adequately ventilated, enclosed compartments or spaces containing diesel fuel tanks and...

  16. Cellular endocytic compartment localization of expressed canine CD1 molecules

    DEFF Research Database (Denmark)

    Schjærff, Mette; Keller, Stefan M.; Affolter, Verena K.

    2016-01-01

    CD1 molecules are glycoproteins present primarily on dendritic cells (DCs), which recognize and presenta variety of foreign- and self-lipid antigens to T-cells. Humans have five different CD1 isoforms that sur-vey distinct cellular compartments allowing for recognition of a large repertoire...... onlya diminished GFP expression. In conclusion, canine CD1 transfectants show distinct localization patternsthat are similar to human CD1 proteins with the exception of the canine CD1d isoform, which most likelyis non-functional. These findings imply that canine CD1 localization overall resembles human...... CD1 traf-ficking patterns. This knowledge is important for the understanding of lipid antigen-receptor immunityin the dog....

  17. Parvovirus particles and movement in the cellular cytoplasm and effects of the cytoskeleton

    International Nuclear Information System (INIS)

    Lyi, Sangbom Michael; Tan, Min Jie Alvin; Parrish, Colin R.

    2014-01-01

    Cell infection by parvoviruses requires that capsids be delivered from outside the cell to the cytoplasm, followed by genome trafficking to the nucleus. Here we microinject capsids into cells that lack receptors and followed their movements within the cell over time. In general the capsids remained close to the positions where they were injected, and most particles did not move to the vicinity of or enter the nucleus. When 70 kDa-dextran was injected along with the capsids that did not enter the nucleus in significant amounts. Capsids conjugated to peptides containing the SV40 large T-antigen nuclear localization signal remained in the cytoplasm, although bovine serum albumen conjugated to the same peptide entered the nucleus rapidly. No effects of disruption of microfilaments, intermediate filaments, or microtubules on the distribution of the capsids were observed. These results suggest that movement of intact capsids within cells is primarily associated with passive processes

  18. Parvovirus particles and movement in the cellular cytoplasm and effects of the cytoskeleton

    Energy Technology Data Exchange (ETDEWEB)

    Lyi, Sangbom Michael; Tan, Min Jie Alvin, E-mail: tanmja@gis.a-star.edu.sg; Parrish, Colin R., E-mail: crp3@cornell.edu

    2014-05-15

    Cell infection by parvoviruses requires that capsids be delivered from outside the cell to the cytoplasm, followed by genome trafficking to the nucleus. Here we microinject capsids into cells that lack receptors and followed their movements within the cell over time. In general the capsids remained close to the positions where they were injected, and most particles did not move to the vicinity of or enter the nucleus. When 70 kDa-dextran was injected along with the capsids that did not enter the nucleus in significant amounts. Capsids conjugated to peptides containing the SV40 large T-antigen nuclear localization signal remained in the cytoplasm, although bovine serum albumen conjugated to the same peptide entered the nucleus rapidly. No effects of disruption of microfilaments, intermediate filaments, or microtubules on the distribution of the capsids were observed. These results suggest that movement of intact capsids within cells is primarily associated with passive processes.

  19. Microbial investigations of deep geological compartments

    International Nuclear Information System (INIS)

    Barsotti, V.; Sergeant, C.; Vesvres, M.H.; Joulian, C.; Coulon, S.; Le Marrec, C.; Garrido, F.

    2010-01-01

    Document available in extended abstract form only. Deep sedimentary rocks are now considered to contain a significant part of the total bacterial population, but are microbiologically unexplored. The drilling down to the base of the Triassic (1980 meters deep) in the geological formations of the eastern Paris Basin performed by ANDRA (EST433) in 2008 provides us a good opportunity to explore the deep biosphere. We conditioned and sub-sampled on the coring site, in as aseptic conditions as possible, the nine cores: two in the Callovo-Oxfordian clay, two in the Dogger, five in the Triassic compartments. In addition to storage at atmospheric pressure, a portion of the five Triassic samples was placed in a 190 bars pressurized bars chamber to investigate the influence of the conservation pressure factor on the found microflora. In parallel, in order to evaluate a potential bacterial contamination of the core by the drilling fluids, samples of mud just before each sample drilling were taken and analysed. The microbial exploration we started can be divided in two parts: - A cultural approach in different culture media for six metabolic groups to try to find microbial cells still viable. This type of experiment is difficult because of the small proportion of cultivable species, especially in these extreme environmental samples. - A molecular approach by direct extraction of genomic DNA from the geological samples to explore a larger biodiversity. Here, the limits are the difficulties to extract DNA from these low biomass containing rocks. The five Triassic samples were partly crushed in powder and inoculated in the six culture media with four NaCl concentrations, because this type of rock is known as saline or hyper-saline, and incubated at three temperatures: 30 deg. C, 55 deg. C under agitation and 70 deg. C. First results will be presented. The direct extraction of DNA needs a complete method optimisation to adapt existent procedures (using commercial kit and classical

  20. Collagen/chitosan based two-compartment and bi-functional dermal scaffolds for skin regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Feng [Department of Plastic Surgery and Burns, Shenzhen Second People' s Hospital, Shenzhen 518035 (China); Wang, Mingbo [Key Laboratory of Biomedical Materials and Implants, Research Institute of Tsinghua University in Shenzhen, Shenzhen 518057 (China); She, Zhending [Key Laboratory of Biomedical Materials and Implants, Research Institute of Tsinghua University in Shenzhen, Shenzhen 518057 (China); Shenzhen Lando Biomaterials Co., Ltd., Shenzhen 518057 (China); Fan, Kunwu; Xu, Cheng [Department of Plastic Surgery and Burns, Shenzhen Second People' s Hospital, Shenzhen 518035 (China); Chu, Bin; Chen, Changsheng [Key Laboratory of Biomedical Materials and Implants, Research Institute of Tsinghua University in Shenzhen, Shenzhen 518057 (China); Shi, Shengjun, E-mail: shengjunshi@yahoo.com [The Burns Department of Zhujiang Hospital, Southern Medical University, Guangzhou 510280 (China); Tan, Rongwei, E-mail: tanrw@landobiom.com [Key Laboratory of Biomedical Materials and Implants, Research Institute of Tsinghua University in Shenzhen, Shenzhen 518057 (China); Shenzhen Lando Biomaterials Co., Ltd., Shenzhen 518057 (China)

    2015-07-01

    Inspired from the sophisticated bilayer structures of natural dermis, here, we reported collagen/chitosan based two-compartment and bi-functional dermal scaffolds. Two functions refer to mediating rapid angiogenesis based on recombinant human vascular endothelial growth factor (rhVEGF) and antibacterial from gentamicin, which were encapsulated in PLGA microspheres. The gentamicin and rhVEGF encapsulated PLGA microspheres were further combined with collagen/chitosan mixtures in low (lower layer) and high (upper layer) concentrations, and molded to generate the two-compartment and bi-functional scaffolds. Based on morphology and pore structure analyses, it was found that the scaffold has a distinct double layered porous and connective structure with PLGA microspheres encapsulated. Statistical analysis indicated that the pores in the upper layer and in the lower layer have great variations in diameter, indicative of a two-compartment structure. The release profiles of gentamicin and rhVEGF exceeded 28 and 49 days, respectively. In vitro culture of mouse fibroblasts showed that the scaffold can facilitate cell adhesion and proliferation. Moreover, the scaffold can obviously inhibit proliferation of Staphylococcus aureus and Serratia marcescens, exhibiting its unique antibacterial effect. The two-compartment and bi-functional dermal scaffolds can be a promising candidate for skin regeneration. - Highlights: • The dermal scaffold is inspired from the bilayer structures of natural dermis. • The dermal scaffold has two-compartment structures. • The dermal scaffold containing VEGF and gentamicin encapsulated PLGA microspheres • The dermal scaffold can facilitate cell adhesion and proliferation.

  1. Collagen/chitosan based two-compartment and bi-functional dermal scaffolds for skin regeneration

    International Nuclear Information System (INIS)

    Wang, Feng; Wang, Mingbo; She, Zhending; Fan, Kunwu; Xu, Cheng; Chu, Bin; Chen, Changsheng; Shi, Shengjun; Tan, Rongwei

    2015-01-01

    Inspired from the sophisticated bilayer structures of natural dermis, here, we reported collagen/chitosan based two-compartment and bi-functional dermal scaffolds. Two functions refer to mediating rapid angiogenesis based on recombinant human vascular endothelial growth factor (rhVEGF) and antibacterial from gentamicin, which were encapsulated in PLGA microspheres. The gentamicin and rhVEGF encapsulated PLGA microspheres were further combined with collagen/chitosan mixtures in low (lower layer) and high (upper layer) concentrations, and molded to generate the two-compartment and bi-functional scaffolds. Based on morphology and pore structure analyses, it was found that the scaffold has a distinct double layered porous and connective structure with PLGA microspheres encapsulated. Statistical analysis indicated that the pores in the upper layer and in the lower layer have great variations in diameter, indicative of a two-compartment structure. The release profiles of gentamicin and rhVEGF exceeded 28 and 49 days, respectively. In vitro culture of mouse fibroblasts showed that the scaffold can facilitate cell adhesion and proliferation. Moreover, the scaffold can obviously inhibit proliferation of Staphylococcus aureus and Serratia marcescens, exhibiting its unique antibacterial effect. The two-compartment and bi-functional dermal scaffolds can be a promising candidate for skin regeneration. - Highlights: • The dermal scaffold is inspired from the bilayer structures of natural dermis. • The dermal scaffold has two-compartment structures. • The dermal scaffold containing VEGF and gentamicin encapsulated PLGA microspheres • The dermal scaffold can facilitate cell adhesion and proliferation

  2. Carbon fuel cells with carbon corrosion suppression

    Science.gov (United States)

    Cooper, John F [Oakland, CA

    2012-04-10

    An electrochemical cell apparatus that can operate as either a fuel cell or a battery includes a cathode compartment, an anode compartment operatively connected to the cathode compartment, and a carbon fuel cell section connected to the anode compartment and the cathode compartment. An effusion plate is operatively positioned adjacent the anode compartment or the cathode compartment. The effusion plate allows passage of carbon dioxide. Carbon dioxide exhaust channels are operatively positioned in the electrochemical cell to direct the carbon dioxide from the electrochemical cell.

  3. Identifiability Results for Several Classes of Linear Compartment Models.

    Science.gov (United States)

    Meshkat, Nicolette; Sullivant, Seth; Eisenberg, Marisa

    2015-08-01

    Identifiability concerns finding which unknown parameters of a model can be estimated, uniquely or otherwise, from given input-output data. If some subset of the parameters of a model cannot be determined given input-output data, then we say the model is unidentifiable. In this work, we study linear compartment models, which are a class of biological models commonly used in pharmacokinetics, physiology, and ecology. In past work, we used commutative algebra and graph theory to identify a class of linear compartment models that we call identifiable cycle models, which are unidentifiable but have the simplest possible identifiable functions (so-called monomial cycles). Here we show how to modify identifiable cycle models by adding inputs, adding outputs, or removing leaks, in such a way that we obtain an identifiable model. We also prove a constructive result on how to combine identifiable models, each corresponding to strongly connected graphs, into a larger identifiable model. We apply these theoretical results to several real-world biological models from physiology, cell biology, and ecology.

  4. Enteral Feeding in Abdominal Compartment Syndrome

    Directory of Open Access Journals (Sweden)

    Ye. V Grigoryev

    2009-01-01

    Full Text Available Objective: to substantiate the choice of a gastrointestinal tract (GIT function support regimen as a mode for correction of the abdominal compartment syndrome (ACS. Subjects and methods. Forty-three patients with different causes of inadequate GIT function of various origin and ACS (disseminated peritonitis (45%, pancreatitis (24%, and severe concomitant injury (31% were examined. Group 1 (control received complete parenteral nutritional feeding (n=23; APACHE II scores, 21±4; calculated probability of fatal outcome, 33.5%. In Group II (study, complete parenteral feeding in the first 24 hours after stabilization was supplemented with GIT function support with Pepsisorb (Nutricia in doses of 500, 1000, and 1500 ml on days 1, 2, and 3, respectively (n=20; APACHE II scores, 20±6; calculated probability of fatal outcome, 37.1%. During early enteral nutritional support, the SOFA score was significantly less than that in Group 1 on days 2—3; the oxygenation index significantly increased on day 3; the value of intra-abdominal hypertension decreased to the control values. The positive effect of the GIT function support regimen on regression of the multiple organ dysfunction syndrome (MODS was confirmed by the lowered levels of biological markers (von Willebrand factor (WF and endothelin-1 as markers of endothelial damage of MODS. Correlation analysis showed a direct correlation between the markers of endothelial damage and the SOFA scores (r=0.34; p=0.05 for WF and r=0.49;p=0.03 for endothelin. Conclusion. The GIT function support regimen via early enteral alimentation with Peptisorb, which was initiated in the first 24 hours after admission, is able to level off the manifestations of the early stages of the abdominal compartment syndrome, with the acceptable values of oxygen balance and water-electrolyte and osmotic homeostasis being achieved. Key words: abdominal compartment syndrome, nutritional support, biological markers, oxygenation index

  5. [Intraabdominal hypertension and abdominal compartment syndrome

    DEFF Research Database (Denmark)

    Sonne, M.; Hilligsø, Jens Georg

    2008-01-01

    Intraabdominal hypertension (IAH) and abdominal compartment syndrome (ACS) are rare conditions with high mortality. IAH is an intraabdominal pressure (IAP) above 12 mmHg and ACS an IAP above 20 mmHg with evidence of organ dysfunction. IAP is measured indirectly via the bladder or stomach. Various...... medical and surgical conditions increase the intraabdominal volume. When the content exceeds the compliance of the abdominal wall, the IAP rises. Increased IAP affects the functioning of the brain, lungs, circulation, kidneys, and bowel. The treatment of ACS is a reduction of IAP Udgivelsesdato: 2008/2/11...

  6. Lessons from Animal Models of Cytoplasmic Intermediate Filament Proteins.

    Science.gov (United States)

    Bouameur, Jamal-Eddine; Magin, Thomas M

    Cytoplasmic intermediate filaments (IFs) represent a major cytoskeletal network contributing to cell shape, adhesion and migration as well as to tissue resilience and renewal in numerous bilaterians, including mammals. The observation that IFs are dispensable in cultured mammalian cells, but cause tissue-specific, life-threatening disorders, has pushed the need to investigate their function in vivo. In keeping with human disease, the deletion or mutation of murine IF genes resulted in highly specific pathologies. Epidermal keratins, together with desmin, are essential to protect corresponding tissues against mechanical force but also participate in stabilizing cell adhesion and in inflammatory signalling. Surprisingly, other IF proteins contribute to tissue integrity to a much lesser extent than anticipated, pointing towards their role in stress situations. In support, the overexpression of small chaperones or the interference with inflammatory signalling in several settings has been shown to rescue severe tissue pathologies that resulted from the expression of mutant IF proteins. It stills remains an open issue whether the wide range of IF disorders share similar pathomechanisms. Moreover, we lack an understanding how IF proteins participate in signalling processes. Now, with a large number of mouse models in hand, the next challenge will be to develop organotypic cell culture models to dissect pathomechanisms at the molecular level, to employ Crispr/Cas-mediated genome engineering to optimize models and, finally, to combine available animal models with medicinal chemistry for the development of molecular therapies.

  7. Endogenous RGS14 is a cytoplasmic-nuclear shuttling protein that localizes to juxtanuclear membranes and chromatin-rich regions of the nucleus

    Science.gov (United States)

    Hepler, John R.

    2017-01-01

    Regulator of G protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates G protein and H-Ras/MAPkinase signaling pathways to regulate synaptic plasticity important for hippocampal learning and memory. However, to date, little is known about the subcellular distribution and roles of endogenous RGS14 in a neuronal cell line. Most of what is known about RGS14 cellular behavior is based on studies of tagged, recombinant RGS14 ectopically overexpressed in unnatural host cells. Here, we report for the first time a comprehensive assessment of the subcellular distribution and dynamic localization of endogenous RGS14 in rat B35 neuroblastoma cells. Using confocal imaging and 3D-structured illumination microscopy, we find that endogenous RGS14 localizes to subcellular compartments not previously recognized in studies of recombinant RGS14. RGS14 localization was observed most notably at juxtanuclear membranes encircling the nucleus, at nuclear pore complexes (NPC) on both sides of the nuclear envelope and within intranuclear membrane channels, and within both chromatin-poor and chromatin-rich regions of the nucleus in a cell cycle-dependent manner. In addition, a subset of nuclear RGS14 localized adjacent to active RNA polymerase II. Endogenous RGS14 was absent from the plasma membrane in resting cells; however, the protein could be trafficked to the plasma membrane from juxtanuclear membranes in endosomes derived from ER/Golgi, following constitutive activation of endogenous RGS14 G protein binding partners using AlF4¯. Finally, our findings show that endogenous RGS14 behaves as a cytoplasmic-nuclear shuttling protein confirming what has been shown previously for recombinant RGS14. Taken together, the findings highlight possible cellular roles for RGS14 not previously recognized that are distinct from the regulation of conventional GPCR-G protein signaling, in particular undefined roles for RGS14 in the nucleus. PMID:28934222

  8. A cataract-causing connexin 50 mutant is mislocalized to the ER due to loss of the fourth transmembrane domain and cytoplasmic domain.

    Science.gov (United States)

    Somaraju Chalasani, Madhavi Latha; Muppirala, Madhavi; G Ponnam, Surya Prakash; Kannabiran, Chitra; Swarup, Ghanshyam

    2013-01-01

    Mutations in the eye lens gap junction protein connexin 50 cause cataract. Earlier we identified a frameshift mutant of connexin 50 (c.670insA; p.Thr203AsnfsX47) in a family with autosomal recessive cataract. The mutant protein is smaller and contains 46 aberrant amino acids at the C-terminus after amino acid 202. Here, we have analysed this frameshift mutant and observed that it localized to the endoplasmic reticulum (ER) but not in the plasma membrane. Moreover, overexpression of the mutant resulted in disintegration of the ER-Golgi intermediate compartment (ERGIC), reduction in the level of ERGIC-53 protein and breakdown of the Golgi in many cells. Overexpression of the frameshift mutant partially inhibited the transport of wild type connexin 50 to the plasma membrane. A deletion mutant lacking the aberrant sequence showed predominant localization in the ER and inhibited anterograde protein transport suggesting, therefore, that the aberrant sequence is not responsible for improper localization of the frameshift mutant. Further deletion analysis showed that the fourth transmembrane domain and a membrane proximal region (231-294 amino acids) of the cytoplasmic domain are needed for transport from the ER and localization to the plasma membrane. Our results show that a frameshift mutant of connexin 50 mislocalizes to the ER and causes disintegration of the ERGIC and Golgi. We have also identified a sequence of connexin 50 crucial for transport from the ER and localization to the plasma membrane.

  9. Regulation of H+ Extrusion and Cytoplasmic pH in Maize Root Tips Acclimated to a Low-Oxygen Environment.

    Science.gov (United States)

    Xia, J. H.; Roberts, JKM.

    1996-05-01

    We tested the hypothesis that H+ extrusion contributes to cytoplasmic pH regulation and tolerance of anoxia in maize (Zea mays) root tips. We studied root tips of whole seedlings that were acclimated to a low-oxygen environment by pretreatment in 3% (v/v) O2. Acclimated root tips characteristically regulate cytoplasmic pH near neutrality and survive prolonged anoxia, whereas nonacclimated tips undergo severe cytoplasmic acidosis and die much more quickly. We show that the plasma membrane H+-ATPase can operate under anoxia and that net H+ extrusion increases when cytoplasmic pH falls. However, at an external pH near 6.0, H+ extrusion contributes little to cytoplasmic pH regulation. At more acidic external pH values, net H+ flux into root tips increases dramatically, leading to a decrease in cytoplasmic pH and reduced tolerance of anoxia. We present evidence that, under these conditions, H+ pumps are activated to partly offset acidosis due to H+ influx and, thereby, contribute to cytoplasmic pH regulation and tolerance of anoxia. The regulation of H+ extrusion under anoxia is discussed with respect to the acclimation response and mechanisms of intracellular pH regulation in aerobic plant cells.

  10. Differentiation of the lateral compartment of the cochlea requires a temporally restricted FGF20 signal.

    Directory of Open Access Journals (Sweden)

    Sung-Ho Huh

    2012-01-01

    Full Text Available A large proportion of age-related hearing loss is caused by loss or damage to outer hair cells in the organ of Corti. The organ of Corti is the mechanosensory transducing apparatus in the inner ear and is composed of inner hair cells, outer hair cells, and highly specialized supporting cells. The mechanisms that regulate differentiation of inner and outer hair cells are not known. Here we report that fibroblast growth factor 20 (FGF20 is required for differentiation of cells in the lateral cochlear compartment (outer hair and supporting cells within the organ of Corti during a specific developmental time. In the absence of FGF20, mice are deaf and lateral compartment cells remain undifferentiated, postmitotic, and unresponsive to Notch-dependent lateral inhibition. These studies identify developmentally distinct medial (inner hair and supporting cells and lateral compartments in the developing organ of Corti. The viability and hearing loss in Fgf20 knockout mice suggest that FGF20 may also be a deafness-associated gene in humans.

  11. Polarized sphingolipid transport from the subapical compartment : Evidence for distinct sphingolipid domains

    NARCIS (Netherlands)

    van Ijzendoorn, SCD; Hoekstra, D

    1999-01-01

    In polarized HepG2 cells, the sphingolipids glucosylceramide and sphingomyelin (SM), transported along the reverse transcytotic pathway, are sorted in subapical compartments (SACs), and subsequently targeted to either apical or basolateral plasma membrane domains, respectively. In the present study,

  12. Dynamics of highly polydisperse colloidal suspensions as a model system for bacterial cytoplasm.

    Science.gov (United States)

    Hwang, Jiye; Kim, Jeongmin; Sung, Bong June

    2016-08-01

    There are various kinds of macromolecules in bacterial cell cytoplasm. The size polydispersity of the macromolecules is so significant that the crystallization and the phase separation could be suppressed, thus stabilizing the liquid state of bacterial cytoplasm. On the other hand, recent experiments suggested that the macromolecules in bacterial cytoplasm should exhibit glassy dynamics, which should be also affected significantly by the size polydispersity of the macromolecules. In this work, we investigate the anomalous and slow dynamics of highly polydisperse colloidal suspensions, of which size distribution is chosen to mimic Escherichia coli cytoplasm. We find from our Langevin dynamics simulations that the diffusion coefficient (D_{tot}) and the displacement distribution functions (P(r,t)) averaged over all colloids of different sizes do not show anomalous and glassy dynamic behaviors until the system volume fraction ϕ is increased up to 0.82. This indicates that the intrinsic polydispersity of bacterial cytoplasm should suppress the glass transition and help maintain the liquid state of the cytoplasm. On the other hand, colloids of each kind show totally different dynamic behaviors depending on their size. The dynamics of colloids of different size becomes non-Gaussian at a different range of ϕ, which suggests that a multistep glass transition should occur. The largest colloids undergo the glass transition at ϕ=0.65, while the glass transition does not occur for smaller colloids in our simulations even at the highest value of ϕ. We also investigate the distribution (P(θ,t)) of the relative angles of displacement for macromolecules and find that macromolecules undergo directionally correlated motions in a sufficiently dense system.

  13. A Mechanism for Cytoplasmic Streaming: Kinesin-Driven Alignment of Microtubules and Fast Fluid Flows.

    Science.gov (United States)

    Monteith, Corey E; Brunner, Matthew E; Djagaeva, Inna; Bielecki, Anthony M; Deutsch, Joshua M; Saxton, William M

    2016-05-10

    The transport of cytoplasmic components can be profoundly affected by hydrodynamics. Cytoplasmic streaming in Drosophila oocytes offers a striking example. Forces on fluid from kinesin-1 are initially directed by a disordered meshwork of microtubules, generating minor slow cytoplasmic flows. Subsequently, to mix incoming nurse cell cytoplasm with ooplasm, a subcortical layer of microtubules forms parallel arrays that support long-range, fast flows. To analyze the streaming mechanism, we combined observations of microtubule and organelle motions with detailed mathematical modeling. In the fast state, microtubules tethered to the cortex form a thin subcortical layer and undergo correlated sinusoidal bending. Organelles moving in flows along the arrays show velocities that are slow near the cortex and fast on the inward side of the subcortical microtubule layer. Starting with fundamental physical principles suggested by qualitative hypotheses, and with published values for microtubule stiffness, kinesin velocity, and cytoplasmic viscosity, we developed a quantitative coupled hydrodynamic model for streaming. The fully detailed mathematical model and its simulations identify key variables that can shift the system between disordered (slow) and ordered (fast) states. Measurements of array curvature, wave period, and the effects of diminished kinesin velocity on flow rates, as well as prior observations on f-actin perturbation, support the model. This establishes a concrete mechanistic framework for the ooplasmic streaming process. The self-organizing fast phase is a result of viscous drag on kinesin-driven cargoes that mediates equal and opposite forces on cytoplasmic fluid and on microtubules whose minus ends are tethered to the cortex. Fluid moves toward plus ends and microtubules are forced backward toward their minus ends, resulting in buckling. Under certain conditions, the buckling microtubules self-organize into parallel bending arrays, guiding varying directions

  14. Antineutrophil cytoplasm antibody: positivity and clinical correlation.

    Science.gov (United States)

    Martínez Téllez, Goitybell; Torres Rives, Bárbara; Rangel Velázquez, Suchiquil; Sánchez Rodríguez, Vicky; Ramos Ríos, María Antonia; Fuentes Smith, Lisset Evelyn

    2015-01-01

    To determine positivity and clinical correlation of anti-neutrophil cytoplasmic antibodies (ANCA), taking into account the interference of antinuclear antibodies (ANA). A prospective study was conducted in the Laboratory of Immunology of the National Cuban Center of Medical Genetic during one year. Two hounded sixty-seven patients with indication for ANCA determination were included. ANCA and ANA determinations with different cut off points and assays were determined by indirect immunofluorescense. Anti proteinase 3 and antimyeloperoxidase antibodies were determined by ELISA. Most positivity for ANCA was seen in patients with ANCA associated, primary small-vessel vasculitides, rheumatoid arthritis and systemic lupus erythematosus. Presence of ANCA without positivity for proteinase 3 and myeloperoxidase was higher in patients with ANA and little relation was observed between the perinuclear pattern confirmed in formalin and specificity by myeloperoxidase. Highest sensibility and specificity values for vasculitides diagnostic were achieved by ANCA determination using indirect immunofluorescense with a cut off 1/80 and confirming antigenic specificities with ELISA. ANCA can be present in a great number of chronic inflammatory or autoimmune disorders in the population studied. This determination using indirect immunofluorescence and following by ELISA had a great value for vasculitis diagnosis. Anti mieloperoxidasa assay has a higher utility than the formalin assay when ANA is present. Copyright © 2013 Elsevier España, S.L.U. All rights reserved.

  15. Snake-like chromatin in conjunctival cells of normal elderly persons and of patients with primary Sjögren's syndrome and other connective tissue diseases

    DEFF Research Database (Denmark)

    Bjerrum, Kirsten Birgitte

    1995-01-01

    Ophthalmology, snake-like chromatin, cytoplasm ratio, keratoconjunctivitis sicca, nucleus, goblet cell......Ophthalmology, snake-like chromatin, cytoplasm ratio, keratoconjunctivitis sicca, nucleus, goblet cell...

  16. Modeling malware propagation using a carrier compartment

    Science.gov (United States)

    Hernández Guillén, J. D.; Martín del Rey, A.

    2018-03-01

    The great majority of mathematical models proposed to simulate malware spreading are based on systems of ordinary differential equations. These are compartmental models where the devices are classified according to some types: susceptible, exposed, infectious, recovered, etc. As far as we know, there is not any model considering the special class of carrier devices. This type is constituted by the devices whose operative systems is not targeted by the malware (for example, iOS devices for Android malware). In this work a novel mathematical model considering this new compartment is considered. Its qualitative study is presented and a detailed analysis of the efficient control measures is shown by studying the basic reproductive number.

  17. The upper hand on compartment syndrome.

    LENUS (Irish Health Repository)

    Dolan, Roisin T

    2012-11-01

    Metacarpal fractures are common injuries, accounting for approximately 30% to 40% of all hand fractures and with a lifetime incidence of 2.5%. Traditionally regarded as an innocuous injury, metacarpal fractures tend to be associated with successful outcomes after closed reduction and immobilization. Hand compartment syndrome (HCS) is a rare clinical entity with potential devastating consequences in terms of loss of function and quality-of-life outcomes. We discuss the case of a 44-year-old woman presenting with multiple closed metacarpal fractures as a result of low-energy trauma, complicated by acute HCS. We review the presentation, clinical assessment, and optimal surgical management of acute HCS with reference to international literature.

  18. An experimental study on crib fires in a closed compartment

    Directory of Open Access Journals (Sweden)

    Dhurandher Bhisham Kumar

    2017-01-01

    Full Text Available An experimental investigation on burning behavior of fire in closed compartments is presented. Fire experiments were performed in a closed compartment of interior dimensions 4 × 4 × 4 m (length × width × height with ply board cribs as fire source. The parameters including the gas temperature, mass loss rate, heat flux, flame temperature, and compartment pressure were measured during the experiments. Experimental results indicated that the providing sudden ventilation to the closed compartment had great influence on the behavior of fire. The mass loss rate of the burning crib increased by 150% due to sudden ventilation which results in the increase in heat release rate by 198 kW. From the perspective of total heat flux, compartment pressure, and gas temperatures closed compartment with sudden ventilation were more hazardous.

  19. PERMANGANATE FIXATION OF THE GOLGI COMPLEX AND OTHER CYTOPLASMIC STRUCTURES OF MAMMALIAN TESTES

    Science.gov (United States)

    Mollenhauer, Hilton H.; Zebrun, William

    1960-01-01

    Observations on the fine structure of KMnO4-fixed testes of small mammals (guinea pig, rat, and mouse) reveal certain morphological differences between the spermatogenic and Sertoli cells which have not been demonstrated in the same tissue fixed with OsO4. Aggregates of minute circular profiles, much smaller than the spherical Golgi vesicles, are described in close association with the Golgi complex of developing spermatids. Groups of dense flattened vesicles, individually surrounded by a membrane of different dimensions than that which bounds most of the other cell organelles, appear dispersed within the cytoplasm of some spermatogenic cells. Flattened vesicles of greater density than those belonging to the Golgi complex are reported confined to the inner Golgi zone of developing guinea pig spermatids between the Golgi cisternae and the head cap. The profiles of endoplasmic reticulum within spermatocytes appear shorter, wider, and more tortuous than those of Sertoli cells. Minute cytoplasmic particles approximately 300 A in diameter and of high electron opacity appear randomly disposed in some Sertoli cells. Groups of irregular-shaped ovoid bodies within the developing spermatids are described as resembling portions of cytoplasm from closely adjacent spermatids. Interpretation is presented regarding the fine structure of KMnO4-fixed testes in view of what has already been reported for mammalian testes fixed in OsO4. PMID:13771855

  20. Aconitum pseudo-laeve var. erectum Inhibits Receptor Activator of Nuclear Factor Kappa-B Ligand-Induced Osteoclastogenesis via the c-Fos/nuclear Factor of Activated T-Cells, Cytoplasmic 1 Signaling Pathway and Prevents Lipopolysaccharide-Induced Bone Loss in Mice

    Directory of Open Access Journals (Sweden)

    Jong Min Baek

    2014-08-01

    Full Text Available Aconitum pseudo-laeve var. erectum (APE has been widely shown in herbal medicine to have a therapeutic effect on inflammatory conditions. However, there has been no evidence on whether the extract of APE is involved in the biological bone metabolism process, particularly osteoclast-mediated bone resorption. In this study, we confirmed that the administration of APE could restore normal skeletal conditions in a murine model of lipopolysaccharide (LPS-induced bone loss via a decrease in the receptor activator of nuclear factor kappa-B ligand (RANKL/osteoprotegerin (OPG ratio and osteoclast number. We then investigated the effect of APE on the RANKL-induced formation and function of osteoclasts to elucidate its underlying molecular mechanisms. APE suppressed the formation of tartrate-resistant acid phosphatase (TRAP-positive cells, as well as the bone-resorbing activity of mature osteoclasts. Furthermore, APE attenuated nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1 and c-Fos without affecting any early signal pathway of osteoclastogenesis. Subsequently, APE significantly downregulated the expression of various genes exclusively expressed in osteoclasts. These results demonstrate that APE restores LPS-induced bone loss through a decrease of the serum RANKL/OPG ratio, and inhibits osteoclast differentiation and function, suggesting the promise of APE as a potential cure for various osteoclast-associated bone diseases.

  1. A human cadaver fascial compartment pressure measurement model.

    Science.gov (United States)

    Messina, Frank C; Cooper, Dylan; Huffman, Gretchen; Bartkus, Edward; Wilbur, Lee

    2013-10-01

    Fresh human cadavers provide an effective model for procedural training. Currently, there are no realistic models to teach fascial compartment pressure measurement. We created a human cadaver fascial compartment pressure measurement model and studied its feasibility with a pre-post design. Three faculty members, following instructions from a common procedure textbook, used a standard handheld intra-compartment pressure monitor (Stryker(®), Kalamazoo, MI) to measure baseline pressures ("unembalmed") in the anterior, lateral, deep posterior, and superficial posterior compartments of the lower legs of a fresh human cadaver. The right femoral artery was then identified by superficial dissection, cannulated distally towards the lower leg, and connected to a standard embalming machine. After a 5-min infusion, the same three faculty members re-measured pressures ("embalmed") of the same compartments on the cannulated right leg. Unembalmed and embalmed readings for each compartment, and baseline readings for each leg, were compared using a two-sided paired t-test. The mean baseline compartment pressures did not differ between the right and left legs. Using the embalming machine, compartment pressure readings increased significantly over baseline for three of four fascial compartments; all in mm Hg (±SD): anterior from 40 (±9) to 143 (±44) (p = 0.08); lateral from 22 (±2.5) to 160 (±4.3) (p cadaver using a standard embalming machine. Set-up is minimal and the model can be incorporated into teaching curricula. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Rapid and Sustained Nuclear-Cytoplasmic ERK Oscillations Induced by Epidermal Growth Factor

    Energy Technology Data Exchange (ETDEWEB)

    Shankaran, Harish; Ippolito, Danielle L.; Chrisler, William B.; Resat, Haluk; Bollinger, Nikki; Opresko, Lee K.; Wiley, H. S.

    2009-12-01

    Mathematical modeling has predicted that ERK activity should oscillate in response to cell stimulation, but this has never been observed. To explore this inconsistency, we expressed an ERK1-GFP fusion protein in mammary epithelial cells. Following EGF stimulation, we observed rapid and continuous ERK oscillations between the nucleus and cytoplasm with a periodicity of approximately 15 minutes. These oscillations were remarkably persistent (>45 cycles), displayed an asymmetric waveform, and were highly dependent on cell density, essentially disappearing at confluency. We conclude that the ERK pathway is an intrinsic oscillator. Although the functional implications of the observed oscillations are uncertain, this property can be used to continuously monitor ERK activity in single cells.

  3. Antineutrophil Cytoplasmic Antibodies, Autoimmune Neutropenia, and Vasculitis

    Science.gov (United States)

    Grayson, Peter C.; Sloan, J. Mark; Niles, John L.; Monach, Paul A.; Merkel, Peter A.

    2011-01-01

    Objectives Reports of an association between antineutrophil cytoplasmic antibodies (ANCA) and autoimmune neutropenia have rarely included cases of proven vasculitis. A case of ANCA-associated vasculitis (AAV) with recurrent neutropenia is described and relevant literature on the association between ANCA, neutropenia, and vasculitis is reviewed. Methods Longitudinal clinical assessments and laboratory findings are described in a patient with AAV and recurrent episodes of profound neutropenia from December 2008 – October 2010. A PubMed database search of the medical literature was performed for papers published from 1960 through October 2010 to identify all reported cases of ANCA and neutropenia. Results A 49 year-old man developed recurrent neutropenia, periodic fevers, arthritis, biopsy-proven cutaneous vasculitis, sensorineural hearing loss, epididymitis, and positive tests for ANCA with specificity for antibodies to both proteinase 3 and myeloperoxidase. Antineutrophil membrane antibodies were detected during an acute neutropenic phase and were not detectable in a post-recovery sample, whereas ANCA titers did not seem to correlate with neutropenia. An association between ANCA and neutropenia has been reported in 74 cases from 24 studies in the context of drug/toxin exposure, underlying autoimmune disease, or chronic neutropenia without underlying autoimmune disease. In these cases, the presence of atypical ANCA patterns and other antibodies were common; however, vasculitis was uncommon and when it occurred was usually limited to the skin and in cases of underlying toxin exposure. Conclusions ANCA is associated with autoimmune neutropenia, but systemic vasculitis rarely occurs in association with ANCA and neutropenia. The interaction between neutrophils and ANCA may provide insight into understanding both autoimmune neutropenia and AAV. PMID:21507463