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Sample records for cells requires fusion-mediated

  1. Ordered chromatin changes and human X chromosome reactivation by cell fusion-mediated pluripotent reprogramming

    Science.gov (United States)

    Cantone, Irene; Bagci, Hakan; Dormann, Dirk; Dharmalingam, Gopuraja; Nesterova, Tatyana; Brockdorff, Neil; Rougeulle, Claire; Vallot, Celine; Heard, Edith; Chaligne, Ronan; Merkenschlager, Matthias; Fisher, Amanda G.

    2016-01-01

    Erasure of epigenetic memory is required to convert somatic cells towards pluripotency. Reactivation of the inactive X chromosome (Xi) has been used to model epigenetic reprogramming in mouse, but human studies are hampered by Xi epigenetic instability and difficulties in tracking partially reprogrammed iPSCs. Here we use cell fusion to examine the earliest events in the reprogramming-induced Xi reactivation of human female fibroblasts. We show that a rapid and widespread loss of Xi-associated H3K27me3 and XIST occurs in fused cells and precedes the bi-allelic expression of selected Xi-genes by many heterokaryons (30–50%). After cell division, RNA-FISH and RNA-seq analyses confirm that Xi reactivation remains partial and that induction of human pluripotency-specific XACT transcripts is rare (1%). These data effectively separate pre- and post-mitotic events in reprogramming-induced Xi reactivation and reveal a complex hierarchy of epigenetic changes that are required to reactivate the genes on the human Xi chromosome. PMID:27507283

  2. Ca2+ -regulated lysosome fusion mediates angiotensin II-induced lipid raft clustering in mesenteric endothelial cells.

    Science.gov (United States)

    Han, Wei-Qing; Chen, Wen-Dong; Zhang, Ke; Liu, Jian-Jun; Wu, Yong-Jie; Gao, Ping-Jin

    2016-04-01

    It has been reported that intracellular Ca2+ is involved in lysosome fusion and membrane repair in skeletal cells. Given that angiotensin II (Ang II) elicits an increase in intracellular Ca2+ and that lysosome fusion is a crucial mediator of lipid raft (LR) clustering, we hypothesized that Ang II induces lysosome fusion and activates LR formation in rat mesenteric endothelial cells (MECs). We found that Ang II acutely increased intracellular Ca2+ content, an effect that was inhibited by the extracellular Ca2+ chelator ethylene glycol tetraacetic acid (EGTA) and the inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release inhibitor 2-aminoethoxydiphenyl borate (2-APB). Further study showed that EGTA almost completely blocked Ang II-induced lysosome fusion, the translocation of acid sphingomyelinase (ASMase) to LR clusters, ASMase activation and NADPH (nicotinamide adenine dinucleotide phosphate) oxidase activation. In contrast, 2-APB had a slight inhibitory effect. Functionally, both the lysosome inhibitor bafilomycin A1 and the ASMase inhibitor amitriptyline reversed Ang II-induced impairment of vasodilation. We conclude that Ca2+ -regulated lysosome fusion mediates the Ang II-induced regulation of the LR-redox signaling pathway and mesenteric endothelial dysfunction.

  3. Human mesenchymal stem cells reprogram adult cardiomyocytes toward a progenitor-like state through partial cell fusion and mitochondria transfer : Cell fusion-mediated cardiomyocyte reprogramming.

    OpenAIRE

    Acquistapace, Adrien; Bru, Thierry; Lesault, Pierre-François; Figeac, Florence; Coudert, Amélie,; Le Coz, Olivier; Christov, Christo; Baudin, Xavier; Auber, Fréderic; Yiou, René; Dubois-Randé, Jean-Luc; Rodriguez, Anne-Marie

    2011-01-01

    International audience Because stem cells are often found to improve repair tissue including heart without evidence of engraftment or differentiation, mechanisms underlying wound healing are still elusive. Several studies have reported that stem cells can fuse with cardiomyocytes either by permanent or partial cell fusion processes. However, the respective physiological impact of these two processes remains unknown in part because of the lack of knowledge of the resulting hybrid cells. To ...

  4. Using a split luciferase assay (SLA) to measure the kinetics of cell-cell fusion mediated by herpes simplex virus glycoproteins.

    Science.gov (United States)

    Saw, Wan Ting; Matsuda, Zene; Eisenberg, Roselyn J; Cohen, Gary H; Atanasiu, Doina

    2015-11-15

    Herpes simplex virus (HSV) entry and cell-cell fusion require the envelope proteins gD, gH/gL and gB. We propose that receptor-activated conformational changes to gD activate gH/gL, which then triggers gB (the fusogen) into an active form. To study this dynamic process, we have adapted a dual split protein assay originally developed to study the kinetics of human immunodeficiency virus (HIV) mediated fusion. This assay uses a chimera of split forms of renilla luciferase (RL) and green fluorescent protein (GFP). Effector cells are co-transfected with the glycoproteins and one of the split reporters. Receptor-bearing target cells are transfected with the second reporter. Co-culture results in fusion and restoration of RL, which can convert a membrane permeable substrate into a luminescent product, thereby enabling one to monitor initiation and extent of fusion in live cells in real time. Restoration of GFP can also be studied by fluorescence microscopy. Two sets of split reporters have been developed: the original one allows one to measure fusion kinetics over hours whereas the more recent version was designed to enhance the sensitivity of RL activity allowing one to monitor both initiation and rates of fusion in minutes. Here, we provide a detailed, step-by-step protocol for the optimization of the assay (which we call the SLA for split luciferase assay) using the HSV system. We also show several examples of the power of this assay to examine both the initiation and kinetics of cell-cell fusion by wild type forms of gD, gB, gH/gL of both serotypes of HSV as well as the effect of mutations and antibodies that alter the kinetics of fusion. The SLA can be applied to other viral systems that carry out membrane fusion.

  5. Imaging multiple intermediates of single-virus membrane fusion mediated by distinct fusion proteins.

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    Joo, Kye-Il; Tai, April; Lee, Chi-Lin; Wong, Clement; Wang, Pin

    2010-09-01

    Membrane fusion plays an essential role in the entry of enveloped viruses into target cells. The merging of viral and target cell membranes is catalyzed by viral fusion proteins, which involves multiple sequential steps in the fusion process. However, the fusion mechanisms mediated by different fusion proteins involve multiple transient intermediates that have not been well characterized. Here, we report a synthetic virus platform that allows us to better understand the different fusion mechanisms driven by the diverse types fusion proteins. The platform consists of lentiviral particles coenveloped with a surface antibody, which serves as the binding protein, along with a fusion protein derived from either influenza virus (HAmu) or Sindbis virus (SINmu). By using a single virus tracking technique, we demonstrated that both HAmu- and SINmu-bearing viruses enter cells through clathrin-dependent endocytosis, but they required different endosomal trafficking routes to initiate viral fusion. Direct observation of single viral fusion events clearly showed that hemifusion mediated by SINmu upon exposure to low pH occurs faster than that mediated by HAmu. Monitoring sequential fusion processes by dual labeling the outer and inner leaflets of viral membranes also revealed that the SINmu-mediated hemifusion intermediate is relatively long-lived as compared with that mediated by HAmu. Taken together, we have demonstrated that the combination of this versatile viral platform with the techniques of single virus tracking can be a powerful tool for revealing molecular details of fusion mediated by various fusion proteins.

  6. Point mutations in EBV gH that abrogate or differentially affect B cell and epithelial cell fusion

    OpenAIRE

    Wu, Liguo; Lindsey M Hutt-Fletcher

    2007-01-01

    Cell fusion mediated by Epstein-Barr virus requires three conserved glycoproteins, gB and gHgL, but activation is cell type specific. B cell fusion requires interaction between MHC-class II and a fourth virus glycoprotein, gp42, which complexes non-covalently with gHgL. Epithelial cell fusion requires interaction between gHgL and a novel epithelial cell coreceptor and is blocked by excess gp42. We show here that gp42 interacts directly with gH and that point mutations in the region of gH reco...

  7. Schwann cell myelination requires Dynein function

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    Langworthy Melissa M

    2012-11-01

    Full Text Available Abstract Background Interaction of Schwann cells with axons triggers signal transduction that drives expression of Pou3f1 and Egr2 transcription factors, which in turn promote myelination. Signal transduction appears to be mediated, at least in part, by cyclic adenosine monophosphate (cAMP because elevation of cAMP levels can stimulate myelination in the absence of axon contact. The mechanisms by which the myelinating signal is conveyed remain unclear. Results By analyzing mutations that disrupt myelination in zebrafish, we learned that Dynein cytoplasmic 1 heavy chain 1 (Dync1h1, which functions as a motor for intracellular molecular trafficking, is required for peripheral myelination. In dync1h1 mutants, Schwann cell progenitors migrated to peripheral nerves but then failed to express Pou3f1 and Egr2 or make myelin membrane. Genetic mosaic experiments revealed that robust Myelin Basic Protein expression required Dync1h1 function within both Schwann cells and axons. Finally, treatment of dync1h1 mutants with a drug to elevate cAMP levels stimulated myelin gene expression. Conclusion Dync1h1 is required for retrograde transport in axons and mutations of Dync1h1 have been implicated in axon disease. Our data now provide evidence that Dync1h1 is also required for efficient myelination of peripheral axons by Schwann cells, perhaps by facilitating signal transduction necessary for myelination.

  8. Membrane Fusion Mediated by pH-Low-Insertion-Peptide (pHLIP)

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    Daniels, Jennifer; Yao, Lan; Engelman, Donald; Andreev, Oleg; Reshetnyak, Yana

    2012-02-01

    Liposomes are traditionally used as drug delivery carriers. The major mechanism of liposome entry into cell is endocytotic. First, the endocytotic pathway of cellular entry is non-specific: the delivery of therapeutics occurs to cells in both diseased and healthy tissues. Second, liposomes are usually trapped in endosome/lysosome, which prevents delivery of therapeutics to cytoplasm. We proposed to use pHLIP (pH-Low-Insertion-Peptide) to promote selective delivery of the liposome content to cytoplasm of cancer cells. We showed that liposomes coated with PEG polymer and pHLIP peptide enhance membrane fusion in acidic environments. pHLIP promotes fusion between lipid bilayer of liposome and plasma membrane or membrane of endosome/lysosome, which results in intracellular delivery of payload. Liposomes composed of 5 % pHLIP and 5 % PEG were ideal for the delivery. Since cancer and other pathological states produce an acid extracellular environment, this allows the liposome to target diseased tissue while avoiding healthy tissue (with neutral pH in extracellular space). The work is supported by NIH grants CA133890 to OAA, DME, YRK.

  9. Replication Termination: Containing Fork Fusion-Mediated Pathologies in Escherichia coli

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    Juachi U. Dimude

    2016-07-01

    Full Text Available Duplication of bacterial chromosomes is initiated via the assembly of two replication forks at a single defined origin. Forks proceed bi-directionally until they fuse in a specialised termination area opposite the origin. This area is flanked by polar replication fork pause sites that allow forks to enter but not to leave. The precise function of this replication fork trap has remained enigmatic, as no obvious phenotypes have been associated with its inactivation. However, the fork trap becomes a serious problem to cells if the second fork is stalled at an impediment, as replication cannot be completed, suggesting that a significant evolutionary advantage for maintaining this chromosomal arrangement must exist. Recently, we demonstrated that head-on fusion of replication forks can trigger over-replication of the chromosome. This over-replication is normally prevented by a number of proteins including RecG helicase and 3’ exonucleases. However, even in the absence of these proteins it can be safely contained within the replication fork trap, highlighting that multiple systems might be involved in coordinating replication fork fusions. Here, we discuss whether considering the problems associated with head-on replication fork fusion events helps us to better understand the important role of the replication fork trap in cellular metabolism.

  10. Requirements for anthrax toxin entry into cells

    OpenAIRE

    Ryan, Patricia Lynn

    2010-01-01

    Bacillus anthracis secretes a harmful exotoxin called anthrax toxin. Anthrax toxin has deleterious effects on several host cell types and is a significant contributor to anthrax pathogenesis. Toxin-deleted strains of B. anthracis are highly attenuated and many of the symptoms of anthrax can be replicated with anthrax toxin alone. Anthrax toxin is an AB-type toxin with two catalytic A moieties. PA, the B moiety, is responsible for receptor binding, pore formation and translocation of the catal...

  11. PCDH10 is required for the tumorigenicity of glioblastoma cells

    International Nuclear Information System (INIS)

    Highlights: • PCDH10 is required for the proliferation, survival and self-renewal of glioblastoma cells. • PCDH10 is required for glioblastoma cell migration and invasion. • PCDH10 is required for the tumorigenicity of glioblastoma cells. • PCDH10 may be a promising target for the therapy of glioblastoma. - Abstract: Protocadherin10 (PCDH10)/OL-protocadherin is a cadherin-related transmembrane protein that has multiple roles in the brain, including facilitating specific cell–cell connections, cell migration and axon guidance. It has recently been reported that PCDH10 functions as a tumor suppressor and that its overexpression inhibits proliferation or invasion of multiple tumor cells. However, the function of PCDH10 in glioblastoma cells has not been elucidated. In contrast to previous reports on other tumors, we show here that suppression of the expression of PCDH10 by RNA interference (RNAi) induces the growth arrest and apoptosis of glioblastoma cells in vitro. Furthermore, we demonstrate that knockdown of PCDH10 inhibits the growth of glioblastoma cells xenografted into immunocompromised mice. These results suggest that PCDH10 is required for the proliferation and tumorigenicity of glioblastoma cells. We speculate that PCDH10 may be a promising target for the therapy of glioblastoma

  12. Internalization of NK cells into tumor cells requires ezrin and leads to programmed cell-in-cell death

    Institute of Scientific and Technical Information of China (English)

    Shan Wang; Zhen Guo; Peng Xia; Tingting Liu; Jufang Wang; Shan Li; Lihua Sun; Jianxin Lu; Qian Wen; Mingqian Zhou; Li Ma; Xia Ding; Xiaoning Wang; Xuebiao Yao

    2009-01-01

    Cytotoxic lymphocytes are key players in the orchestration of immune response and elimination of defective cells. We have previously reported that natural killer (NK) cells enter target tumor cells, leading to either target cell death or self-destruction within tumor cells. However, it has remained elusive as to the fate of NK cells after internaliza-tion and whether the heterotypic cell-in-cell process is different from that of the homotypic cell-in-cell event recently named entosis. Here, we show that NK cells undergo a cell-in-cell process with the ultimate fate of apoptosis within tumor cells and reveal that the internalization process requires the actin cytoskeletal regulator, ezrin. To visualize how NK cells enter into tumor cells, we carried out real-time dual color imaging analyses of NK cell internalization into tumor cells. Surprisingly, most NK cells commit to programmed cell death after their entry into tumor cells, which is distinctively different from entosis observed in the homotypic cell-in-cell process. The apoptotic cell death of the internalized NK cells was evident by activation of caspase 3 and DNA fragmentation. Furthermore, NK cell death after internalization is attenuated by the caspase inhibitor, Z-VAD-FMK, confirming apoptosis as the mode of NK cell death within tumor cells. To determine protein factors essential for the entry of NK cells into tumor cells, we car-ried out siRNA-based knockdown analysis and discovered a critical role of ezrin in NK cell internalization. Impor-tantly, PKA-mediated phosphorylation of ezrin promotes the NK cell internalization process. Our findings suggest a novel regulatory mechanism by which ezrin governs NK cell internalization into tumor cells.

  13. Auxin requirements of sycamore cells in suspension culture.

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    Moloney, M M; Hall, J F; Robinson, G M; Elliott, M C

    1983-04-01

    Sycamore (Acer pseudoplatanus L.) cell suspension cultures (strain OS) require 2,4-dichlorophenoxyacetic acid (2,4-D) in their culture medium for normal growth. If the 2,4-D is omitted, rates of cell division are dramatically reduced and cell lysis may occur. Despite this ;auxin requirement,' it has been shown by gas chromatography-mass spectrometry that the cells synthesize indol-3yl-acetic acid (IAA). Changes in free 2,4-D and IAA in the cells during a culture passage have been monitored.There is a rapid uptake of 2,4-D by the cells during the lag phase leading to a maximum concentration per cell (125 nanograms per 10(6) cells) on day 2 followed by a decline to 45 nanograms per 10(6) cells by day 9 (middle of linear phase). The initial concentration of IAA (0.08 nanograms per 10(6) cells) rises slowly to a peak of 1.4 nanograms per 10(6) cells by day 9 then decreases rapidly to 0.2 nanograms per 10(6) cells by day 15 (early declining phase) and 0.08 nanograms per 10(6) cells by day 23 (early stationary phase).

  14. Desmosome dynamics in migrating epithelial cells requires the actin cytoskeleton

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    Roberts, Brett J.; Pashaj, Anjeza; Johnson, Keith R.; Wahl, James K.

    2011-01-01

    Re-modeling of epithelial tissues requires that the cells in the tissue rearrange their adhesive contacts in order to allow cells to migrate relative to neighboring cells. Desmosomes are prominent adhesive structures found in a variety of epithelial tissues that are believed to inhibit cell migration and invasion. Mechanisms regulating desmosome assembly and stability in migrating cells are largely unknown. In this study we established a cell culture model to examine the fate of desmosomal components during scratch wound migration. Desmosomes are rapidly assembled between epithelial cells at the lateral edges of migrating cells and structures are transported in a retrograde fashion while the structures become larger and mature. Desmosome assembly and dynamics in this system are dependent on the actin cytoskeleton prior to being associated with the keratin intermediate filament cytoskeleton. These studies extend our understanding of desmosome assembly and provide a system to examine desmosome assembly and dynamics during epithelial cell migration. PMID:21945137

  15. Homology with vesicle fusion mediator syntaxin-1a predicts determinants ofepimorphin/syntaxin-2 function in mammary epithelial morphogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Connie S.; Nelson, Celeste M.; Khauv, Davitte; Bennett, Simone; Radisky, Evette S.; Hirai, Yohei; Bissell, Mina J.; Radisky, Derek C.

    2009-06-03

    We have shown that branching morphogenesis of mammary ductal structures requires the action of the morphogen epimorphin/syntaxin-2. Epimorphin, originally identified as an extracellular molecule, is identical to syntaxin-2, an intracellular molecule that is a member of the extensively investigated syntaxin family of proteins that mediate vesicle trafficking. We show here that although epimorphin/syntaxin-2 is highly homologous to syntaxin-1a, only epimorphin/syntaxin-2 can stimulate mammary branching morphogenesis. We construct a homology model of epimorphin/syntaxin-2 based on the published structure of syntaxin-1a, and we use this model to identify the structural motif responsible for the morphogenic activity. We identify four residues located within the cleft between helices B and C that differ between syntaxin-1a and epimorphin/syntaxin-2; through site-directed mutagenesis of these four amino acids, we confer the properties of epimorphin for cell adhesion, gene activation, and branching morphogenesis onto the inactive syntaxin-1a template. These results provide a dramatic demonstration of the use of structural information about one molecule to define a functional motif of a second molecule that is related at the sequence level but highly divergent functionally.

  16. Verifying cell loss requirements in high-speed communication networks

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    Kerry W. Fendick

    1998-01-01

    Full Text Available In high-speed communication networks it is common to have requirements of very small cell loss probabilities due to buffer overflow. Losses are measured to verify that the cell loss requirements are being met, but it is not clear how to interpret such measurements. We propose methods for determining whether or not cell loss requirements are being met. A key idea is to look at the stream of losses as successive clusters of losses. Often clusters of losses, rather than individual losses, should be regarded as the important “loss events”. Thus we propose modeling the cell loss process by a batch Poisson stochastic process. Successive clusters of losses are assumed to arrive according to a Poisson process. Within each cluster, cell losses do not occur at a single time, but the distance between losses within a cluster should be negligible compared to the distance between clusters. Thus, for the purpose of estimating the cell loss probability, we ignore the spaces between successive cell losses in a cluster of losses. Asymptotic theory suggests that the counting process of losses initiating clusters often should be approximately a Poisson process even though the cell arrival process is not nearly Poisson. The batch Poisson model is relatively easy to test statistically and fit; e.g., the batch-size distribution and the batch arrival rate can readily be estimated from cell loss data. Since batch (cluster sizes may be highly variable, it may be useful to focus on the number of batches instead of the number of cells in a measurement interval. We also propose a method for approximately determining the parameters of a special batch Poisson cell loss with geometric batch-size distribution from a queueing model of the buffer content. For this step, we use a reflected Brownian motion (RBM approximation of a G/D/1/C queueing model. We also use the RBM model to estimate the input burstiness given the cell loss rate. In addition, we use the RBM model to

  17. Reliable in vitro studies require appropriate ovarian cancer cell lines.

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    Jacob, Francis; Nixdorf, Sheri; Hacker, Neville F; Heinzelmann-Schwarz, Viola A

    2014-01-01

    Ovarian cancer is the fifth most common cause of cancer death in women and the leading cause of death from gynaecological malignancies. Of the 75% women diagnosed with locally advanced or disseminated disease, only 30% will survive five years following treatment. This poor prognosis is due to the following reasons: limited understanding of the tumor origin, unclear initiating events and early developmental stages of ovarian cancer, lack of reliable ovarian cancer-specific biomarkers, and drug resistance in advanced cases. In the past, in vitro studies using cell line models have been an invaluable tool for basic, discovery-driven cancer research. However, numerous issues including misidentification and cross-contamination of cell lines have hindered research efforts. In this study we examined all ovarian cancer cell lines available from cell banks. Hereby, we identified inconsistencies in the reporting, difficulties in the identification of cell origin or clinical data of the donor patients, restricted ethnic and histological type representation, and a lack of tubal and peritoneal cancer cell lines. We recommend that all cell lines should be distributed via official cell banks only with strict guidelines regarding the minimal available information required to improve the quality of ovarian cancer research in future. PMID:24936210

  18. Requirements for Peptide-induced T Cell Receptor Downregulation on Naive CD8+ T Cells

    OpenAIRE

    Cai, Zeling; Kishimoto, Hidehiro; Brunmark, Anders; Jackson, Michael R.; Peterson, Per A.; Sprent, Jonathan

    1997-01-01

    The requirements for inducing downregulation of α/β T cell receptor (TCR) molecules on naive major histocompatibility complex class I–restricted T cells was investigated with 2C TCR transgenic mice and defined peptides as antigen. Confirming previous results, activation of 2C T cells in response to specific peptides required CD8 expression on the responder cells and was heavily dependent upon costimulation provided by either B7-1 or ICAM-1 on antigen-presenting cells (APC). These stringent re...

  19. Establishment of human papillomavirus infection requires cell cycle progression.

    Directory of Open Access Journals (Sweden)

    Dohun Pyeon

    2009-02-01

    Full Text Available Human papillomaviruses (HPVs are DNA viruses associated with major human cancers. As such there is a strong interest in developing new means, such as vaccines and microbicides, to prevent HPV infections. Developing the latter requires a better understanding of the infectious life cycle of HPVs. The HPV infectious life cycle is closely linked to the differentiation state of the stratified epithelium it infects, with progeny virus only made in the terminally differentiating suprabasal compartment. It has long been recognized that HPV must first establish its infection within the basal layer of stratified epithelium, but why this is the case has not been understood. In part this restriction might reflect specificity of expression of entry receptors. However, this hypothesis could not fully explain the differentiation restriction of HPV infection, since many cell types can be infected with HPVs in monolayer cell culture. Here, we used chemical biology approaches to reveal that cell cycle progression through mitosis is critical for HPV infection. Using infectious HPV16 particles containing the intact viral genome, G1-synchronized human keratinocytes as hosts, and early viral gene expression as a readout for infection, we learned that the recipient cell must enter M phase (mitosis for HPV infection to take place. Late M phase inhibitors had no effect on infection, whereas G1, S, G2, and early M phase cell cycle inhibitors efficiently prevented infection. We conclude that host cells need to pass through early prophase for successful onset of transcription of the HPV encapsidated genes. These findings provide one reason why HPVs initially establish infections in the basal compartment of stratified epithelia. Only this compartment of the epithelium contains cells progressing through the cell cycle, and therefore it is only in these cells that HPVs can establish their infection. By defining a major condition for cell susceptibility to HPV infection, these

  20. Materials requirements for high-efficiency silicon solar cells

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    Wolf, M.

    1985-01-01

    To achieve higher Si solar cell efficiencies (greater than 20%), better single-crystal Si must be produced. It is believed possible to bring Cz (Czochralski) Si up to the same low recombination level as FZ (Float Zone) Si. It is also desirable that solar cell Si meet the following requirements: long minority carrier lifetime (0.2 ohm-cm p-type with tau less than 500 microsec); repeatedly uniform lifetime (not spread from 50 to 1000 microsec); a lifetime that does not decrease during normal device processing; a silicon wafer sheet that is flat and stays throughout normal device processing; uniform and reasonable mechanical strength; and, manufacture at low cost (less than $50/sq m).

  1. IgA production requires B cell interaction with subepithelial dendritic cells in Peyer's patches.

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    Reboldi, Andrea; Arnon, Tal I; Rodda, Lauren B; Atakilit, Amha; Sheppard, Dean; Cyster, Jason G

    2016-05-13

    Immunoglobulin A (IgA) induction primarily occurs in intestinal Peyer's patches (PPs). However, the cellular interactions necessary for IgA class switching are poorly defined. Here we show that in mice, activated B cells use the chemokine receptor CCR6 to access the subepithelial dome (SED) of PPs. There, B cells undergo prolonged interactions with SED dendritic cells (DCs). PP IgA class switching requires innate lymphoid cells, which promote lymphotoxin-β receptor (LTβR)-dependent maintenance of DCs. PP DCs augment IgA production by integrin αvβ8-mediated activation of transforming growth factor-β (TGFβ). In mice where B cells cannot access the SED, IgA responses against oral antigen and gut commensals are impaired. These studies establish the PP SED as a niche supporting DC-B cell interactions needed for TGFβ activation and induction of mucosal IgA responses. PMID:27174992

  2. Discovery of a Splicing Regulator Required for Cell Cycle Progression

    Energy Technology Data Exchange (ETDEWEB)

    Suvorova, Elena S.; Croken, Matthew; Kratzer, Stella; Ting, Li-Min; Conde de Felipe, Magnolia; Balu, Bharath; Markillie, Lye Meng; Weiss, Louis M.; Kim, Kami; White, Michael W.

    2013-02-01

    In the G1 phase of the cell division cycle, eukaryotic cells prepare many of the resources necessary for a new round of growth including renewal of the transcriptional and protein synthetic capacities and building the machinery for chromosome replication. The function of G1 has an early evolutionary origin and is preserved in single and multicellular organisms, although the regulatory mechanisms conducting G1 specific functions are only understood in a few model eukaryotes. Here we describe a new G1 mutant from an ancient family of apicomplexan protozoans. Toxoplasma gondii temperature-sensitive mutant 12-109C6 conditionally arrests in the G1 phase due to a single point mutation in a novel protein containing a single RNA-recognition-motif (TgRRM1). The resulting tyrosine to asparagine amino acid change in TgRRM1 causes severe temperature instability that generates an effective null phenotype for this protein when the mutant is shifted to the restrictive temperature. Orthologs of TgRRM1 are widely conserved in diverse eukaryote lineages, and the human counterpart (RBM42) can functionally replace the missing Toxoplasma factor. Transcriptome studies demonstrate that gene expression is downregulated in the mutant at the restrictive temperature due to a severe defect in splicing that affects both cell cycle and constitutively expressed mRNAs. The interaction of TgRRM1 with factors of the tri-SNP complex (U4/U6 & U5 snRNPs) indicate this factor may be required to assemble an active spliceosome. Thus, the TgRRM1 family of proteins is an unrecognized and evolutionarily conserved class of splicing regulators. This study demonstrates investigations into diverse unicellular eukaryotes, like the Apicomplexa, have the potential to yield new insights into important mechanisms conserved across modern eukaryotic kingdoms.

  3. Requirement of cell nucleus for Sindbis virus replication in cultured Aedes albopictus cells.

    Science.gov (United States)

    Erwin, C; Brown, D T

    1983-02-01

    The ability of Sindbis virus to grow in enucleated BHK-21 (vertebrate) and Aedes albopictus (invertebrate) cells was tested to determine the dependence of this virus upon nuclear function in these two phylogenetically unrelated hosts. Although both cell types could be demonstrated to produce viable cytoplasts (enucleated cells) which produced virus-specific antigen subsequent to infection. BHK cytoplasts produced a significant number of progeny virions, whereas mosquito cytoplasts did not. The production of vesicular stomatitis virus in mosquito cells was not significantly reduced by enucleation. That such a host function was not essential for vesicular stomatitis virus growth in insect cells is supported by the observation that the production of this virus by mosquito cells is not actinomycin D sensitive. This result agrees with a previously published report in which it was shown that Sindbis virus maturation in invertebrate cells is inhibited by actinomycin D, indicating a possible requirement for host cell nuclear function (Scheefers-Borchel et al., Virology, 110:292-301, 1981).

  4. ACE2 is required for daughter cell-specific G1 delay in Saccharomyces cerevisiae

    OpenAIRE

    Laabs, Tracy L.; Markwardt, David D.; Slattery, Matthew G.; Newcomb, Laura L.; Stillman, David J.; Heideman, Warren

    2003-01-01

    Saccharomyces cerevisiae cells reproduce by budding to yield a mother cell and a smaller daughter cell. Although both mother and daughter begin G1 simultaneously, the mother cell progresses through G1 more rapidly. Daughter cell G1 delay has long been thought to be due to a requirement for attaining a certain critical cell size before passing the commitment point in the cell cycle known as START. We present an alternative model in which the daughter cell-specific Ace2 ...

  5. LSD1 is Required for Hair Cell Regeneration in Zebrafish.

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    He, Yingzi; Tang, Dongmei; Cai, Chengfu; Chai, Renjie; Li, Huawei

    2016-05-01

    Lysine-specific demethylase 1 (LSD1/KDM1A) plays an important role in complex cellular processes such as differentiation, proliferation, apoptosis, and cell cycle progression. It has recently been demonstrated that during development, downregulation of LSD1 inhibits cell proliferation, modulates the expression of cell cycle regulators, and reduces hair cell formation in the zebrafish lateral line, which suggests that LSD1-mediated epigenetic regulation plays a key role in the development of hair cells. However, the role of LSD1 in hair cell regeneration after hair cell loss remains poorly understood. Here, we demonstrate the effect of LSD1 on hair cell regeneration following neomycin-induced hair cell loss. We show that the LSD1 inhibitor trans-2-phenylcyclopropylamine (2-PCPA) significantly decreases the regeneration of hair cells in zebrafish after neomycin damage. In addition, immunofluorescent staining demonstrates that 2-PCPA administration suppresses supporting cell proliferation and alters cell cycle progression. Finally, in situ hybridization shows that 2-PCPA significantly downregulates the expression of genes related to Wnt/β-catenin and Fgf activation. Altogether, our data suggest that downregulation of LSD1 significantly decreases hair cell regeneration after neomycin-induced hair cell loss through inactivation of the Wnt/β-catenin and Fgf signaling pathways. Thus, LSD1 plays a critical role in hair cell regeneration and might represent a novel biomarker and potential therapeutic approach for the treatment of hearing loss. PMID:26008620

  6. Large stationary fuel cell systems: Status and dynamic requirements

    Science.gov (United States)

    Bischoff, Manfred

    Molten carbonate fuel cell demonstrations to-date, have been able to show the highest fuel-to-electricity conversion efficiencies (>50%) of any stand-alone fuel cell type. The primary developer of this type of fuel cell in United States is Fuel Cell Energy Corporation (FCE), the developer and manufacturer of the Direct FuelCell ™ concept. FCE and MTU CFC Solutions in Germany, a licensee of FCE have demonstrated carbonate fuel cells from 10 kW to 2 MW of electrical output on a variety of fuels. IHI in Japan are also developing carbonate fuel cells for stationary power and have recently successfully demonstrated the technology in Kawagoe, Japan. In Italy, Ansaldo fuel cell have demonstrated a 100 kW carbonate fuel cell in Milan. In Korea, the Ministry of Commerce, Industry and Energy has committed to install 300 fuel cell units, sized 250 kW to 1 MW, for distributed power generation by 2012. Carbonate fuel cell technology is more fuel flexible than lower temperature fuel cell technologies and is well suited for on-site stationary CHP applications as well as to marine, military, and traction applications. The present paper gives an overview about the commercialisation efforts for the molten carbonate fuel cell technology.

  7. Metabolic profiling of hypoxic cells revealed a catabolic signature required for cell survival.

    Directory of Open Access Journals (Sweden)

    Christian Frezza

    Full Text Available Hypoxia is one of the features of poorly vascularised areas of solid tumours but cancer cells can survive in these areas despite the low oxygen tension. The adaptation to hypoxia requires both biochemical and genetic responses that culminate in a metabolic rearrangement to counter-balance the decrease in energy supply from mitochondrial respiration. The understanding of metabolic adaptations under hypoxia could reveal novel pathways that, if targeted, would lead to specific death of hypoxic regions. In this study, we developed biochemical and metabolomic analyses to assess the effects of hypoxia on cellular metabolism of HCT116 cancer cell line. We utilized an oxygen fluorescent probe in anaerobic cuvettes to study oxygen consumption rates under hypoxic conditions without the need to re-oxygenate the cells and demonstrated that hypoxic cells can maintain active, though diminished, oxidative phosphorylation even at 1% oxygen. These results were further supported by in situ microscopy analysis of mitochondrial NADH oxidation under hypoxia. We then used metabolomic methodologies, utilizing liquid chromatography-mass spectrometry (LC-MS, to determine the metabolic profile of hypoxic cells. This approach revealed the importance of synchronized and regulated catabolism as a mechanism of adaptation to bioenergetic stress. We then confirmed the presence of autophagy under hypoxic conditions and demonstrated that the inhibition of this catabolic process dramatically reduced the ATP levels in hypoxic cells and stimulated hypoxia-induced cell death. These results suggest that under hypoxia, autophagy is required to support ATP production, in addition to glycolysis, and that the inhibition of autophagy might be used to selectively target hypoxic regions of tumours, the most notoriously resistant areas of solid tumours.

  8. Sialic Acids on Varicella-Zoster Virus Glycoprotein B Are Required for Cell-Cell Fusion.

    Science.gov (United States)

    Suenaga, Tadahiro; Matsumoto, Maki; Arisawa, Fuminori; Kohyama, Masako; Hirayasu, Kouyuki; Mori, Yasuko; Arase, Hisashi

    2015-08-01

    Varicella-zoster virus (VZV) is a member of the human Herpesvirus family that causes varicella (chicken pox) and zoster (shingles). VZV latently infects sensory ganglia and is also responsible for encephalomyelitis. Myelin-associated glycoprotein (MAG), a member of the sialic acid (SA)-binding immunoglobulin-like lectin family, is mainly expressed in neural tissues. VZV glycoprotein B (gB) associates with MAG and mediates membrane fusion during VZV entry into host cells. The SA requirements of MAG when associating with its ligands vary depending on the specific ligand, but it is unclear whether the SAs on gB are involved in the association with MAG. In this study, we found that SAs on gB are essential for the association with MAG as well as for membrane fusion during VZV infection. MAG with a point mutation in the SA-binding site did not bind to gB and did not mediate cell-cell fusion or VZV entry. Cell-cell fusion and VZV entry mediated by the gB-MAG interaction were blocked by sialidase treatment. N-glycosylation or O-glycosylation inhibitors also inhibited the fusion and entry mediated by gB-MAG interaction. Furthermore, gB with mutations in N-glycosylation sites, i.e. asparagine residues 557 and 686, did not associate with MAG, and the cell-cell fusion efficiency was low. Fusion between the viral envelope and cellular membrane is essential for host cell entry by herpesviruses. Therefore, these results suggest that SAs on gB play important roles in MAG-mediated VZV infection. PMID:26105052

  9. Rab24 is required for normal cell division.

    Science.gov (United States)

    Militello, Rodrigo D; Munafó, Daniela B; Berón, Walter; López, Luis A; Monier, Solange; Goud, Bruno; Colombo, María I

    2013-05-01

    Rab24 is an atypical member of the Rab GTPase family whose distribution in interphase cells has been characterized; however, its function remains largely unknown. In this study, we have analyzed the distribution of Rab24 throughout cell division. We have observed that Rab24 was located at the mitotic spindle in metaphase, at the midbody during telophase and in the furrow during cytokinesis. We have also observed partial co-localization of Rab24 and tubulin and demonstrated its association to microtubules. Interestingly, more than 90% of transiently transfected HeLa cells with Rab24 presented abnormal nuclear connections (i.e., chromatin bridges). Furthermore, in CHO cells stably transfected with GFP-Rab24wt, we observed a large percentage of binucleated and multinucleated cells. In addition, these cells presented an extremely large size and multiple failures in mitosis, as aberrant spindle formation (metaphase), delayed chromosomes (telophase) and multiple cytokinesis. A marked increase in binucleated, multinucleated and multilobulated nucleus formation was observed in HeLa cells depleted of Rab24. We also present evidence that a fraction of Rab24 associates with microtubules. In addition, Rab24 knock down resulted in misalignment of chromosomes and abnormal spindle formation in metaphase leading to the appearance of delayed chromosomes during late telophase and failures in cytokinesis. Our findings suggest that an adequate level of Rab24 is necessary for normal cell division. In summary, Rab24 modulates several mitotic events, including chromosome segregation and cytokinesis, perhaps through the interaction with microtubules. PMID:23387408

  10. Renin secretion from permeabilized juxtaglomerular cells requires a permeant cation

    DEFF Research Database (Denmark)

    Jensen, B L; Ellekvist, Peter; Skøtt, O

    1999-01-01

    The cytosolic concentration of chloride correlates directly with renin secretion from renal juxtaglomerular granular (JG) cells. In the present study, the mechanism by which chloride stimulates renin release was investigated in a preparation of permeabilized rat glomeruli with attached JG cells. ...

  11. PEM fuel cell bipolar plate material requirements for transportation applications

    Energy Technology Data Exchange (ETDEWEB)

    Borup, R.L.; Stroh, K.R.; Vanderborgh, N.E. [Los Alamos National Lab., NM (United States)] [and others

    1996-04-01

    Cost effective bipolar plates are currently under development to help make proton exchange membrane (PEM) fuel cells commercially viable. Bipolar plates separate individual cells of the fuel cell stack, and thus must supply strength, be electrically conductive, provide for thermal control of the fuel stack, be a non-porous materials separating hydrogen and oxygen feed streams, be corrosion resistant, provide gas distribution for the feed streams and meet fuel stack cost targets. Candidate materials include conductive polymers and metal plates with corrosion resistant coatings. Possible metals include aluminium, titanium, iron/stainless steel and nickel.

  12. Folliculostellate Cells Are Required for Laminin Release from Gonadotrophs in Rat Anterior Pituitary

    International Nuclear Information System (INIS)

    The anterior pituitary gland is organized tissue comprising hormone-producing cells and folliculostellate (FS) cells. FS cells interconnect to form a meshwork, and their cytoplasmic processes are anchored by a basement membrane containing laminin. Recently, we developed a three-dimensional (3D) cell culture that reproduces this FS cell architecture. In this study of the novel function of FS cells, we used transgenic rats that express green fluorescent protein in FS cells for the 3D culture. Anterior pituitary cells were cultured with different proportions of FS cells (0%, 5%, 10%, and 20%). Anterior pituitary cells containing 5–20% FS cells formed round/oval cell aggregates, whereas amorphous cell aggregates were formed in the absence of FS cells. Interestingly, immunohistochemistry showed laminin-immunopositive cells instead of extracellular laminin deposition in FS cell-deficient cell aggregates. Double-immunostaining revealed that these laminin-immunopositive cells were gonadotrophs. Laminin mRNA expression did not differ in relation to the presence or absence of FS cells. When anterior pituitary cells with no FS cells were cultured with FS cell-conditioned medium, the proportion of laminin-immunopositive cells was lower than in control. These results suggest that a humoral factor from FS cells is required for laminin release from gonadotrophs

  13. Cell-autonomous requirement for TCF1 and LEF1 in the development of Natural Killer T cells.

    Science.gov (United States)

    Berga-Bolaños, Rosa; Zhu, Wandi S; Steinke, Farrah C; Xue, Hai-Hui; Sen, Jyoti Misra

    2015-12-01

    Natural killer T (NKT) cells develop from common CD4(+) CD8(+) thymocyte precursors. Transcriptional programs that regulate the development of NKT cells in the thymus development remain to be fully delineated. Here, we demonstrate a cell-intrinsic requirement for transcription factors TCF1 and LEF1 for the development of all subsets of NKT cells. Conditional deletion of TCF1 alone results in a substantial reduction in NKT cells. The remaining NKT cells are eliminated when TCF1 and LEF1 are both deleted. These data reveal an essential role for TCF1 and LEF1 in development of NKT cells.

  14. Design requirements for high-efficiency high concentration ratio space solar cells

    Science.gov (United States)

    Rauschenbach, H.; Patterson, R.

    1980-01-01

    A miniaturized Cassegrainian concentrator system concept was developed for low cost, multikilowatt space solar arrays. The system imposes some requirements on solar cells which are new and different from those imposed for conventional applications. The solar cells require a circular active area of approximately 4 mm in diameter. High reliability contacts are required on both front and back surfaces. The back area must be metallurgically bonded to a heat sink. The cell should be designed to achieve the highest practical efficiency at 100 AMO suns and at 80 C. The cell design must minimize losses due to nonuniform illumination intensity and nonnormal light incidence. The primary radiation concern is the omnidirectional proton environment.

  15. Mind bomb 1 is required for pancreatic ß-cell formation

    DEFF Research Database (Denmark)

    Horn, Signe; Kobberup, Sune; Jørgensen, Mette C;

    2012-01-01

    the insulin producing ß-cells. However, signals that regulate proximodistal (P-D) patterning and thus formation of ß-cell progenitors are unknown. Here we show that Mind bomb 1 (Mib1) is required for correct P-D patterning of the developing pancreas and ß-cell formation. We found that endoderm...

  16. Transparent electrode requirements for thin film solar cell modules

    KAUST Repository

    Rowell, Michael W.

    2011-01-01

    The transparent conductor (TC) layer in thin film solar cell modules has a significant impact on the power conversion efficiency. Reflection, absorption, resistive losses and lost active area either from the scribed interconnect region in monolithically integrated modules or from the shadow losses of a metal grid in standard modules typically reduce the efficiency by 10-25%. Here, we perform calculations to show that a competitive TC must have a transparency of at least 90% at a sheet resistance of less than 10 Ω/sq (conductivity/absorptivity ≥ 1 Ω -1) for monolithically integrated modules. For standard modules, losses are much lower and the performance of alternative lower cost TC materials may already be sufficient to replace conducting oxides in this geometry. © 2011 The Royal Society of Chemistry.

  17. Programmed cell death activated by Rose Bengal in Arabidopsis thaliana cell suspension cultures requires functional chloroplasts.

    Science.gov (United States)

    Gutiérrez, Jorge; González-Pérez, Sergio; García-García, Francisco; Daly, Cara T; Lorenzo, Oscar; Revuelta, José L; McCabe, Paul F; Arellano, Juan B

    2014-07-01

    Light-grown Arabidopsis thaliana cell suspension culture (ACSC) were subjected to mild photooxidative damage with Rose Bengal (RB) with the aim of gaining a better understanding of singlet oxygen-mediated defence responses in plants. Additionally, ACSC were treated with H2O2 at concentrations that induced comparable levels of protein oxidation damage. Under low to medium light conditions, both RB and H2O2 treatments activated transcriptional defence responses and inhibited photosynthetic activity, but they differed in that programmed cell death (PCD) was only observed in cells treated with RB. When dark-grown ACSC were subjected to RB in the light, PCD was suppressed, indicating that the singlet oxygen-mediated signalling pathway in ACSC requires functional chloroplasts. Analysis of up-regulated transcripts in light-grown ACSC, treated with RB in the light, showed that both singlet oxygen-responsive transcripts and transcripts with a key role in hormone-activated PCD (i.e. ethylene and jasmonic acid) were present. A co-regulation analysis proved that ACSC treated with RB exhibited higher correlation with the conditional fluorescence (flu) mutant than with other singlet oxygen-producing mutants or wild-type plants subjected to high light. However, there was no evidence for the up-regulation of EDS1, suggesting that activation of PCD was not associated with the EXECUTER- and EDS1-dependent signalling pathway described in the flu mutant. Indigo Carmine and Methylene Violet, two photosensitizers unable to enter chloroplasts, did not activate transcriptional defence responses in ACSC; however, whether this was due to their location or to their inherently low singlet oxygen quantum efficiencies was not determined.

  18. Continuous requirement for the T cell receptor for regulatory T cell function

    OpenAIRE

    Levine, Andrew G; Arvey, Aaron; Jin, Wei; Rudensky, Alexander Y.

    2014-01-01

    Foxp3+ regulatory T cells (Treg cells) maintain immunological tolerance and their deficiency results in fatal multi-organ autoimmunity. Although heightened T cell receptor (TCR) signaling is critical for the differentiation of Treg cells, the role of TCR signaling in Treg cell function remains largely unknown. Here we demonstrate inducible ablation of the TCR results in Treg cell dysfunction which cannot be attributed to impaired Foxp3 expression, decreased expression of Treg cell signature g...

  19. Fuel cell systems for passenger cars - opportunities and requirements

    Energy Technology Data Exchange (ETDEWEB)

    Tachtler, J. [BMW, Munich (Germany); Bourne, C. [Rover Group, Coventry (United Kingdom)

    1996-12-31

    From the point of view of energy density, handling and economy, present-day motor fuels are superior to all known alternatives. The internal combustion engine powered by them satisfies the requirements of customers to an excellent degree. The search for alternatives can therefore only be justified if emissions can be avoided totally and non-fossil primary energy sources can be used or at least partially our dependence on mineral oil can be reduced. What was long suspected has been increasingly confirmed, not least by developments at BMW: electricity (stored in batteries) and hydrogen offer the best prerequisites for achieving these goals in the long term. These forms of energy can be produced in sufficient quantities and with relatively little effect on the environment. They promise to produce an absolute minimum of pollutants when used in vehicles. Natural gas, which is very similar to hydrogen, and hybrid systems, that would compensate for battery risks, could perform a valuable function in the transitional phase.

  20. Memory CD4+ T cells are required for optimal NK cell effector functions against the opportunistic fungal pathogen Pneumocystis murina.

    Science.gov (United States)

    Kelly, Michelle N; Zheng, Mingquan; Ruan, Sanbao; Kolls, Jay; D'Souza, Alain; Shellito, Judd E

    2013-01-01

    Little is known about the role of NK cells or their interplay with other immune cells during opportunistic infections. Using our murine model of Pneumocystis pneumonia, we found that loss of NK cells during immunosuppression results in substantial Pneumocystis lung burden. During early infection of C57B/6 CD4(+) T cell-depleted mice, there were significantly fewer NK cells in the lung tissue compared with CD4(+) T cell-intact animals, and the NK cells present demonstrated decreased upregulation of the activation marker NKp46 and production of the effector cytokine, IFN-γ. Furthermore, coincubation studies revealed a significant increase in fungal killing when NK cells were combined with CD4(+) T cells compared with either cell alone, which was coincident with a significant increase in perforin production by NK cells. Finally, however, we found through adoptive transfer that memory CD4(+) T cells are required for significant NK cell upregulation of the activation marker NK group 2D and production of IFN-γ, granzyme B, and perforin during Pneumocystis infection. To the best of our knowledge, this study is the first to demonstrate a role for NK cells in immunity to Pneumocystis pneumonia, as well as to establish a functional relationship between CD4(+) T cells and NK cells in the host response to an opportunistic fungal pathogen.

  1. Steroids are required for epidermal cell fate establishment in Arabidopsis roots.

    Science.gov (United States)

    Kuppusamy, Kavitha T; Chen, Andrew Y; Nemhauser, Jennifer L

    2009-05-12

    The simple structure of Arabidopsis roots provides an excellent model system to study epidermal cell fate specification. Epidermal cells in contact with 2 underlying cortical cells differentiate into hair cells (H cells; trichoblasts), whereas cells that contact only a single cortical cell differentiate into mature hairless cells (N cells; atrichoblasts). This position-dependent patterning, in combination with the constrained orientation of cell divisions, results in hair and nonhair cell files running longitudinally along the root epidermis. Here, we present strong evidence that steroid hormones called brassinosteroids (BRs) are required to maintain position-dependent fate specification in roots. We show that BRs are required for normal expression levels and patterns of WEREWOLF (WER) and GLABRA2 (GL2), master regulators of epidermal patterning. Loss of BR signaling results in loss of hair cells in H positions, likely as a consequence of reduced expression of CAPRICE (CPC), a direct downstream target of WER. Our observations demonstrate that in addition to their well-known role in cell expansion, BRs play an essential role in directing cell fate.

  2. Langerhans cells are not required for epidermal V gamma 3 T cell homeostasis and function

    NARCIS (Netherlands)

    Taveirne, Sylvie; De Colvenaer, Veerle; Van Den Broeck, Tina; Van Ammel, Els; Bennett, Clare L.; Taghon, Tom; Vandekerckhove, Bart; Plum, Jean; Clausen, Bjorn E.; Kaplan, Daniel H.; Leclercq, Georges

    2011-01-01

    This study tested the hypothesis that V gamma 3 TCR-bearing T cells are influenced by LCs. V gamma 3 T cells and LCs are located in the epidermis of mice. V gamma 3 T cells represent the main T cell population in the skin epithelium and play a crucial role in maintaining the skin integrity, whereas

  3. TORC1 is required to balance cell proliferation and cell death in planarians

    OpenAIRE

    Tu, Kimberly C; Pearson, Bret J.; Alvarado, Alejandro Sánchez

    2012-01-01

    Multicellular organisms are equipped with cellular mechanisms that enable them to replace differentiated cells lost to normal physiological turnover, injury, and for some such as planarians, even amputation. This process of tissue homeostasis is generally mediated by adult stem cells (ASCs), tissue-specific stem cells responsible for maintaining anatomical form and function. To do so, ASCs must modulate the balance between cell proliferation, i.e. in response to nutrients, and that of cell de...

  4. Prkci is required for a non-autonomous signal that coordinates cell polarity during cavitation.

    Science.gov (United States)

    Mah, In Kyoung; Soloff, Rachel; Izuhara, Audrey K; Lakeland, Daniel L; Wang, Charles; Mariani, Francesca V

    2016-08-01

    Polarized epithelia define boundaries, spaces, and cavities within organisms. Cavitation, a process by which multicellular hollow balls or tubes are produced, is typically associated with the formation of organized epithelia. In order for these epithelial layers to form, cells must ultimately establish a distinct apical-basal polarity. Atypical PKCs have been proposed to be required for apical-basal polarity in diverse species. Here we show that while cells null for the Prkci isozyme exhibit some polarity characteristics, they fail to properly segregate apical-basal proteins, form a coordinated ectodermal epithelium, or participate in normal cavitation. A failure to cavitate could be due to an overgrowth of interior cells or to an inability of interior cells to die. Null cells however, do not have a marked change in proliferation rate and are still capable of undergoing cell death, suggesting that alterations in these processes are not the predominant cause of the failed cavitation. Overexpression of BMP4 or EZRIN can partially rescue the phenotype possibly by promoting cell death, polarity, and differentiation. However, neither is sufficient to provide the required cues to generate a polarized epithelium and fully rescue cavitation. Interestingly, when wildtype and Prkci(-/-) ES cells are mixed together, a polarized ectodermal epithelium forms and cavitation is rescued, likely due to the ability of wildtype cells to produce non-autonomous polarity cues. We conclude that Prkci is not required for cells to respond to these cues, though it is required to produce them. Together these findings indicate that environmental cues can facilitate the formation of polarized epithelia and that cavitation requires the proper coordination of multiple basic cellular processes including proliferation, differentiation, cell death, and apical-basal polarization. PMID:27312576

  5. The polycomb group protein Suz12 is required for embryonic stem cell differentiation

    DEFF Research Database (Denmark)

    Pasini, Diego; Bracken, Adrian P; Hansen, Jacob Bo Højberg;

    2007-01-01

    results in early lethality of mouse embryos. Here, we demonstrate that Suz12(-/-) mouse embryonic stem (ES) cells can be established and expanded in tissue culture. The Suz12(-/-) ES cells are characterized by global loss of H3K27 trimethylation (H3K27me3) and higher expression levels of differentiation......-specific genes. Moreover, Suz12(-/-) ES cells are impaired in proper differentiation, resulting in a lack of repression of ES cell markers as well as activation of differentiation-specific genes. Finally, we demonstrate that the PcGs are actively recruited to several genes during ES cell differentiation, which...... despite an increase in H3K27me3 levels is not always sufficient to prevent transcriptional activation. In summary, we demonstrate that Suz12 is required for the establishment of specific expression programs required for ES cell differentiation. Furthermore, we provide evidence that PcGs have different...

  6. Human CD4+ T cells require exogenous cystine for glutathione and DNA synthesis

    DEFF Research Database (Denmark)

    Levring, Trine B; Kongsbak-Wismann, Martin; Rode, Anna Kathrine Obelitz;

    2015-01-01

    aim of this study was to elucidate why activated human T cells require exogenous Cys2 in order to proliferate. We activated purified naïve human CD4+ T cells and found that glutathione (GSH) levels and DNA synthesis were dependent on Cys2 and increased in parallel with increasing concentrations of Cys...

  7. Change in Cell Shape Is Required for Matrix Metalloproteinase-Induced Epithelial-Mesenchymal Transition of Mammary Epithelial Cells

    Science.gov (United States)

    Nelson, Celeste M.; Khauv, Davitte; Bissell, Mina J.; Radisky, Derek C.

    2010-01-01

    Cell morphology dictates response to a wide variety of stimuli, controlling cell metabolism, differentiation, proliferation, and death. Epithelial-mesenchymal transition (EMT) is a developmental process in which epithelial cells acquire migratory characteristics, and in the process convert from a “cuboidal” epithelial structure into an elongated mesenchymal shape. We had shown previously that matrix metalloproteinase-3 (MMP3) can stimulate EMT of cultured mouse mammary epithelial cells through a process that involves increased expression of Rac1b, a protein that stimulates alterations in cytoskeletal structure. We show here that cells treated with MMP-3 or induced to express Rac1b spread to cover a larger surface, and that this induction of cell spreading is a requirement of MMP-3/Rac1b-induced EMT. We find that limiting cell spreading, either by increasing cell density or by culturing cells on precisely defined micropatterned substrata, blocks expression of characteristic markers of EMT in cells treated with MMP-3. These effects are not caused by general disruptions in cell signaling pathways, as TGF-β-induced EMT is not affected by similar limitations on cell spreading. Our data reveal a previously unanticipated cell shape-dependent mechanism that controls this key phenotypic alteration and provide insight into the distinct mechanisms activated by different EMT-inducing agents. PMID:18506791

  8. Change in cell shape is required for matrix metalloproteinase-induced epithelial-mesenchymal transition of mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Nelson, Celeste M.; Khauv, Davitte; Bissell, Mina J.; Radisky, Derek C.

    2008-06-26

    Cell morphology dictates response to a wide variety of stimuli, controlling cell metabolism, differentiation, proliferation, and death. Epithelial-mesenchymal transition (EMT) is a developmental process in which epithelial cells acquire migratory characteristics, and in the process convert from a 'cuboidal' epithelial structure into an elongated mesenchymal shape. We had shown previously that matrix metalloproteinase-3 (MMP3) can stimulate EMT of cultured mouse mammary epithelial cells through a process that involves increased expression of Rac1b, a protein that stimulates alterations in cytoskeletal structure. We show here that cells treated with MMP-3 or induced to express Rac1b spread to cover a larger surface, and that this induction of cell spreading is a requirement of MMP-3/Rac1b-induced EMT. We find that limiting cell spreading, either by increasing cell density or by culturing cells on precisely defined micropatterned substrata, blocks expression of characteristic markers of EMT in cells treated with MMP-3. These effects are not caused by general disruptions in cell signaling pathways, as TGF-{beta}-induced EMT is not affected by similar limitations on cell spreading. Our data reveal a previously unanticipated cell shape-dependent mechanism that controls this key phenotypic alteration and provide insight into the distinct mechanisms activated by different EMT-inducing agents.

  9. c-di-GMP induction of Dictyostelium cell death requires the polyketide DIF-1.

    Science.gov (United States)

    Song, Yu; Luciani, Marie-Françoise; Giusti, Corinne; Golstein, Pierre

    2015-02-15

    Cell death in the model organism Dictyostelium, as studied in monolayers in vitro, can be induced by the polyketide DIF-1 or by the cyclical dinucleotide c-di-GMP. c-di-GMP, a universal bacterial second messenger, can trigger innate immunity in bacterially infected animal cells and is involved in developmental cell death in Dictyostelium. We show here that c-di-GMP was not sufficient to induce cell death in Dictyostelium cell monolayers. Unexpectedly, it also required the DIF-1 polyketide. The latter could be exogenous, as revealed by a telling synergy between c-di-GMP and DIF-1. The required DIF-1 polyketide could also be endogenous, as shown by the inability of c-di-GMP to induce cell death in Dictyostelium HMX44A cells and DH1 cells upon pharmacological or genetic inhibition of DIF-1 biosynthesis. In these cases, c-di-GMP-induced cell death was rescued by complementation with exogenous DIF-1. Taken together, these results demonstrated that c-di-GMP could trigger cell death in Dictyostelium only in the presence of the DIF-1 polyketide or its metabolites. This identified another element of control to this cell death and perhaps also to c-di-GMP effects in other situations and organisms.

  10. Zebrafish neural tube morphogenesis requires Scribble-dependent oriented cell divisions.

    Science.gov (United States)

    Žigman, Mihaela; Trinh, Le A; Fraser, Scott E; Moens, Cecilia B

    2011-01-11

    How control of subcellular events in single cells determines morphogenesis on the scale of the tissue is largely unresolved. The stereotyped cross-midline mitoses of progenitors in the zebrafish neural keel provide a unique experimental paradigm for defining the role and control of single-cell orientation for tissue-level morphogenesis in vivo. We show here that the coordinated orientation of individual progenitor cell division in the neural keel is the cellular determinant required for morphogenesis into a neural tube epithelium with a single straight lumen. We find that Scribble is required for oriented cell division and that its function in this process is independent of canonical apicobasal and planar polarity pathways. We identify a role for Scribble in controlling clustering of α-catenin foci in dividing progenitors. Loss of either Scrib or N-cadherin results in abnormally oriented mitoses, reduced cross-midline cell divisions, and similar neural tube defects. We propose that Scribble-dependent nascent cell-cell adhesion clusters between neuroepithelial progenitors contribute to define orientation of their cell division. Finally, our data demonstrate that while oriented mitoses of individual cells determine neural tube architecture, the tissue can in turn feed back on its constituent cells to define their polarization and cell division orientation to ensure robust tissue morphogenesis.

  11. DMRT1 Is Required for Mouse Spermatogonial Stem Cell Maintenance and Replenishment.

    Science.gov (United States)

    Zhang, Teng; Oatley, Jon; Bardwell, Vivian J; Zarkower, David

    2016-09-01

    Male mammals produce sperm for most of postnatal life and therefore require a robust germ line stem cell system, with precise balance between self-renewal and differentiation. Prior work established doublesex- and mab-3-related transcription factor 1 (Dmrt1) as a conserved transcriptional regulator of male sexual differentiation. Here we investigate the role of Dmrt1 in mouse spermatogonial stem cell (SSC) homeostasis. We find that Dmrt1 maintains SSCs during steady state spermatogenesis, where it regulates expression of Plzf, another transcription factor required for SSC maintenance. We also find that Dmrt1 is required for recovery of spermatogenesis after germ cell depletion. Committed progenitor cells expressing Ngn3 normally do not contribute to SSCs marked by the Id4-Gfp transgene, but do so when spermatogonia are chemically depleted using busulfan. Removal of Dmrt1 from Ngn3-positive germ cells blocks the replenishment of Id4-GFP-positive SSCs and recovery of spermatogenesis after busulfan treatment. Our data therefore reveal that Dmrt1 supports SSC maintenance in two ways: allowing SSCs to remain in the stem cell pool under normal conditions; and enabling progenitor cells to help restore the stem cell pool after germ cell depletion. PMID:27583450

  12. Ki-67 is required for maintenance of cancer stem cells but not cell proliferation

    Science.gov (United States)

    Cidado, Justin; Wong, Hong Yuen; Rosen, D. Marc; Cimino-Mathews, Ashley; Garay, Joseph P.; Fessler, Abigail G.; Rasheed, Zeshaan A.; Hicks, Jessica; Cochran, Rory L.; Croessmann, Sarah; Zabransky, Daniel J.; Mohseni, Morassa; Beaver, Julia A.; Chu, David; Cravero, Karen; Christenson, Eric S.; Medford, Arielle; Mattox, Austin; De Marzo, Angelo M.; Argani, Pedram; Chawla, Ajay; Hurley, Paula J.; Lauring, Josh; Park, Ben Ho

    2016-01-01

    Ki-67 expression is correlated with cell proliferation and is a prognostic marker for various cancers; however, its function is unknown. Here we demonstrate that genetic disruption of Ki-67 in human epithelial breast and colon cancer cells depletes the cancer stem cell niche. Ki-67 null cells had a proliferative disadvantage compared to wildtype controls in colony formation assays and displayed increased sensitivity to various chemotherapies. Ki-67 null cancer cells showed decreased and delayed tumor formation in xenograft assays, which was associated with a reduction in cancer stem cell markers. Immunohistochemical analyses of human breast cancers revealed that Ki-67 expression is maintained at equivalent or greater levels in metastatic sites of disease compared to matched primary tumors, suggesting that maintenance of Ki-67 expression is associated with metastatic/clonogenic potential. These results elucidate Ki-67's role in maintaining the cancer stem cell niche, which has potential diagnostic and therapeutic implications for human malignancies. PMID:26823390

  13. Platelet-derived stromal cell-derived factor-1 is required for the transformation of circulating monocytes into multipotential cells.

    Directory of Open Access Journals (Sweden)

    Noriyuki Seta

    Full Text Available BACKGROUND: We previously described a primitive cell population derived from human circulating CD14(+ monocytes, named monocyte-derived multipotential cells (MOMCs, which are capable of differentiating into mesenchymal and endothelial lineages. To generate MOMCs in vitro, monocytes are required to bind to fibronectin and be exposed to soluble factor(s derived from circulating CD14(- cells. The present study was conducted to identify factors that induce MOMC differentiation. METHODS: We cultured CD14(+ monocytes on fibronectin in the presence or absence of platelets, CD14(- peripheral blood mononuclear cells, platelet-conditioned medium, or candidate MOMC differentiation factors. The transformation of monocytes into MOMCs was assessed by the presence of spindle-shaped adherent cells, CD34 expression, and the potential to differentiate in vitro into mesenchymal and endothelial lineages. RESULTS: The presence of platelets or platelet-conditioned medium was required to generate MOMCs from monocytes. A screening of candidate platelet-derived soluble factors identified stromal cell-derived factor (SDF-1 as a requirement for generating MOMCs. Blocking an interaction between SDF-1 and its receptor CXCR4 inhibited MOMC generation, further confirming SDF-1's critical role in this process. Finally, circulating MOMC precursors were found to reside in the CD14(+CXCR4(high cell population. CONCLUSION: The interaction of SDF-1 with CXCR4 is essential for the transformation of circulating monocytes into MOMCs.

  14. Exocyst-Dependent Membrane Addition Is Required for Anaphase Cell Elongation and Cytokinesis in Drosophila.

    Directory of Open Access Journals (Sweden)

    Maria Grazia Giansanti

    2015-11-01

    Full Text Available Mitotic and cytokinetic processes harness cell machinery to drive chromosomal segregation and the physical separation of dividing cells. Here, we investigate the functional requirements for exocyst complex function during cell division in vivo, and demonstrate a common mechanism that directs anaphase cell elongation and cleavage furrow progression during cell division. We show that onion rings (onr and funnel cakes (fun encode the Drosophila homologs of the Exo84 and Sec8 exocyst subunits, respectively. In onr and fun mutant cells, contractile ring proteins are recruited to the equatorial region of dividing spermatocytes. However, cytokinesis is disrupted early in furrow ingression, leading to cytokinesis failure. We use high temporal and spatial resolution confocal imaging with automated computational analysis to quantitatively compare wild-type versus onr and fun mutant cells. These results demonstrate that anaphase cell elongation is grossly disrupted in cells that are compromised in exocyst complex function. Additionally, we observe that the increase in cell surface area in wild type peaks a few minutes into cytokinesis, and that onr and fun mutant cells have a greatly reduced rate of surface area growth specifically during cell division. Analysis by transmission electron microscopy reveals a massive build-up of cytoplasmic astral membrane and loss of normal Golgi architecture in onr and fun spermatocytes, suggesting that exocyst complex is required for proper vesicular trafficking through these compartments. Moreover, recruitment of the small GTPase Rab11 and the PITP Giotto to the cleavage site depends on wild-type function of the exocyst subunits Exo84 and Sec8. Finally, we show that the exocyst subunit Sec5 coimmunoprecipitates with Rab11. Our results are consistent with the exocyst complex mediating an essential, coordinated increase in cell surface area that potentiates anaphase cell elongation and cleavage furrow ingression.

  15. Exocyst-Dependent Membrane Addition Is Required for Anaphase Cell Elongation and Cytokinesis in Drosophila.

    Science.gov (United States)

    Giansanti, Maria Grazia; Vanderleest, Timothy E; Jewett, Cayla E; Sechi, Stefano; Frappaolo, Anna; Fabian, Lacramioara; Robinett, Carmen C; Brill, Julie A; Loerke, Dinah; Fuller, Margaret T; Blankenship, J Todd

    2015-11-01

    Mitotic and cytokinetic processes harness cell machinery to drive chromosomal segregation and the physical separation of dividing cells. Here, we investigate the functional requirements for exocyst complex function during cell division in vivo, and demonstrate a common mechanism that directs anaphase cell elongation and cleavage furrow progression during cell division. We show that onion rings (onr) and funnel cakes (fun) encode the Drosophila homologs of the Exo84 and Sec8 exocyst subunits, respectively. In onr and fun mutant cells, contractile ring proteins are recruited to the equatorial region of dividing spermatocytes. However, cytokinesis is disrupted early in furrow ingression, leading to cytokinesis failure. We use high temporal and spatial resolution confocal imaging with automated computational analysis to quantitatively compare wild-type versus onr and fun mutant cells. These results demonstrate that anaphase cell elongation is grossly disrupted in cells that are compromised in exocyst complex function. Additionally, we observe that the increase in cell surface area in wild type peaks a few minutes into cytokinesis, and that onr and fun mutant cells have a greatly reduced rate of surface area growth specifically during cell division. Analysis by transmission electron microscopy reveals a massive build-up of cytoplasmic astral membrane and loss of normal Golgi architecture in onr and fun spermatocytes, suggesting that exocyst complex is required for proper vesicular trafficking through these compartments. Moreover, recruitment of the small GTPase Rab11 and the PITP Giotto to the cleavage site depends on wild-type function of the exocyst subunits Exo84 and Sec8. Finally, we show that the exocyst subunit Sec5 coimmunoprecipitates with Rab11. Our results are consistent with the exocyst complex mediating an essential, coordinated increase in cell surface area that potentiates anaphase cell elongation and cleavage furrow ingression. PMID:26528720

  16. Latent KSHV Infected Endothelial Cells Are Glutamine Addicted and Require Glutaminolysis for Survival.

    Directory of Open Access Journals (Sweden)

    Erica L Sanchez

    2015-07-01

    Full Text Available Kaposi's Sarcoma-associated Herpesvirus (KSHV is the etiologic agent of Kaposi's Sarcoma (KS. KSHV establishes a predominantly latent infection in the main KS tumor cell type, the spindle cell, which is of endothelial cell origin. KSHV requires the induction of multiple metabolic pathways, including glycolysis and fatty acid synthesis, for the survival of latently infected endothelial cells. Here we demonstrate that latent KSHV infection leads to increased levels of intracellular glutamine and enhanced glutamine uptake. Depletion of glutamine from the culture media leads to a significant increase in apoptotic cell death in latently infected endothelial cells, but not in their mock-infected counterparts. In cancer cells, glutamine is often required for glutaminolysis to provide intermediates for the tri-carboxylic acid (TCA cycle and support for the production of biosynthetic and bioenergetic precursors. In the absence of glutamine, the TCA cycle intermediates alpha-ketoglutarate (αKG and pyruvate prevent the death of latently infected cells. Targeted drug inhibition of glutaminolysis also induces increased cell death in latently infected cells. KSHV infection of endothelial cells induces protein expression of the glutamine transporter, SLC1A5. Chemical inhibition of SLC1A5, or knockdown by siRNA, leads to similar cell death rates as glutamine deprivation and, similarly, can be rescued by αKG. KSHV also induces expression of the heterodimeric transcription factors c-Myc-Max and related heterodimer MondoA-Mlx. Knockdown of MondoA inhibits expression of both Mlx and SLC1A5 and induces a significant increase in cell death of only cells latently infected with KSHV, again, fully rescued by the supplementation of αKG. Therefore, during latent infection of endothelial cells, KSHV activates and requires the Myc/MondoA-network to upregulate the glutamine transporter, SLC1A5, leading to increased glutamine uptake for glutaminolysis. These findings

  17. Modification of T cell responses by stem cell mobilization requires direct signaling of the T cell by G-CSF and IL-10

    DEFF Research Database (Denmark)

    MacDonald, Kelli P.A.; Le Texier, Laetitia; Zhang, Ping;

    2014-01-01

    The majority of allogeneic stem cell transplants are currently undertaken using G-CSF mobilized peripheral blood stem cells. G-CSF has diverse biological effects on a broad range of cells and IL-10 is a key regulator of many of these effects. Using mixed radiation chimeras in which......, stem cell mobilization with the CXCR4 antagonist AMD3100 did not alter the donor T cell's ability to induce acute GVHD. These studies provide an explanation for the effects of G-CSF on T cell function and demonstrate that IL-10 is required to license regulatory function but T cell production of IL-10...... is not itself required for the attenuation GVHD. Although administration of CXCR4 antagonists is an efficient means of stem cell mobilization, this fails to evoke the immunomodulatory effects seen during G-CSF mobilization. These data provide a compelling rationale for considering the immunological benefits...

  18. Programmed cell cycle arrest is required for infection of corn plants by the fungus Ustilago maydis.

    Science.gov (United States)

    Castanheira, Sónia; Mielnichuk, Natalia; Pérez-Martín, José

    2014-12-01

    Ustilago maydis is a plant pathogen that requires a specific structure called infective filament to penetrate the plant tissue. Although able to grow, this filament is cell cycle arrested on the plant surface. This cell cycle arrest is released once the filament penetrates the plant tissue. The reasons and mechanisms for this cell cycle arrest are unknown. Here, we have tried to address these questions. We reached three conclusions from our studies. First, the observed cell cycle arrest is the result of the cooperation of at least two distinct mechanisms: one involving the activation of the DNA damage response (DDR) cascade; and the other relying on the transcriptional downregulation of Hsl1, a kinase that modulates the G2/M transition. Second, a sustained cell cycle arrest during the infective filament step is necessary for the virulence in U. maydis, as a strain unable to arrest the cell cycle was severely impaired in its ability to infect corn plants. Third, production of the appressorium, a structure required for plant penetration, is incompatible with an active cell cycle. The inability to infect plants by strains defective in cell cycle arrest seems to be caused by their failure to induce the appressorium formation process. In summary, our findings uncover genetic circuits to arrest the cell cycle during the growth of this fungus on the plant surface, thus allowing the penetration into plant tissue.

  19. ABCC4 is required for cell proliferation and tumorigenesis in non-small cell lung cancer

    Directory of Open Access Journals (Sweden)

    Zhao X

    2014-02-01

    Full Text Available Xiaoting Zhao, Yinan Guo, Wentao Yue, Lina Zhang, Meng Gu, Yue Wang Department of Cellular and Molecular Biology, Beijing TB and Thoracic Tumor Research Institute/Beijing Chest Hospital, Capital Medical University, Beijing, People's Republic of China Background: Multidrug resistance protein 4 (MRP4, also known as ATP-cassette binding protein 4 (ABCC4, is a member of the MRP/ABCC subfamily of ATP-binding cassette transporters, which are capable of pumping a wide variety of drugs out of the cell. However, little is known about the function of ABCC4 in the proliferation of lung cancer cells. Methods: ABCC4 mRNA and protein levels in lung cancer cell lines were measured by real-time polymerase chain reaction and Western blot, respectively. A lentivirus-mediated RNA interference technique was used to inhibit ABCC4 mRNA expression in A549 and 801D cells. The function of ABCC4 in cell growth was investigated by MTS and colony formation assays. The role of ABCC4 in cell cycle progression was evaluated by flow cytometry and Western blot analysis. ABCC4 mRNA levels in 30 pairs of tumors and corresponding matched adjacent normal tissues from non-small cell lung cancer patients were detected by real-time polymerase chain reaction. Results: ABCC4 was highly expressed in lung cancer cell lines. ABCC4 expression was markedly downregulated in A549 and 801D cells using the RNA interference technique. Suppression of ABCC4 expression inhibited cell growth. The percentage of cells in G1 phase was increased when ABCC4 expression was suppressed. Phosphorylation of retinoblastoma protein was weakened, originating in the downregulation of ABCC4. ABCC4 mRNA was highly expressed in lung cancer tissue and lung cancer cell lines. Conclusion: ABCC4 may play an important role in the control of A549 and 801D cell growth. ABCC4 is a potential target for lung cancer therapy. Keywords: ABCC4, cell proliferation, lung cancer, cell cycle

  20. Tbx16 and Msgn1 are required to establish directional cell migration of zebrafish mesodermal progenitors.

    Science.gov (United States)

    Manning, Alyssa J; Kimelman, David

    2015-10-15

    The epithelial to mesenchymal transition (EMT) is an essential process that occurs repeatedly during embryogenesis whereby stably adherent cells convert to an actively migrating state. While much is known about the factors and events that initiate the EMT, the steps that cells undergo to become directionally migratory are far less well understood. Zebrafish embryos lacking the transcription factors Tbx16/Spadetail and Mesogenin1 (Msgn1) are a valuable system for investigating the EMT. Mesodermal cells in these embryos are unable to perform the EMT necessary to leave the most posterior end of the body (the tailbud) and join the pre-somitic mesoderm, a process that is conserved in all vertebrates. It has previously been very difficult to study this EMT in vertebrates because of the multiple cell types in the tailbud and the morphogenetic changes the whole embryo undergoes. Here, we describe a novel tissue explant system for imaging the mesodermal cell EMT in vivo that allows us to investigate the requirements for cells to acquire migratory properties during the EMT with high spatio-temporal resolution. This method revealed that, despite the inability of tbx16;msgn1-deficient cells to leave the tailbud, actin-based protrusions form surprisingly normally in these cells and they become highly motile. However, tbx16;msgn1-deficient cells have specific cell-autonomous defects in the persistence and anterior direction of migration because the lamellipodia they form are not productive in driving anteriorward migration. Additionally, we show that mesoderm morphogenesis and differentiation are separable and that there is a migratory cue that directs mesodermal cell migration that is independent of Tbx16 and Msgn1. This work defines changes that cells undergo as they complete the EMT and provides new insight into the mechanisms required in vivo for cells to become mesenchymal.

  1. JNK controls the onset of mitosis in planarian stem cells and triggers apoptotic cell death required for regeneration and remodeling.

    Directory of Open Access Journals (Sweden)

    María Almuedo-Castillo

    2014-06-01

    Full Text Available Regeneration of lost tissues depends on the precise interpretation of molecular signals that control and coordinate the onset of proliferation, cellular differentiation and cell death. However, the nature of those molecular signals and the mechanisms that integrate the cellular responses remain largely unknown. The planarian flatworm is a unique model in which regeneration and tissue renewal can be comprehensively studied in vivo. The presence of a population of adult pluripotent stem cells combined with the ability to decode signaling after wounding enable planarians to regenerate a complete, correctly proportioned animal within a few days after any kind of amputation, and to adapt their size to nutritional changes without compromising functionality. Here, we demonstrate that the stress-activated c-jun-NH2-kinase (JNK links wound-induced apoptosis to the stem cell response during planarian regeneration. We show that JNK modulates the expression of wound-related genes, triggers apoptosis and attenuates the onset of mitosis in stem cells specifically after tissue loss. Furthermore, in pre-existing body regions, JNK activity is required to establish a positive balance between cell death and stem cell proliferation to enable tissue renewal, remodeling and the maintenance of proportionality. During homeostatic degrowth, JNK RNAi blocks apoptosis, resulting in impaired organ remodeling and rescaling. Our findings indicate that JNK-dependent apoptotic cell death is crucial to coordinate tissue renewal and remodeling required to regenerate and to maintain a correctly proportioned animal. Hence, JNK might act as a hub, translating wound signals into apoptotic cell death, controlled stem cell proliferation and differentiation, all of which are required to coordinate regeneration and tissue renewal.

  2. p73 is required for ependymal cell maturation and neurogenic SVZ cytoarchitecture.

    Science.gov (United States)

    Gonzalez-Cano, L; Fuertes-Alvarez, S; Robledinos-Anton, N; Bizy, A; Villena-Cortes, A; Fariñas, I; Marques, M M; Marin, Maria C

    2016-07-01

    The adult subventricular zone (SVZ) is a highly organized microenvironment established during the first postnatal days when radial glia cells begin to transform into type B-cells and ependymal cells, all of which will form regenerative units, pinwheels, along the lateral wall of the lateral ventricle. Here, we identify p73, a p53 homologue, as a critical factor controlling both cell-type specification and structural organization of the developing mouse SVZ. We describe that p73 deficiency halts the transition of the radial glia into ependymal cells, leading to the emergence of immature cells with abnormal identities in the ventricle and resulting in loss of the ventricular integrity. p73-deficient ependymal cells have noticeably impaired ciliogenesis and they fail to organize into pinwheels, disrupting SVZ niche structure and function. Therefore, p73 is essential for appropriate ependymal cell maturation and the establishment of the neurogenic niche architecture. Accordingly, lack of p73 results in impaired neurogenesis. Moreover, p73 is required for translational planar cell polarity establishment, since p73 deficiency results in profound defects in cilia organization in individual cells and in intercellular patch orientation. Thus, our data reveal a completely new function of p73, independent of p53, in the neurogenic architecture of the SVZ of rodent brain and in the establishment of ependymal planar cell polarity with important implications in neurogenesis. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 730-747, 2016. PMID:26482843

  3. IL-2 inducible T cell kinase (ITK) tunes T regulatory cell development and is required for suppressive function1

    OpenAIRE

    Huang, Weishan; Jeong, Ah-Reum; Kannan, Arun K.; Huang, Lu; August, Avery

    2014-01-01

    ITK is a key signaling mediator downstream of TcR, mediating T cell positive selection, innate T cell and CD4+ Th2/Th17 differentiation. Here we show that ITK also negatively tunes IL-2-induced expansion of Foxp3+ regulatory T cells (Treg). In vivo, Treg abundance is inversely correlated with ITK expression, and iTreg development is inversely dependent on ITK kinase activity. While Treg development normally requires both hematopoietic and thymic MHC class 2 (MHC2) expression, the absence of I...

  4. The Histone H2B Monoubiquitination Regulatory Pathway Is Required for Differentiation of Multipotent Stem Cells

    DEFF Research Database (Denmark)

    Karpiuk, Oleksandra; Najafova, Zeynab; Kramer, Frank;

    2012-01-01

    Extensive changes in posttranslational histone modifications accompany the rewiring of the transcriptional program during stem cell differentiation. However, the mechanisms controlling the changes in specific chromatin modifications and their function during differentiation remain only poorly...... understood. We show that histone H2B monoubiquitination (H2Bub1) significantly increases during differentiation of human mesenchymal stem cells (hMSCs) and various lineage-committed precursor cells and in diverse organisms. Furthermore, the H2B ubiquitin ligase RNF40 is required for the induction of...... during the transition from an inactive to an active chromatin conformation. Thus, these data indicate that H2Bub1 is required for maintaining multipotency of hMSCs and plays a central role in controlling stem cell differentiation....

  5. Nutritional requirements for methyl orange decolourisation by freely suspended cells and growing cells of Lactobacillus casei TISTR 1500

    Directory of Open Access Journals (Sweden)

    Phisit Seesuriyachan

    2011-01-01

    Full Text Available Lactobacillus casei TISTR 1500 possesses cytoplasmic azoreductase and can breakdown azo bonds under microaerophilic condition. It was found previously that a growing culture is more tolerant to a high initial dye concentration than freely suspended cells supplied only with sucrose. The present study is aimed at investigating the nutritive requirements for decolourisation by the growing cells and the freely suspended cells using Plackett-Burmann experimental design. In this study, the composition of the medium was found to play an important role in methyl orange decolourisation and biomass production. Sucrose, meat extract and peptone increased methyl orange decolourisation by freely suspended cells, whereas sodium acetate exerted a negative effect on decolourisation. In addition, it was observed that the yeast and meat extracts enhanced the degradation of the dye by the growing cells. Sucrose was an important factor in biomass production by freely suspended cells and growing cells. On the other hand, dipotassium hydrogen phosphate and sodium acetate decreased the biomass production. These findings promote the understanding and knowledge about the requirements of azo dye decolourisation by Lactobacillus casei.

  6. The p38/MK2/Hsp25 pathway is required for BMP-2-induced cell migration.

    Directory of Open Access Journals (Sweden)

    Cristina Gamell

    Full Text Available BACKGROUND: Bone morphogenetic proteins (BMPs have been shown to participate in the patterning and specification of several tissues and organs during development and to regulate cell growth, differentiation and migration in different cell types. BMP-mediated cell migration requires activation of the small GTPase Cdc42 and LIMK1 activities. In our earlier report we showed that activation of LIMK1 also requires the activation of PAKs through Cdc42 and PI3K. However, the requirement of additional signaling is not clearly known. METHODOLOGY/PRINCIPAL FINDINGS: Activation of p38 MAPK has been shown to be relevant for a number of BMP-2's physiological effects. We report here that BMP-2 regulation of cell migration and actin cytoskeleton remodelling are dependent on p38 activity. BMP-2 treatment of mesenchymal cells results in activation of the p38/MK2/Hsp25 signaling pathway downstream from the BMP receptors. Moreover, chemical inhibition of p38 signaling or genetic ablation of either p38α or MK2 blocks the ability to activate the downstream effectors of the pathway and abolishes BMP-2-induction of cell migration. These signaling effects on p38/MK2/Hsp25 do not require the activity of either Cdc42 or PAK, whereas p38/MK2 activities do not significantly modify the BMP-2-dependent activation of LIMK1, measured by either kinase activity or with an antibody raised against phospho-threonine 508 at its activation loop. Finally, phosphorylated Hsp25 colocalizes with the BMP receptor complexes in lamellipodia and overexpression of a phosphorylation mutant form of Hsp25 is able to abolish the migration of cells in response to BMP-2. CONCLUSIONS: These results indicate that Cdc42/PAK/LIMK1 and p38/MK2/Hsp25 pathways, acting in parallel and modulating specific actin regulatory proteins, play a critical role in integrating responses during BMP-induced actin reorganization and cell migration.

  7. Innate lymphoid cell development requires TOX-dependent generation of a common ILC progenitor

    OpenAIRE

    Seehus, Corey R.; Aliahmad, Parinaz; de la Torre, Brian; Iliev, Iliyan D.; Spurka, Lindsay; Funari, Vincent A; Kaye, Jonathan

    2015-01-01

    Diverse innate lymphoid cell (ILC) subtypes have been defined, based on effector function and transcription factor expression. ILCs derive from common lymphoid progenitors, although the transcriptional pathways leading to ILC lineage specification remain poorly characterized. Here we demonstrate that transcriptional regulator TOX is required for the in vivo differentiation of common lymphoid progenitors to ILC lineage-restricted cells. In vitro modeling demonstrates that TOX deficiency result...

  8. Transparent conducting oxide electrodes requirements for high efficiency micromorph solar cells

    OpenAIRE

    Boccard, Mathieu; Cuony, Peter; SöDerströM, Karin; Bugnon, Grégory; Despeisse, Matthieu; Battaglia, Corsin; Ding, Laura; NICOLAY, Sylvain; Ballif, Christophe

    2010-01-01

    The requirements for a micromorph tandem cell front transparent conductive oxide (TCO) are multiple. This essential layer needs a high transparency, excellent conduction, strong light scattering into silicon and good surface morphology for the subsequent growth of silicon cells. These parameters are all linked and trade-offs have to be found for optimal layer. The optimum combination, taking into account current achievable materials properties, is still unclear. Concerning transparency, we st...

  9. Ethanolamine requirement of mammary epithelial cells is due to reduced activity of base exchange enzyme

    International Nuclear Information System (INIS)

    Epithelial cells and some of their transformed derivatives require ethanolamine (Etn) to proliferate normally in defined culture medium. The amount of cellular phosphatidylethanolamine (PtdEtn) is considerably reduced when these cells are cultured without Etn. Using Etn-responsive and -nonresponsive rat mammary carcinoma cell lines, the biochemical mechanism of Etn-responsiveness of investigated. The incorporation of [3H]serine into phosphatidylserine (PtdSer) and PtdEtn in Etn-responsive cells was 60 and 37%, respectively, of those in Etn-nonresponsive cells. There was no significant difference between the two cell types in the activities of enzymes involved in PtdEtn synthesis via CDP-Etn. The activity of PtdSer decarboxylase was also very similar in these two cell types. When these cells were cultured in the presence of [32P]PtdEtn, the rate of accumulation of [32P]-labeled PtdSer from the radioactive PtdEtn was considerably reduced in Etn-responsive cells as compared to Etn-nonresponsive cells. Whereas there was no significant difference in the accumulation of the labeled PtdSer from [32P]phosphatidylcholine. These results demonstrate that the Etn-responsiveness is due to a limited ability to synthesize PtdSer resulting from a limited base exchange activity utilizing PtdEtn

  10. Cell-to-Cell Transmission of HIV-1 Is Required to Trigger Pyroptotic Death of Lymphoid-Tissue-Derived CD4 T Cells

    Directory of Open Access Journals (Sweden)

    Nicole L.K. Galloway

    2015-09-01

    Full Text Available The progressive depletion of CD4 T cells underlies clinical progression to AIDS in untreated HIV-infected subjects. Most dying CD4 T cells correspond to resting nonpermissive cells residing in lymphoid tissues. Death is due to an innate immune response against the incomplete cytosolic viral DNA intermediates accumulating in these cells. The viral DNA is detected by the IFI16 sensor, leading to inflammasome assembly, caspase-1 activation, and the induction of pyroptosis, a highly inflammatory form of programmed cell death. We now show that cell-to-cell transmission of HIV is obligatorily required for activation of this death pathway. Cell-free HIV-1 virions, even when added in large quantities, fail to activate pyroptosis. These findings underscore the infected CD4 T cells as the major killing units promoting progression to AIDS and highlight a previously unappreciated role for the virological synapse in HIV pathogenesis.

  11. Cell-to-Cell Transmission of HIV-1 Is Required to Trigger Pyroptotic Death of Lymphoid-Tissue-Derived CD4 T Cells.

    Science.gov (United States)

    Galloway, Nicole L K; Doitsh, Gilad; Monroe, Kathryn M; Yang, Zhiyuan; Muñoz-Arias, Isa; Levy, David N; Greene, Warner C

    2015-09-01

    The progressive depletion of CD4 T cells underlies clinical progression to AIDS in untreated HIV-infected subjects. Most dying CD4 T cells correspond to resting nonpermissive cells residing in lymphoid tissues. Death is due to an innate immune response against the incomplete cytosolic viral DNA intermediates accumulating in these cells. The viral DNA is detected by the IFI16 sensor, leading to inflammasome assembly, caspase-1 activation, and the induction of pyroptosis, a highly inflammatory form of programmed cell death. We now show that cell-to-cell transmission of HIV is obligatorily required for activation of this death pathway. Cell-free HIV-1 virions, even when added in large quantities, fail to activate pyroptosis. These findings underscore the infected CD4 T cells as the major killing units promoting progression to AIDS and highlight a previously unappreciated role for the virological synapse in HIV pathogenesis.

  12. Bmi1 is required for hepatic progenitor cell expansion and liver tumor development.

    Directory of Open Access Journals (Sweden)

    Lingling Fan

    Full Text Available Bmi1 is a polycomb group transcriptional repressor and it has been implicated in regulating self-renewal and proliferation of many types of stem or progenitor cells. In addition, Bmi1 has been shown to function as an oncogene in multiple tumor types. In this study, we investigated the functional significance of Bmi1 in regulating hepatic oval cells, the major type of bipotential progenitor cells in adult liver, as well as the role of Bmi1 during hepatocarcinogenesis using Bmi1 knockout mice. We found that loss of Bmi1 significantly restricted chemically induced oval cell expansion in the mouse liver. Concomitant deletion of Ink4a/Arf in Bmi1 deficient mice completely rescued the oval cell expansion phenotype. Furthermore, ablation of Bmi1 delayed hepatocarcinogenesis induced by AKT and Ras co-expression. This antineoplastic effect was accompanied by the loss of hepatic oval cell marker expression in the liver tumor samples. In summary, our data demonstrated that Bmi1 is required for hepatic oval cell expansion via deregulating the Ink4a/Arf locus in mice. Our study also provides the evidence, for the first time, that Bmi1 expression is required for liver cancer development in vivo, thus representing a promising target for innovative treatments against human liver cancer.

  13. CRTC2 is required for β-cell function and proliferation.

    Science.gov (United States)

    Eberhard, Chandra E; Fu, Accalia; Reeks, Courtney; Screaton, Robert A

    2013-07-01

    Previous work in insulinoma cell lines has established that calcineurin plays a critical role in the activation of cAMP-responsive element binding protein (Creb), a key transcription factor required for β-cell function and survival, by dephosphorylating the Creb coactivator Creb-regulated transcription coactivator (Crtc)2 at 2 regulatory sites, Ser171 and Ser275. Here, we report that Crtc2 is essential both for glucose-stimulated insulin secretion and cell survival in the β-cell. Endogenous Crtc2 activation is achieved via increasing glucose levels to the physiological feeding range, indicating that Crtc2 is a sensor that couples ambient glucose concentrations to Creb activity in the β-cell. Immunosuppressant drugs such as cyclosporin A and tacrolimus that target the protein phosphatase calcineurin are commonly administered after organ transplantation. Chronic use is associated with reduced insulin secretion and new onset diabetes, suggestive of pancreatic β-cell dysfunction. Importantly, we show that overexpression of a Crtc2 mutant rendered constitutively active by introduction of nonphosphorylatable alanine residues at Ser171 and Ser275 permits Creb target gene activation under conditions when calcineurin is inhibited. Taken together, these data suggest that promoting Crtc2-Creb activity is required for β-cell function and proliferation and promoting this pathway could ameliorate symptoms of new onset diabetes after transplantation.

  14. Skp2 is required for Aurora B activation in cell mitosis and spindle checkpoint.

    Science.gov (United States)

    Wu, Juan; Huang, Yu-Fan; Zhou, Xin-Ke; Zhang, Wei; Lian, Yi-Fan; Lv, Xiao-Bin; Gao, Xiu-Rong; Lin, Hui-Kuan; Zeng, Yi-Xin; Huang, Jian-Qing

    2015-01-01

    The Aurora B kinase plays a critical role in cell mitosis and spindle checkpoint. Here, we showed that the ubiquitin E3-ligase protein Skp2, also as a cell-cycle regulatory protein, was required for the activation of Aurora B and its downstream protein. When we restored Skp2 knockdown Hela cells with Skp2 and Skp2-LRR E3 ligase dead mutant we found that Skp2 could rescue the defect in the activation of Aurora B, but the mutant failed to do so. Furthermore, we discovered that Skp2 could interact with Aurora B and trigger Aurora B Lysine (K) 63-linked ubiquitination. Finally, we demonstrated the essential role of Skp2 in cell mitosis progression and spindle checkpoint, which was Aurora B dependent. Our results identified a novel ubiquitinated substrate of Skp2, and also indicated that Aurora B ubiquitination might serve as an important event for Aurora B activation in cell mitosis and spindle checkpoint.

  15. The phosphatase domains of CD45 are required for ligand induced T-cell receptor downregulation

    DEFF Research Database (Denmark)

    Kastrup, J; Lauritsen, Jens Peter Holst; Menné, C;

    2000-01-01

    Down-regulation of the T-cell receptor (TCR) plays an important role in modulating T-cell responses, both during T-cell development and in mature T cells. At least two distinct pathways exist for TCR down-regulation: down-regulation following TCR ligation; and down-regulation following activation...... of protein kinase C (PKC). Ligand-induced TCR down-regulation is dependent on protein tyrosine kinase (PTK) activity and seems to be closely related to T-cell activation. In addition, previous studies have indicated that ligand-induced TCR down-regulation is dependent on the expression of CD45, a...... transmembrane protein tyrosine phosphatase. The role of the different domains of CD45 in TCR down-regulation was investigated in this study. We found that the phosphatase domains of CD45 are required for efficient ligand-induced TCR down-regulation. In contrast, the extracellular domain of CD45 is dispensable...

  16. Lineage-Specific Early Differentiation of Human Embryonic Stem Cells Requires a G2 Cell Cycle Pause.

    Science.gov (United States)

    Van Oudenhove, Jennifer J; Grandy, Rodrigo A; Ghule, Prachi N; Del Rio, Roxana; Lian, Jane B; Stein, Janet L; Zaidi, Sayyed K; Stein, Gary S

    2016-07-01

    Human embryonic stem cells (hESCs) have an abbreviated G1 phase of the cell cycle that allows rapid proliferation and maintenance of pluripotency. Lengthening of G1 corresponds to loss of pluripotency during differentiation. However, precise mechanisms that link alterations in the cell cycle and early differentiation remain to be defined. We investigated initial stages of mesendodermal lineage commitment in hESCs, and observed a cell cycle pause. Transcriptome profiling identified several genes with known roles in regulation of the G2/M transition that were differentially expressed early during lineage commitment. WEE1 kinase, which blocks entry into mitosis by phosphorylating CDK1 at Y15, was the most highly expressed of these genes. Inhibition of CDK1 phosphorylation by a specific inhibitor of WEE1 restored cell cycle progression by preventing the G2 pause. Directed differentiation of hESCs revealed that cells paused during commitment to the endo- and mesodermal, but not ectodermal, lineages. Functionally, WEE1 inhibition during meso- and endodermal differentiation selectively decreased expression of definitive endodermal markers SOX17 and FOXA2. Our findings identify a novel G2 cell cycle pause that is required for endodermal differentiation and provide important new mechanistic insights into early events of lineage commitment. Stem Cells 2016;34:1765-1775. PMID:26946228

  17. Stage-specific requirement for cyclin D1 in glial progenitor cells of the cerebral cortex.

    Science.gov (United States)

    Nobs, Lionel; Baranek, Constanze; Nestel, Sigrun; Kulik, Akos; Kapfhammer, Josef; Nitsch, Cordula; Atanasoski, Suzana

    2014-05-01

    Despite the vast abundance of glial progenitor cells in the mouse brain parenchyma, little is known about the molecular mechanisms driving their proliferation in the adult. Here we unravel a critical role of the G1 cell cycle regulator cyclin D1 in controlling cell division of glial cells in the cortical grey matter. We detect cyclin D1 expression in Olig2-immunopositive (Olig2+) oligodendrocyte progenitor cells, as well as in Iba1+ microglia and S100β+ astrocytes in cortices of 3-month-old mice. Analysis of cyclin D1-deficient mice reveals a cell and stage-specific molecular control of cell cycle progression in the various glial lineages. While proliferation of fast dividing Olig2+ cells at early postnatal stages becomes gradually dependent on cyclin D1, this particular G1 regulator is strictly required for the slow divisions of Olig2+/NG2+ oligodendrocyte progenitors in the adult cerebral cortex. Further, we find that the population of mature oligodendrocytes is markedly reduced in the absence of cyclin D1, leading to a significant decrease in the number of myelinated axons in both the prefrontal cortex and the corpus callosum of 8-month-old mutant mice. In contrast, the pool of Iba1+ cells is diminished already at postnatal day 3 in the absence of cyclin D1, while the number of S100β+ astrocytes remains unchanged in the mutant.

  18. Pineal photoreceptor cells are required for maintaining the circadian rhythms of behavioral visual sensitivity in zebrafish.

    Directory of Open Access Journals (Sweden)

    Xinle Li

    Full Text Available In non-mammalian vertebrates, the pineal gland functions as the central pacemaker that regulates the circadian rhythms of animal behavior and physiology. We generated a transgenic zebrafish line [Tg(Gnat2:gal4-VP16/UAS:nfsB-mCherry] in which the E. coli nitroreductase is expressed in pineal photoreceptor cells. In developing embryos and young adults, the transgene is expressed in both retinal and pineal photoreceptor cells. During aging, the expression of the transgene in retinal photoreceptor cells gradually diminishes. By 8 months of age, the Gnat2 promoter-driven nitroreductase is no longer expressed in retinal photoreceptor cells, but its expression in pineal photoreceptor cells persists. This provides a tool for selective ablation of pineal photoreceptor cells, i.e., by treatments with metronidazole. In the absence of pineal photoreceptor cells, the behavioral visual sensitivity of the fish remains unchanged; however, the circadian rhythms of rod and cone sensitivity are diminished. Brief light exposures restore the circadian rhythms of behavioral visual sensitivity. Together, the data suggest that retinal photoreceptor cells respond to environmental cues and are capable of entraining the circadian rhythms of visual sensitivity; however, they are insufficient for maintaining the rhythms. Cellular signals from the pineal photoreceptor cells may be required for maintaining the circadian rhythms of visual sensitivity.

  19. Breast cancer lung metastasis requires expression of chemokine receptor CCR4 and regulatory T cells.

    Science.gov (United States)

    Olkhanud, Purevdorj B; Baatar, Dolgor; Bodogai, Monica; Hakim, Fran; Gress, Ronald; Anderson, Robin L; Deng, Jie; Xu, Mai; Briest, Susanne; Biragyn, Arya

    2009-07-15

    Cancer metastasis is a leading cause of cancer morbidity and mortality. More needs to be learned about mechanisms that control this process. In particular, the role of chemokine receptors in metastasis remains controversial. Here, using a highly metastatic breast cancer (4T1) model, we show that lung metastasis is a feature of only a proportion of the tumor cells that express CCR4. Moreover, the primary tumor growing in mammary pads activates remotely the expression of TARC/CCL17 and MDC/CCL22 in the lungs. These chemokines acting through CCR4 attract both tumor and immune cells. However, CCR4-mediated chemotaxis was not sufficient to produce metastasis, as tumor cells in the lung were efficiently eliminated by natural killer (NK) cells. Lung metastasis required CCR4(+) regulatory T cells (Treg), which directly killed NK cells using beta-galactoside-binding protein. Thus, strategies that abrogate any part of this process should improve the outcome through activation of effector cells and prevention of tumor cell migration. We confirm this prediction by killing CCR4(+) cells through delivery of TARC-fused toxins or depleting Tregs and preventing lung metastasis. PMID:19567680

  20. TRPM7 is required for ovarian cancer cell growth, migration and invasion

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jing; Liao, Qian-jin [The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha 410013 (China); Zhang, Yi [Department of Obstetrics and Gynaecology, Xiangya Hospital, Central South University, Changsha 410078 (China); Zhou, Hui; Luo, Chen-hui; Tang, Jie; Wang, Ying; Tang, Yan; Zhao, Min; Zhao, Xue-heng [The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha 410013 (China); Zhang, Qiong-yu [Department of Basic Medical Science, Yongzhou Vocational Technical College, Yong Zhou 425100 (China); Xiao, Ling, E-mail: lingxiaocsu@126.com [Department of Histology and Embryology, School of Basic Medical Sciences, Central South University, Changsha 410013 (China); Institute of Clinical Pharmacology, Central South University, Changsha 410018 (China)

    2014-11-28

    Highlights: • Silence of TRPM7 in ovarian cancer cells inhibits cell proliferation, migration and invasion. • Silence of TRPM7 decreases phosphorylation levels of Akt, Src and p38 in ovarian cancer cells. • Silence of TRPM7 increases expression of filamentous actin and number of focal adhesions in ovarian cancer cells. - Abstract: Our previous study demonstrated that the melastatin-related transient receptor potential channel 7 (TRPM7) was highly expressed in ovarian carcinomas and its overexpression was significantly associated with poor prognosis in ovarian cancer patients. However, the function of TRPM7 in ovarian cancer is mostly unknown. In this study, we examined the roles of TRPM7 in ovarian cancer cell proliferation, migration and invasion. We found that short hairpin RNA interference-mediated silence of TRPM7 significantly inhibited cell proliferation, colony formation, migration and invasion in multiple ovarian cancer cell lines. Mechanistic investigation revealed that silence of TRPM7 decreased phosphorylation levels of Akt, Src and p38 and increased filamentous actin and focal adhesion number in ovarian cancer cells. Thus, our results suggest that TRPM7 is required for proliferation, migration and invasion of ovarian cancer cells through regulating multiple signaling transduction pathways and the formation of focal adhesions.

  1. 14-3-3ε Is required for germ cell migration in Drosophila.

    Directory of Open Access Journals (Sweden)

    K Kirki Tsigkari

    Full Text Available Although 14-3-3 proteins participate in multiple biological processes, isoform-specific specialized functions, as well as functional redundancy are emerging with tissue and developmental stage-specificity. Accordingly, the two 14-3-3ε proteins in Drosophila exhibit functional specificity and redundancy. Homozygotes for loss of function alleles of D14-3-3ε contain significantly fewer germ line cells (pole cells in their gonads, a phenotype not shared by mutants in the other 14-3-3 gene leo. We show that although D14-3-3ε is enriched within pole cells it is required in mesodermal somatic gonad precursor cells which guide pole cells in their migration through the mesoderm and coalesce with them to form the embryonic gonad. Loss of D14-3-3ε results in defective pole cell migration, reduced pole cell number. We present evidence that D14-3-3ε loss results in reduction or loss of the transcription factor Zfh-1, one of the main regulatory molecules of the pole cell migration, from the somatic gonad precursor cells.

  2. Fast Vesicle Fusion in Living Cells Requires at Least Three SNARE Complexes

    DEFF Research Database (Denmark)

    Mohrmann, Ralf; de Wit, Heidi; Verhage, Matthijs;

    2010-01-01

    relationship for fast (synchronous) fusion and a near-linear relationship for overall release. Thus, fast fusion typically observed in synapses and neurosecretory cells requires at least three functional SNARE complexes, while slower release might occur with fewer. Heterogeneity in SNARE-complex number may...

  3. The development of innate lymphoid cells requires TOX-dependent generation of a common innate lymphoid cell progenitor.

    Science.gov (United States)

    Seehus, Corey R; Aliahmad, Parinaz; de la Torre, Brian; Iliev, Iliyan D; Spurka, Lindsay; Funari, Vincent A; Kaye, Jonathan

    2015-06-01

    Diverse innate lymphoid cell (ILC) subtypes have been defined on the basis of effector function and transcription factor expression. ILCs derive from common lymphoid progenitors, although the transcriptional pathways that lead to ILC-lineage specification remain poorly characterized. Here we found that the transcriptional regulator TOX was required for the in vivo differentiation of common lymphoid progenitors into ILC lineage-restricted cells. In vitro modeling demonstrated that TOX deficiency resulted in early defects in the survival or proliferation of progenitor cells, as well as ILC differentiation at a later stage. In addition, comparative transcriptome analysis of bone marrow progenitors revealed that TOX-deficient cells failed to upregulate many genes of the ILC program, including genes that are targets of Notch, which indicated that TOX is a key determinant of early specification to the ILC lineage.

  4. Differential requirement for MEK Partner 1 in DU145 prostate cancer cell migration

    Directory of Open Access Journals (Sweden)

    Bailey Evangeline M

    2009-11-01

    Full Text Available Abstract ERK signaling regulates focal adhesion disassembly during cell movement, and increased ERK signaling frequently contributes to enhanced motility of human tumor cells. We previously found that the ERK scaffold MEK Partner 1 (MP1 is required for focal adhesion disassembly in fibroblasts. Here we test the hypothesis that MP1-dependent ERK signaling regulates motility of DU145 prostate cancer cells. We find that MP1 is required for motility on fibronectin, but not for motility stimulated by serum or EGF. Surprisingly, MP1 appears not to function through its known binding partners MEK1 or PAK1, suggesting the existence of a novel pathway by which MP1 can regulate motility on fibronectin. MP1 may function by regulating the stability or expression of paxillin, a key regulator of motility.

  5. Dystroglycan is required for polarizing the epithelial cells and the oocyte in Drosophila

    DEFF Research Database (Denmark)

    Deng, Wu-Min; Schneider, Martina; Frock, Richard;

    2003-01-01

    , and plays a role in linking the ECM to the actin cytoskeleton; however, how these interactions are regulated and their basic cellular functions are poorly understood. Using mosaic analysis and RNAi in the model organism Drosophila melanogaster, we show that Dystroglycan is required cell...... localization of these same markers. In Dystroglycan germline clones early oocyte polarity markers fail to be localized to the posterior, and oocyte cortical F-actin organization is abnormal. Dystroglycan is also required non-cell-autonomously to organize the planar polarity of basal actin in follicle cells......, possibly by organizing the Laminin ECM. These data suggest that the primary function of Dystroglycan in oogenesis is to organize cellular polarity; and this study sets the stage for analyzing the Dystroglycan complex by using the power of Drosophila molecular genetics....

  6. FGF7 and cell density are required for final differentiation of pancreatic amylase-positive cells from human ES cells.

    Science.gov (United States)

    Takizawa-Shirasawa, Sakiko; Yoshie, Susumu; Yue, Fengming; Mogi, Akimi; Yokoyama, Tadayuki; Tomotsune, Daihachiro; Sasaki, Katsunori

    2013-12-01

    The major molecular signals of pancreatic exocrine development are largely unknown. We examine the role of fibroblast growth factor 7 (FGF7) in the final induction of pancreatic amylase-containing exocrine cells from induced-pancreatic progenitor cells derived from human embryonic stem (hES) cells. Our protocol consisted in three steps: Step I, differentiation of definitive endoderm (DE) by activin A treatment of hES cell colonies; Step II, differentiation of pancreatic progenitor cells by re-plating of the cells of Step I onto 24-well plates at high density and stimulation with all-trans retinoic acid; Step III, differentiation of pancreatic exocrine cells with a combination of FGF7, glucagon-like peptide 1 and nicotinamide. The expression levels of pancreatic endodermal markers such as Foxa2, Sox17 and gut tube endoderm marker HNF1β were up-regulated in both Step I and II. Moreover, in Step III, the induced cells expressed pancreatic markers such as amylase, carboxypeptidase A and chymotrypsinogen B, which were similar to those in normal human pancreas. From day 8 in Step III, cells immunohistochemically positive for amylase and for carboxypeptidase A, a pancreatic exocrine cell product, were induced by FGF7. Pancreatic progenitor Pdx1-positive cells were localized in proximity to the amylase-positive cells. In the absence of FGF7, few amylase-positive cells were identified. Thus, our three-step culture protocol for human ES cells effectively induces the differentiation of amylase- and carboxypeptidase-A-containing pancreatic exocrine cells.

  7. Notch signal reception is required in vascular smooth muscle cells for ductus arteriosus closure.

    Science.gov (United States)

    Krebs, Luke T; Norton, Christine R; Gridley, Thomas

    2016-02-01

    The ductus arteriosus is an arterial vessel that shunts blood flow away from the lungs during fetal life, but normally occludes after birth to establish the adult circulation pattern. Failure of the ductus arteriosus to close after birth is termed patent ductus arteriosus, and is one of the most common congenital heart defects. Our previous work demonstrated that vascular smooth muscle cell expression of the Jag1 gene, which encodes a ligand for Notch family receptors, is essential for postnatal closure of the ductus arteriosus in mice. However, it was not known what cell population was responsible for receiving the Jag1-mediated signal. Here we show, using smooth muscle cell-specific deletion of the Rbpj gene, which encodes a transcription factor that mediates all canonical Notch signaling, that Notch signal reception in the vascular smooth muscle cell compartment is required for ductus arteriosus closure. These data indicate that homotypic vascular smooth muscle cell interactions are required for proper contractile smooth muscle cell differentiation and postnatal closure of the ductus arteriosus in mice.

  8. A mex3 homolog is required for differentiation during planarian stem cell lineage development.

    Science.gov (United States)

    Zhu, Shu Jun; Hallows, Stephanie E; Currie, Ko W; Xu, ChangJiang; Pearson, Bret J

    2015-01-01

    Neoblasts are adult stem cells (ASCs) in planarians that sustain cell replacement during homeostasis and regeneration of any missing tissue. While numerous studies have examined genes underlying neoblast pluripotency, molecular pathways driving postmitotic fates remain poorly defined. In this study, we used transcriptional profiling of irradiation-sensitive and irradiation-insensitive cell populations and RNA interference (RNAi) functional screening to uncover markers and regulators of postmitotic progeny. We identified 32 new markers distinguishing two main epithelial progenitor populations and a planarian homolog to the MEX3 RNA-binding protein (Smed-mex3-1) as a key regulator of lineage progression. mex3-1 was required for generating differentiated cells of multiple lineages, while restricting the size of the stem cell compartment. We also demonstrated the utility of using mex3-1(RNAi) animals to identify additional progenitor markers. These results identified mex3-1 as a cell fate regulator, broadly required for differentiation, and suggest that mex3-1 helps to mediate the balance between ASC self-renewal and commitment. PMID:26114597

  9. Requirement for Dlgh-1 in planar cell polarity and skeletogenesis during vertebrate development.

    Directory of Open Access Journals (Sweden)

    Charlene Rivera

    Full Text Available The development of specialized organs is tightly linked to the regulation of cell growth, orientation, migration and adhesion during embryogenesis. In addition, the directed movements of cells and their orientation within the plane of a tissue, termed planar cell polarity (PCP, appear to be crucial for the proper formation of the body plan. In Drosophila embryogenesis, Discs large (dlg plays a critical role in apical-basal cell polarity, cell adhesion and cell proliferation. Craniofacial defects in mice carrying an insertional mutation in Dlgh-1 suggest that Dlgh-1 is required for vertebrate development. To determine what roles Dlgh-1 plays in vertebrate development, we generated mice carrying a null mutation in Dlgh-1. We found that deletion of Dlgh-1 caused open eyelids, open neural tube, and misorientation of cochlear hair cell stereociliary bundles, indicative of defects in planar cell polarity (PCP. Deletion of Dlgh-1 also caused skeletal defects throughout the embryo. These findings identify novel roles for Dlgh-1 in vertebrates that differ from its well-characterized roles in invertebrates and suggest that the Dlgh-1 null mouse may be a useful animal model to study certain human congenital birth defects.

  10. Requirement for HIV-1 TAR sequences for Tat activation in rodent cells.

    Science.gov (United States)

    Sutton, J A; Braddock, M; Kingsman, A J; Kingsman, S M

    1995-01-10

    HIV-1 gene expression is activated via an interaction between the virally encoded Tat protein and a target RNA, TAR. TAR is located at the immediate 5' end of all viral mRNAs and comprises a partially base-paired stem with a tripyrimidine bulge in the upper stem and a hexanucleotide loop. In vitro, Tat binds specifically to the bulge and upper stem region with no requirement for the loop. In contrast, when Tat activation is analyzed in primate cells, mutations in the loop abolish activation, suggesting a critical role for loop binding cellular factors. However, in rodent cells the reverse is true. Messages with a mutation in the TAR loop are activated whereas messages harboring a wild-type TAR sequence are not activated. By testing the effect of mutations in the bulge and stem in the context of mutation in the loop we now show that this loop-independent activation by Tat in rodent cells requires the critical bulge-stem sequences needed for Tat binding in vitro. These data suggest that in rodent cells, as in vitro, Tat does not require a loop binding cofactor. In rodent cells containing human chromosome 12 (CHO12), however, Tat activation is both bulge and loop dependent. It appears that rodent cells, but not CHO12 cells, are refractory to the normal Tat/TAR activation pathway not by virtue of lacking a loop binding cofactor, but rather by the presence of a loop binding inhibitor of either Tat binding or the activation process. PMID:7530399

  11. ATM Kinase Is Required for Telomere Elongation in Mouse and Human Cells

    Directory of Open Access Journals (Sweden)

    Stella Suyong Lee

    2015-11-01

    Full Text Available Short telomeres induce a DNA damage response, senescence, and apoptosis, thus maintaining telomere length equilibrium is essential for cell viability. Telomerase addition of telomere repeats is tightly regulated in cells. To probe pathways that regulate telomere addition, we developed the ADDIT assay to measure new telomere addition at a single telomere in vivo. Sequence analysis showed telomerase-specific addition of repeats onto a new telomere occurred in just 48 hr. Using the ADDIT assay, we found that ATM is required for addition of new repeats onto telomeres in mouse cells. Evaluation of bulk telomeres, in both human and mouse cells, showed that blocking ATM inhibited telomere elongation. Finally, the activation of ATM through the inhibition of PARP1 resulted in increased telomere elongation, supporting the central role of the ATM pathway in regulating telomere addition. Understanding this role of ATM may yield new areas for possible therapeutic intervention in telomere-mediated disease.

  12. Oct1 and OCA-B are selectively required for CD4 memory T cell function.

    Science.gov (United States)

    Shakya, Arvind; Goren, Alon; Shalek, Alex; German, Cody N; Snook, Jeremy; Kuchroo, Vijay K; Yosef, Nir; Chan, Raymond C; Regev, Aviv; Williams, Matthew A; Tantin, Dean

    2015-11-16

    Epigenetic changes are crucial for the generation of immunological memory. Failure to generate or maintain these changes will result in poor memory responses. Similarly, augmenting or stabilizing the correct epigenetic states offers a potential method of enhancing memory. Yet the transcription factors that regulate these processes are poorly defined. We find that the transcription factor Oct1 and its cofactor OCA-B are selectively required for the in vivo generation of CD4(+) memory T cells. More importantly, the memory cells that are formed do not respond properly to antigen reencounter. In vitro, both proteins are required to maintain a poised state at the Il2 target locus in resting but previously stimulated CD4(+) T cells. OCA-B is also required for the robust reexpression of multiple other genes including Ifng. ChIPseq identifies ∼50 differentially expressed direct Oct1 and OCA-B targets. We identify an underlying mechanism involving OCA-B recruitment of the histone lysine demethylase Jmjd1a to targets such as Il2, Ifng, and Zbtb32. The findings pinpoint Oct1 and OCA-B as central mediators of CD4(+) T cell memory. PMID:26481684

  13. Memory CD8(+) T Cells Require Increased Concentrations of Acetate Induced by Stress for Optimal Function.

    Science.gov (United States)

    Balmer, Maria L; Ma, Eric H; Bantug, Glenn R; Grählert, Jasmin; Pfister, Simona; Glatter, Timo; Jauch, Annaïse; Dimeloe, Sarah; Slack, Emma; Dehio, Philippe; Krzyzaniak, Magdalena A; King, Carolyn G; Burgener, Anne-Valérie; Fischer, Marco; Develioglu, Leyla; Belle, Réka; Recher, Mike; Bonilla, Weldy V; Macpherson, Andrew J; Hapfelmeier, Siegfried; Jones, Russell G; Hess, Christoph

    2016-06-21

    How systemic metabolic alterations during acute infections impact immune cell function remains poorly understood. We found that acetate accumulates in the serum within hours of systemic bacterial infections and that these increased acetate concentrations are required for optimal memory CD8(+) T cell function in vitro and in vivo. Mechanistically, upon uptake by memory CD8(+) T cells, stress levels of acetate expanded the cellular acetyl-coenzyme A pool via ATP citrate lyase and promoted acetylation of the enzyme GAPDH. This context-dependent post-translational modification enhanced GAPDH activity, catalyzing glycolysis and thus boosting rapid memory CD8(+) T cell responses. Accordingly, in a murine Listeria monocytogenes model, transfer of acetate-augmented memory CD8(+) T cells exerted superior immune control compared to control cells. Our results demonstrate that increased systemic acetate concentrations are functionally integrated by CD8(+) T cells and translate into increased glycolytic and functional capacity. The immune system thus directly relates systemic metabolism with immune alertness. PMID:27212436

  14. DipM, a new factor required for peptidoglycan remodelling during cell division in Caulobacter crescentus.

    Science.gov (United States)

    Möll, Andrea; Schlimpert, Susan; Briegel, Ariane; Jensen, Grant J; Thanbichler, Martin

    2010-07-01

    In bacteria, cytokinesis is dependent on lytic enzymes that facilitate remodelling of the cell wall during constriction. In this work, we identify a thus far uncharacterized periplasmic protein, DipM, that is required for cell division and polarity in Caulobacter crescentus. DipM is composed of four peptidoglycan binding (LysM) domains and a C-terminal lysostaphin-like (LytM) peptidase domain. It binds to isolated murein sacculi in vitro, and is recruited to the site of constriction through interaction with the cell division protein FtsN. Mutational analyses showed that the LysM domains are necessary and sufficient for localization of DipM, while its peptidase domain is essential for function. Consistent with a role in cell wall hydrolysis, DipM was found to interact with purified murein sacculi in vitro and to induce cell lysis upon overproduction. Its inactivation causes severe defects in outer membrane invagination, resulting in a significant delay between cytoplasmic compartmentalization and final separation of the daughter cells. Overall, these findings indicate that DipM is a periplasmic component of the C. crescentus divisome that facilitates remodelling of the peptidoglycan layer and, thus, coordinated constriction of the cell envelope during the division process.

  15. TCF1 Is Required for the T Follicular Helper Cell Response to Viral Infection

    Directory of Open Access Journals (Sweden)

    Tuoqi Wu

    2015-09-01

    Full Text Available T follicular helper (TFH and T helper 1 (Th1 cells generated after viral infections are critical for the control of infection and the development of immunological memory. However, the mechanisms that govern the differentiation and maintenance of these two distinct lineages during viral infection remain unclear. We found that viral-specific TFH and Th1 cells showed reciprocal expression of the transcriptions factors TCF1 and Blimp1 early after infection, even before the differential expression of the canonical TFH marker CXCR5. Furthermore, TCF1 was intrinsically required for the TFH cell response to viral infection; in the absence of TCF1, the TFH cell response was severely compromised, and the remaining TCF1-deficient TFH cells failed to maintain TFH-associated transcriptional and metabolic signatures, which were distinct from those in Th1 cells. Mechanistically, TCF1 functioned through forming negative feedback loops with IL-2 and Blimp1. Our findings demonstrate an essential role of TCF1 in TFH cell responses to viral infection.

  16. Kif11 dependent cell cycle progression in radial glial cells is required for proper neurogenesis in the zebrafish neural tube.

    Science.gov (United States)

    Johnson, Kimberly; Moriarty, Chelsea; Tania, Nessy; Ortman, Alissa; DiPietrantonio, Kristina; Edens, Brittany; Eisenman, Jean; Ok, Deborah; Krikorian, Sarah; Barragan, Jessica; Golé, Christophe; Barresi, Michael J F

    2014-03-01

    Radial glia serve as the resident neural stem cells in the embryonic vertebrate nervous system, and their proliferation must be tightly regulated to generate the correct number of neuronal and glial cell progeny in the neural tube. During a forward genetic screen, we recently identified a zebrafish mutant in the kif11 loci that displayed a significant increase in radial glial cell bodies at the ventricular zone of the spinal cord. Kif11, also known as Eg5, is a kinesin-related, plus-end directed motor protein responsible for stabilizing and separating the bipolar mitotic spindle. We show here that Gfap+ radial glial cells express kif11 in the ventricular zone and floor plate. Loss of Kif11 by mutation or pharmacological inhibition with S-trityl-L-cysteine (STLC) results in monoastral spindle formation in radial glial cells, which is characteristic of mitotic arrest. We show that M-phase radial glia accumulate over time at the ventricular zone in kif11 mutants and STLC treated embryos. Mathematical modeling of the radial glial accumulation in kif11 mutants not only confirmed an ~226× delay in mitotic exit (likely a mitotic arrest), but also predicted two modes of increased cell death. These modeling predictions were supported by an increase in the apoptosis marker, anti-activated Caspase-3, which was also found to be inversely proportional to a decrease in cell proliferation. In addition, treatment with STLC at different stages of neural development uncovered two critical periods that most significantly require Kif11 function for stem cell progression through mitosis. We also show that loss of Kif11 function causes specific reductions in oligodendroglia and secondary interneurons and motorneurons, suggesting these later born populations require proper radial glia division. Despite these alterations to cell cycle dynamics, survival, and neurogenesis, we document unchanged cell densities within the neural tube in kif11 mutants, suggesting that a mechanism of

  17. Kif11 dependent cell cycle progression in radial glial cells is required for proper neurogenesis in the zebrafish neural tube.

    Science.gov (United States)

    Johnson, Kimberly; Moriarty, Chelsea; Tania, Nessy; Ortman, Alissa; DiPietrantonio, Kristina; Edens, Brittany; Eisenman, Jean; Ok, Deborah; Krikorian, Sarah; Barragan, Jessica; Golé, Christophe; Barresi, Michael J F

    2014-03-01

    Radial glia serve as the resident neural stem cells in the embryonic vertebrate nervous system, and their proliferation must be tightly regulated to generate the correct number of neuronal and glial cell progeny in the neural tube. During a forward genetic screen, we recently identified a zebrafish mutant in the kif11 loci that displayed a significant increase in radial glial cell bodies at the ventricular zone of the spinal cord. Kif11, also known as Eg5, is a kinesin-related, plus-end directed motor protein responsible for stabilizing and separating the bipolar mitotic spindle. We show here that Gfap+ radial glial cells express kif11 in the ventricular zone and floor plate. Loss of Kif11 by mutation or pharmacological inhibition with S-trityl-L-cysteine (STLC) results in monoastral spindle formation in radial glial cells, which is characteristic of mitotic arrest. We show that M-phase radial glia accumulate over time at the ventricular zone in kif11 mutants and STLC treated embryos. Mathematical modeling of the radial glial accumulation in kif11 mutants not only confirmed an ~226× delay in mitotic exit (likely a mitotic arrest), but also predicted two modes of increased cell death. These modeling predictions were supported by an increase in the apoptosis marker, anti-activated Caspase-3, which was also found to be inversely proportional to a decrease in cell proliferation. In addition, treatment with STLC at different stages of neural development uncovered two critical periods that most significantly require Kif11 function for stem cell progression through mitosis. We also show that loss of Kif11 function causes specific reductions in oligodendroglia and secondary interneurons and motorneurons, suggesting these later born populations require proper radial glia division. Despite these alterations to cell cycle dynamics, survival, and neurogenesis, we document unchanged cell densities within the neural tube in kif11 mutants, suggesting that a mechanism of

  18. Computational identification of a p38SAPK regulated transcription factor network required for tumor cell quiescence

    OpenAIRE

    Adam, Alejandro P.; George, Ajish; Schewe, Denis; Bragado, Paloma; Iglesias, Bibiana V.; Ranganathan, Aparna C.; Kourtidis, Antonis; Conklin, Douglas S.; Julio A Aguirre-Ghiso

    2009-01-01

    The stress activated kinase p38 plays key roles in tumor suppression and induction of tumor cell dormancy. However, the mechanisms behind these functions remain poorly understood. Using computational tools we identified a transcription factor (TF) network regulated by p38α/β and required for human squamous carcinoma cell quiescence in vivo. We found that p38 transcriptionally regulates a core network of 46 genes that includes 16 TFs. Activation of p38 induced the expression of the TFs p53 and...

  19. Utx Is Required for Proper Induction of Ectoderm and Mesoderm during Differentiation of Embryonic Stem Cells

    DEFF Research Database (Denmark)

    Morales Torres, Cristina; Laugesen, Anne; Helin, Kristian

    2013-01-01

    for the activation of lineage choice genes in response to developmental signals. To further understand the function of Utx in pluripotency and differentiation we generated Utx knockout embryonic stem cells (ESCs). Here we show that Utx is not required for the proliferation of ESCs, however, Utx contributes......Embryonic development requires chromatin remodeling for dynamic regulation of gene expression patterns to ensure silencing of pluripotent transcription factors and activation of developmental regulators. Demethylation of H3K27me3 by the histone demethylases Utx and Jmjd3 is important...

  20. Merkel cell tumor of the skin treated with localized radiotherapy: are widely negative margins required?

    Science.gov (United States)

    Trombetta, Mark; Packard, Matthew; Velosa, Claudia; Silverman, Jan; Werts, Day; Parda, David

    2011-03-30

    Merkel's cell carcinoma is a rare cutaneous tumor that can affect a wide variety of sites throughout the body. Commonly, it affects the skin alone and the management of limited disease can be confusing since the natural history of the disease involves distant metastasis. Traditional management has required wide local excision with negative margins of resection. We describe a case treated with local therapy alone and review the literature to suggest that complete microscopic excision may not be required if adjuvant radiotherapy is used.

  1. Merkel cell tumor of the skin treated with localized radiotherapy: are widely negative margins required?

    Directory of Open Access Journals (Sweden)

    David Parda

    2011-03-01

    Full Text Available Merkel’s cell carcinoma is a rare cutaneous tumor that can affect a wide variety of sites throughout the body. Commonly, it affects the skin alone and the management of limited disease can be confusing since the natural history of the disease involves distant metastasis. Traditional management has required wide local excision with negative margins of resection. We describe a case treated with local therapy alone and review the literature to suggest that complete microscopic excision may not be required if adjuvant radiotherapy is used.

  2. Cell-intrinsic in vivo requirement for the E47-p21 pathway in long-term hematopoietic stem cells.

    Science.gov (United States)

    Santos, Patricia M; Ding, Ying; Borghesi, Lisa

    2014-01-01

    Major regulators of long-term hematopoietic stem cell (LT-HSC) self-renewal and proliferation have been identified, but knowledge of their in vivo interaction in a linear pathway is lacking. In this study, we show a direct genetic link between the transcription factor E47 and the major cell cycle regulator p21 in controlling LT-HSC integrity in vivo under repopulation stress. Numerous studies have shown that E47 activates p21 transcription in hematopoietic subsets in vitro, and we now reveal the in vivo relevance of the E47-p21 pathway by reducing the gene dose of each factor individually (E47(het) or p21(het)) versus in tandem (E47(het)p21(het)). E47(het)p21(het) LT-HSCs and downstream short-term hematopoietic stem cells exhibit hyperproliferation and preferential susceptibility to mitotoxin compared to wild-type or single haploinsufficient controls. In serial adoptive transfers that rigorously challenge self-renewal, E47(het)p21(het) LT-HSCs dramatically and progressively decline, indicating the importance of cell-intrinsic E47-p21 in preserving LT-HSCs under stress. Transient numeric recovery of downstream short-term hematopoietic stem cells enabled the production of functionally competent myeloid but not lymphoid cells, as common lymphoid progenitors were decreased, and peripheral lymphocytes were virtually ablated. Thus, we demonstrate a developmental compartment-specific and lineage-specific requirement for the E47-p21 pathway in maintaining LT-HSCs, B cells, and T cells under hematopoietic repopulation stress in vivo.

  3. CD8+ T cell priming by dendritic cell vaccines requires antigen transfer to endogenous antigen presenting cells.

    Directory of Open Access Journals (Sweden)

    Alice W Yewdall

    Full Text Available Immunotherapeutic strategies to stimulate anti-tumor immunity are promising approaches for cancer treatment. A major barrier to their success is the immunosuppressive microenvironment of tumors, which inhibits the functions of endogenous dendritic cells (DCs that are necessary for the generation of anti-tumor CD8+ T cells. To overcome this problem, autologous DCs are generated ex vivo, loaded with tumor antigens, and activated in this non-suppressive environment before administration to patients. However, DC-based vaccines rarely induce tumor regression.We examined the fate and function of these DCs following their injection using murine models, in order to better understand their interaction with the host immune system. Contrary to previous assumptions, we show that DC vaccines have an insignificant role in directly priming CD8+ T cells, but instead function primarily as vehicles for transferring antigens to endogenous antigen presenting cells, which are responsible for the subsequent activation of T cells.This reliance on endogenous immune cells may explain the limited success of current DC vaccines to treat cancer and offers new insight into how these therapies can be improved. Future approaches should focus on creating DC vaccines that are more effective at directly priming T cells, or abrogating the tumor induced suppression of endogenous DCs.

  4. C. elegans nucleostemin is required for larval growth and germline stem cell division.

    Directory of Open Access Journals (Sweden)

    Michelle M Kudron

    Full Text Available The nucleolus has shown to be integral for many processes related to cell growth and proliferation. Stem cells in particular are likely to depend upon nucleolus-based processes to remain in a proliferative state. A highly conserved nucleolar factor named nucleostemin is proposed to be a critical link between nucleolar function and stem-cell-specific processes. Currently, it is unclear whether nucleostemin modulates proliferation by affecting ribosome biogenesis or by another nucleolus-based activity that is specific to stem cells and/or highly proliferating cells. Here, we investigate nucleostemin (nst-1 in the nematode C. elegans, which enables us to examine nst-1 function during both proliferation and differentiation in vivo. Like mammalian nucleostemin, the NST-1 protein is localized to the nucleolus and the nucleoplasm; however, its expression is found in both differentiated and proliferating cells. Global loss of C. elegans nucleostemin (nst-1 leads to a larval arrest phenotype due to a growth defect in the soma, while loss of nst-1 specifically in the germ line causes germline stem cells to undergo a cell cycle arrest. nst-1 mutants exhibit reduced levels of rRNAs, suggesting defects in ribosome biogenesis. However, NST-1 is generally not present in regions of the nucleolus where rRNA transcription and processing occurs, so this reduction is likely secondary to a different defect in ribosome biogenesis. Transgenic studies indicate that NST-1 requires its N-terminal domain for stable expression and both its G1 GTPase and intermediate domains for proper germ line function. Our data support a role for C. elegans nucleostemin in cell growth and proliferation by promoting ribosome biogenesis.

  5. A Dosage-Dependent Requirement for Sox9 in Pancreatic Endocrine Cell Formation

    OpenAIRE

    Seymour, Philip A.; Freude, Kristine K.; Dubois, Claire L.; Shih, Hung-Ping; Patel, Nisha A.; Sander, Maike

    2008-01-01

    We have previously shown the transcription factor SOX9 to be required for the maintenance of multipotential pancreatic progenitor cells in the early embryonic pancreas. However, the association of pancreatic endocrine defects with the Sox9-haploinsufficiency syndrome campomelic dysplasia (CD) implies additional later roles for Sox9 in endocrine development. Using short-term lineage tracing in mice, we demonstrate here that SOX9 marks a pool of multipotential pancreatic progenitors throughout ...

  6. DREF Is Required for Efficient Growth and Cell Cycle Progression in Drosophila Imaginal Discs

    OpenAIRE

    Hyun, Joogyung; Jasper, Heinrich; Bohmann, Dirk

    2005-01-01

    Based on overexpression studies and target gene analyses, the transcription factor DNA replication-related element factor (DREF) has been proposed to regulate growth and replication in Drosophila melanogaster. Here we present loss-of-function experiments to analyze the contribution of DREF to these processes. RNA interference-mediated extinction of DREF function in vivo demonstrates a requirement for the protein for normal progression through the cell cycle and consequently for growth of imag...

  7. Sensor Needs and Requirements for Fuel Cells and CIDI/SIDI Engines

    Energy Technology Data Exchange (ETDEWEB)

    Glass, R.S.

    2000-03-01

    To reduce U.S. dependence on imported oil, improve urban air quality, and decrease greenhouse gas emissions, the Department of Energy (DOE) is developing advanced vehicle technologies and fuels. Enabling technologies for fuel cell power systems and direct-injection engines are being developed by DOE through the Partnership for a New Generation of Vehicles (PNGV), a government-industry collaboration to produce vehicles having up to three times the fuel economy of conventional mid-size automobiles. Sensors have been identified as a research and development need for both fuel cell and direct-injection systems, because current sensor technologies do not adequately meet requirements. Sensors are needed for emission control, for passenger safety and comfort, to increase system lifetime, and for system performance enhancement through feedback and control. These proceedings document the results of a workshop to define sensor requirements for proton exchange membrane (PEM) fuel cell systems and direct-injection engines for automotive applications. The recommendations from this workshop will be incorporated into the multi-year R&D plan of the DOE Office of Advanced Automotive Technologies. The objectives of the workshop were to: define the requirements for sensors; establish R&D priorities; identify the technical targets and technical barriers; and facilitate collaborations among participants. The recommendations from this workshop will be incorporated into the multi-year R&D plan of the DOE Office of Advanced Automotive Technologies.

  8. A novel isoform of MAP4 organises the paraxial microtubule array required for muscle cell differentiation.

    Science.gov (United States)

    Mogessie, Binyam; Roth, Daniel; Rahil, Zainab; Straube, Anne

    2015-01-01

    The microtubule cytoskeleton is critical for muscle cell differentiation and undergoes reorganisation into an array of paraxial microtubules, which serves as template for contractile sarcomere formation. In this study, we identify a previously uncharacterised isoform of microtubule-associated protein MAP4, oMAP4, as a microtubule organising factor that is crucial for myogenesis. We show that oMAP4 is expressed upon muscle cell differentiation and is the only MAP4 isoform essential for normal progression of the myogenic differentiation programme. Depletion of oMAP4 impairs cell elongation and cell-cell fusion. Most notably, oMAP4 is required for paraxial microtubule organisation in muscle cells and prevents dynein- and kinesin-driven microtubule-microtubule sliding. Purified oMAP4 aligns dynamic microtubules into antiparallel bundles that withstand motor forces in vitro. We propose a model in which the cooperation of dynein-mediated microtubule transport and oMAP4-mediated zippering of microtubules drives formation of a paraxial microtubule array that provides critical support for the polarisation and elongation of myotubes. PMID:25898002

  9. Appressorium formation in the corn smut fungus Ustilago maydis requires a G2 cell cycle arrest.

    Science.gov (United States)

    Castanheira, Sónia; Pérez-Martín, José

    2015-01-01

    Many of the most important plant diseases are caused by fungal pathogens that form specialized cell structures to breach the leaf surface as well as to proliferate inside the plant. To initiate pathogenic development, the fungus responds to a set of inductive cues. Some of them are of extracellular nature (environmental signals) while others respond to intracellular conditions (developmental signals). These signals have to be integrated into a single response that has as a major outcome changes in the morphogenesis of the fungus. The cell cycle regulation is pivotal during these cellular differentiations, and we hypothesized that cell cycle regulation would be likely to provide control points for infection development by fungal pathogens. Although efforts have been done in various fungal systems, there is still limited information available regarding the relationship of these processes with the induction of the virulence programs. Hence, the role of fungal cell cycle regulators -which are wide conserved elements- as true virulence factors, has yet to be defined. Here we discuss the recent finding that the formation of the appressorium, a structure required for plant penetration, in the corn smut fungus Ustilago maydis seems to be incompatible with an active cell cycle and, therefore genetic circuits evolved in this fungus to arrest the cell cycle during the growth of this fungus on plant surface, before the appressorium-mediated penetration into the plant tissue.

  10. Autophagy is Required for the Maintenance of Liver Progenitor Cell Functionality

    Directory of Open Access Journals (Sweden)

    Yiji Cheng

    2015-06-01

    Full Text Available Background: Liver progenitor cells (LPCs are bipotent stem cells existing in the adult liver, which could be activated upon massive liver injury and contribute to liver regeneration. However, mechanisms of maintenance of LPC functionality remain poorly understood. Previous studies found that autophagy was required for the self-renewal and differentiation of several tissue stem cells. Methods: The study compared the level of autophagic activity in LPCs and differentiated hepatocytes. Then, autophagic activity was inhibited in LPCs by lentivirus-mediated autophagy-related gene 5 or Beclin 1 knockdown. Clonogenic assay, cell viability assays, hepatic differentiation assay, and senescence analysis were conducted to assess the role of autophagy in regulating self-renewal, hepatic differentiation and senescence of LPCs. Results: We observed high autophagic activity in LPCs compared with differentiated hepatocytes. We found that inhibition of autophagy impaired the self-renewal, proliferation, and hepatic differentiation capability of LPCs under normal cultural condition, but had little impact on cell viability. Interestingly, while wild-type LPCs remained rarely affected by the toxin, etoposide, inhibition of autophagy induced the senescent phenotype of LPCs. Overexpression of Beclin 1 in Beclin 1-knockdown LPCs restored the functionality of stem cells. Conclusion: Our findings indicate that autophagy may function as a critical regulator of LPC functionality under both physiological and pathological condition.

  11. Msx genes define a population of mural cell precursors required for head blood vessel maturation.

    Science.gov (United States)

    Lopes, Miguel; Goupille, Olivier; Saint Cloment, Cécile; Lallemand, Yvan; Cumano, Ana; Robert, Benoît

    2011-07-01

    Vessels are primarily formed from an inner endothelial layer that is secondarily covered by mural cells, namely vascular smooth muscle cells (VSMCs) in arteries and veins and pericytes in capillaries and veinules. We previously showed that, in the mouse embryo, Msx1(lacZ) and Msx2(lacZ) are expressed in mural cells and in a few endothelial cells. To unravel the role of Msx genes in vascular development, we have inactivated the two Msx genes specifically in mural cells by combining the Msx1(lacZ), Msx2(lox) and Sm22α-Cre alleles. Optical projection tomography demonstrated abnormal branching of the cephalic vessels in E11.5 mutant embryos. The carotid and vertebral arteries showed an increase in caliber that was related to reduced vascular smooth muscle coverage. Taking advantage of a newly constructed Msx1(CreERT2) allele, we demonstrated by lineage tracing that the primary defect lies in a population of VSMC precursors. The abnormal phenotype that ensues is a consequence of impaired BMP signaling in the VSMC precursors that leads to downregulation of the metalloprotease 2 (Mmp2) and Mmp9 genes, which are essential for cell migration and integration into the mural layer. Improper coverage by VSMCs secondarily leads to incomplete maturation of the endothelial layer. Our results demonstrate that both Msx1 and Msx2 are required for the recruitment of a population of neural crest-derived VSMCs.

  12. Nicotinamide phosphoribosyl transferase (Nampt is required for de novo lipogenesis in tumor cells.

    Directory of Open Access Journals (Sweden)

    Sarah C Bowlby

    Full Text Available Tumor cells have increased metabolic requirements to maintain rapid growth. In particular, a highly lipogenic phenotype is a hallmark of many tumor types, including prostate. Cancer cells also have increased turnover of nicotinamide adenine dinucleotide (NAD(+, a coenzyme involved in multiple metabolic pathways. However, a specific role for NAD(+ in tumor cell lipogenesis has yet to be described. Our studies demonstrate a novel role for the NAD(+-biosynthetic enzyme Nicotinamide phosphoribosyltransferase (Nampt in maintaining de novo lipogenesis in prostate cancer (PCa cells. Inhibition of Nampt reduces fatty acid and phospholipid synthesis. In particular, short chain saturated fatty acids and the phosphatidylcholine (PC lipids into which these fatty acids are incorporated were specifically reduced by Nampt inhibition. Nampt blockade resulted in reduced ATP levels and concomitant activation of AMP-activated protein kinase (AMPK and phosphorylation of acetyl-CoA carboxylase (ACC. In spite of this, pharmacological inhibition of AMPK was not sufficient to fully restore fatty acid synthesis. Rather, Nampt blockade also induced protein hyperacetylation in PC-3, DU145, and LNCaP cells, which correlated with the observed decreases in lipid synthesis. Moreover, the sirtuin inhibitor Sirtinol, and the simultaneous knockdown of SIRT1 and SIRT3, phenocopied the effects of Nampt inhibition on fatty acid synthesis. Altogether, these data reveal a novel role for Nampt in the regulation of de novo lipogenesis through the modulation of sirtuin activity in PCa cells.

  13. Oxidation of Alpha-Ketoglutarate Is Required for Reductive Carboxylation in Cancer Cells with Mitochondrial Defects

    Directory of Open Access Journals (Sweden)

    Andrew R. Mullen

    2014-06-01

    Full Text Available Mammalian cells generate citrate by decarboxylating pyruvate in the mitochondria to supply the tricarboxylic acid (TCA cycle. In contrast, hypoxia and other impairments of mitochondrial function induce an alternative pathway that produces citrate by reductively carboxylating α-ketoglutarate (AKG via NADPH-dependent isocitrate dehydrogenase (IDH. It is unknown how cells generate reducing equivalents necessary to supply reductive carboxylation in the setting of mitochondrial impairment. Here, we identified shared metabolic features in cells using reductive carboxylation. Paradoxically, reductive carboxylation was accompanied by concomitant AKG oxidation in the TCA cycle. Inhibiting AKG oxidation decreased reducing equivalent availability and suppressed reductive carboxylation. Interrupting transfer of reducing equivalents from NADH to NADPH by nicotinamide nucleotide transhydrogenase increased NADH abundance and decreased NADPH abundance while suppressing reductive carboxylation. The data demonstrate that reductive carboxylation requires bidirectional AKG metabolism along oxidative and reductive pathways, with the oxidative pathway producing reducing equivalents used to operate IDH in reverse.

  14. Activation of resting human T cells requires prolonged stimulation of protein kinase C

    Energy Technology Data Exchange (ETDEWEB)

    Berry, N.; Ase, K.; Kishimoto, A.; Nishizuka, Y. (Kobe Univ. School of Medicine (Japan))

    1990-03-01

    Purified resting human T cells can be induced to express the {alpha} subunit of the interleukin 2 receptor and to proliferate by treatment with 12-0-tetradecanoylphorbol-13-acetate plus ionomycin but not with 1,2-dioctanoylglycerol plus ionomycin. Determination of the translocation of protein kinase C showed that 12-0-tetradecanoylphorbol-13-acetate plus ionomycin caused a prolonged membrane association of the enzyme for more than 4 hr, whereas 1,2-dioctanoylglycerol plus ionomycin induced a transient membrane association, which was maximal at 20 min. Delivery of multiple additions of 1,2-dioctanoylglycerol plus ionomycin to the T cells resulted in progressively increased expression of the {alpha} subunit of the interleukin 2 receptor and proliferation commensurate with the number of multiple additions delivered, suggesting that prolonged protein kinase C activity is required for T-cell activation.

  15. Hydrogen Monitoring Requirements in the Global Technical Regulation on Hydrogen and Fuel Cell Vehicles: Preprint

    Energy Technology Data Exchange (ETDEWEB)

    Buttner, William; Rivkin, Carl; Burgess, Robert; Hartmann, Kevin; Bubar, Max; Post, Matthew; Boon-Brett, Lois; Weidner, Eveline; Moretto, Pietro

    2016-07-01

    The United Nations Global Technical Regulation (GTR) Number 13 (Global Technical Regulation on Hydrogen and Fuel Cell Vehicles) is the defining document regulating safety requirements in hydrogen vehicles, and in particular fuel cell electric vehicles (FCEV). GTR Number 13 has been formally implemented and will serve as the basis for the national regulatory standards for FCEV safety in North America (Canada, United States), Japan, Korea, and the European Union. The GTR defines safety requirement for these vehicles, including specifications on the allowable hydrogen levels in vehicle enclosures during in-use and post-crash conditions and on the allowable hydrogen emissions levels in vehicle exhaust during certain modes of normal operation. However, in order to be incorporated into national regulations, that is, in order to be binding, methods to verify compliance to the specific requirements must exist. In a collaborative program, the Sensor Laboratories at the National Renewable Energy Laboratory in the United States and the Joint Research Centre, Institute for Energy and Transport in the Netherlands have been evaluating and developing analytical methods that can be used to verify compliance to the hydrogen release requirement as specified in the GTR.

  16. Mitochondria are required for ATM activation by extranuclear oxidative stress in cultured human hepatoblastoma cell line Hep G2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Morita, Akinori, E-mail: morita@tokushima-u.ac.jp [Department of Radiation Medicine, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553 (Japan); Department of Radiological Science, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8509 (Japan); Tanimoto, Keiji; Murakami, Tomoki; Morinaga, Takeshi [Department of Radiation Medicine, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553 (Japan); Hosoi, Yoshio, E-mail: hosoi@med.tohoku.ac.jp [Department of Radiation Medicine, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553 (Japan); Department of Radiation Biology, Graduate School of Medicine, Tohoku University, Sendai 980-8575 (Japan)

    2014-01-24

    Highlights: • Oxidative ATM activation can occur in the absence of nuclear DNA damage response. • The oxidized Hep G2 cells were subjected to subcellular fractionation. • The obtained results suggest that the ATM activation occurs in mitochondria. • ATM failed to respond to oxidative stress in mitochondria-depleted Hep G2 cells. • Mitochondria are required for the oxidative activation of ATM. - Abstract: Ataxia–telangiectasia mutated (ATM) is a serine/threonine protein kinase that plays a central role in DNA damage response (DDR). A recent study reported that oxidized ATM can be active in the absence of DDR. However, the issue of where ATM is activated by oxidative stress remains unclear. Regarding the localization of ATM, two possible locations, namely, mitochondria and peroxisomes are possible. We report herein that ATM can be activated when exposed to hydrogen peroxide without inducing nuclear DDR in Hep G2 cells, and the oxidized cells could be subjected to subcellular fractionation. The first detergent-based fractionation experiment revealed that active, phosphorylated ATM was located in the second fraction, which also contained both mitochondria and peroxisomes. An alternative fractionation method involving homogenization and differential centrifugation, which permits the light membrane fraction containing peroxisomes to be produced, but not mitochondria, revealed that the light membrane fraction contained only traces of ATM. In contrast, the heavy membrane fraction, which mainly contained mitochondrial components, was enriched in ATM and active ATM, suggesting that the oxidative activation of ATM occurs in mitochondria and not in peroxisomes. In Rho 0-Hep G2 cells, which lack mitochondrial DNA and functional mitochondria, ATM failed to respond to hydrogen peroxide, indicating that mitochondria are required for the oxidative activation of ATM. These findings strongly suggest that ATM can be activated in response to oxidative stress in mitochondria

  17. Mitochondria are required for ATM activation by extranuclear oxidative stress in cultured human hepatoblastoma cell line Hep G2 cells

    International Nuclear Information System (INIS)

    Highlights: • Oxidative ATM activation can occur in the absence of nuclear DNA damage response. • The oxidized Hep G2 cells were subjected to subcellular fractionation. • The obtained results suggest that the ATM activation occurs in mitochondria. • ATM failed to respond to oxidative stress in mitochondria-depleted Hep G2 cells. • Mitochondria are required for the oxidative activation of ATM. - Abstract: Ataxia–telangiectasia mutated (ATM) is a serine/threonine protein kinase that plays a central role in DNA damage response (DDR). A recent study reported that oxidized ATM can be active in the absence of DDR. However, the issue of where ATM is activated by oxidative stress remains unclear. Regarding the localization of ATM, two possible locations, namely, mitochondria and peroxisomes are possible. We report herein that ATM can be activated when exposed to hydrogen peroxide without inducing nuclear DDR in Hep G2 cells, and the oxidized cells could be subjected to subcellular fractionation. The first detergent-based fractionation experiment revealed that active, phosphorylated ATM was located in the second fraction, which also contained both mitochondria and peroxisomes. An alternative fractionation method involving homogenization and differential centrifugation, which permits the light membrane fraction containing peroxisomes to be produced, but not mitochondria, revealed that the light membrane fraction contained only traces of ATM. In contrast, the heavy membrane fraction, which mainly contained mitochondrial components, was enriched in ATM and active ATM, suggesting that the oxidative activation of ATM occurs in mitochondria and not in peroxisomes. In Rho 0-Hep G2 cells, which lack mitochondrial DNA and functional mitochondria, ATM failed to respond to hydrogen peroxide, indicating that mitochondria are required for the oxidative activation of ATM. These findings strongly suggest that ATM can be activated in response to oxidative stress in mitochondria

  18. Solar cell development requires effective metrology: lock-in thermography can help

    International Nuclear Information System (INIS)

    The environmental and political benefits of renewable energy sources are understood by any informed observer with an interest in the future sustainability of our planet. Solar cells are getting a lot of attention - not only because they are a clean source of renewable energy, but also because their energy input is essentially free. Through the use of photovoltaic (PV) technology, solar cells convert the sun's rays directly into electricity. According to John Boyd, a technology analyst at Semiconductor Insights, 'a solar array 150 x 150 km could, in principle, meet all of North America's energy needs.' Assuming adequate installation space, and a solution for power grid load balancing, the main problem remaining to be solved is achieving grid parity - the point at which the cost of generating PV power is competitive with that of generating power using existing power plants. Currently, the cost of generating PV power is approximately $0.20/kWh globally. This is still roughly twice the rate of coal-based alternatives. The current generation of silicon solar cells typically achieves conversion efficiencies between 15% and 25%, while typical metallic thin film cells have efficiencies in the 5% to 20% range, depending on materials used. R and D efforts are aimed at increasing the efficiency of both solar cell technologies and reducing PV cell power generation costs to around $0.05/kWh. The primary challenges in reducing the cost of PV power generation exist in the production phase of the development cycle. Too many defects in the semiconducting material structure go undetected before solar cells are put into use. Identifying these defects requires efficient, cost effective test and measurement methods for characterizing a cell's performance and its electronic structure.(author)

  19. Analysis of dynamic requirements for fuel cell systems for vehicle applications

    Science.gov (United States)

    Pischinger, Stefan; Schönfelder, Carsten; Ogrzewalla, Jürgen

    Conventional vehicles with internal combustion engines, as well as battery powered electric vehicles, achieve one of the most important customer requirements; achieving extremely short response times to load changes. Also, fast acceleration times from a cold start to full power in the range of seconds are practicable. New fuel cell-based propulsion systems, as well as auxiliary power units, have to fulfill the same demands to become competitive. This includes heating-up the system to operating temperature as well as the control strategy for start-up. An additional device to supply starting air is necessary, if the compressor motor can only be operated with fuel cell voltage. Since the system components (for example, the air supply or the fuel supply) are not mechanically coupled, as is the case with conventional internal combustion engines, these components have to be controlled by different sensors and actuators. This can be an advantage in optimizing the system, but it also can represent an additional challenge. This paper describes the fuel cell system requirements regarding transient operation and their dependence on system structure. In particular, the requirements for peripheral components such as air supply, fuel supply and the balance of heat in a fuel cell system are examined. Furthermore, the paper outlines the necessity of an electric storage device and its resultant capacity, which will enable faster load changes. Acceleration and deceleration of the vehicle are accomplished through the use of the electric storage device, while the fuel cell system only has to deliver the mean power consumption without higher load peaks. On the basis of system simulation, different concepts are evaluated for use as a propulsion system or APU and, then, critical components are identified. The effects of advanced control strategies regarding the dynamic behavior of the system are demonstrated. Technically, a fuel cell system could be a viable propulsion system alternative

  20. The ligand binding domain of GCNF is not required for repression of pluripotency genes in mouse fetal ovarian germ cells.

    Directory of Open Access Journals (Sweden)

    Leah M Okumura

    Full Text Available In mice, successful development and reproduction require that all cells, including germ cells, transition from a pluripotent to a differentiated state. This transition is associated with silencing of the pluripotency genes Oct4 and Nanog. Interestingly, these genes are repressed at different developmental timepoints in germ and somatic cells. Ovarian germ cells maintain their expression until about embryonic day (E 14.5, whereas somatic cells silence them much earlier, at about E8.0. In both somatic cells and embryonic stem cells, silencing of Oct4 and Nanog requires the nuclear receptor GCNF. However, expression of the Gcnf gene has not been investigated in fetal ovarian germ cells, and whether it is required for silencing Oct4 and Nanog in that context is not known. Here we demonstrate that Gcnf is expressed in fetal ovarian germ cells, peaking at E14.5, when Oct4 and Nanog are silenced. However, conditional ablation of the ligand-binding domain of Gcnf using a ubiquitous, tamoxifen-inducible Cre indicates that Gcnf is not required for the down-regulation of pluripotency genes in fetal ovarian germ cells, nor is it required for initiation of meiosis and oogenesis. These results suggest that the silencing of Oct4 and Nanog in germ cells occurs via a different mechanism from that operating in somatic cells during gastrulation.

  1. Deletion of antigen-specific immature thymocytes by dendritic cells requires LFA-1/ICAM interactions.

    Science.gov (United States)

    Carlow, D A; van Oers, N S; Teh, S J; Teh, H S

    1992-03-15

    An in vitro assay was used for assessing the participation of various cell surface molecules and the efficacy of various cell types in the deletion of Ag-specific immature thymocytes. Thymocytes from mice expressing a transgenic TCR specific for the male Ag presented by the H-2Db class I MHC molecule were used as a target for deletion. In H-2d transgenic mice, cells bearing the transgenic TCR are not subjected to thymic selection as a consequence of the absence of the restricting H-2Db molecule but, nevertheless, express this TCR on the vast majority of immature CD4+8+ thymocytes. In this report we show that CD4+8+ thymocytes from H-2d TCR-transgenic mice are preferentially killed upon in vitro culture with male APC; DC were particularly effective in mediating in vitro deletion when compared with either B cells or T cells. Deletion of CD4+8+ thymocytes by DC was H-2b restricted and could be inhibited by mAb to either LFA-1 alpha or CD8. Partial inhibition was observed with mAb to ICAM-1, whereas mAb to CD4 and LFA-1 beta were without effect. These results are the first direct evidence of LFA-1 involvement in negative selection and provide further direct support for the participation of CD8/class I MHC interactions in this process. Like the requirements for deletion, activation of mature male-specific CD4-8+ T cells from female H-2b TCR-transgenic mice was also largely dependent on Ag presentation by DC and required both LFA-1/ICAM and CD8/class I MHC interactions; these results support the view that activation and deletion may represent maturation stage-dependent consequences of T cells encountering the same APC. Finally, our results also support the hypothesis that negative selection (deletion) does not require previous positive selection because deletion was observed under conditions where positive selection had not occurred.

  2. [Regulatory requirements regarding cell-based medicinal products for human and veterinary use - a comparison].

    Science.gov (United States)

    Kuhlmann-Gottke, Johanna; Duchow, Karin

    2015-11-01

    At present, there is no separate regulatory framework for cell-based medicinal products (CBMP) for veterinary use at the European or German level. Current European and national regulations exclusively apply to the corresponding medicinal products for human use. An increasing number of requests for the regulatory classification of CBMP for veterinary use, such as allogeneic stem cell preparations and dendritic cell-based autologous tumour vaccines, and a rise in scientific advice for companies developing these products, illustrate the need for adequate legislation. Currently, advice is given and decisions are made on a case-by-case basis regarding the regulatory classification and authorisation requirements.Since some of the CBMP - in particular in the area of stem-cell products - are developed in parallel for human and veterinary use, there is an urgent need to create specific legal definitions, regulations, and guidelines for these complex innovative products in the veterinary sector as well. Otherwise, there is a risk that that the current legal grey area regarding veterinary medicinal products will impede therapeutic innovations in the long run. A harmonised EU-wide approach is desirable. Currently the European legislation on veterinary medicinal products is under revision. In this context, veterinary therapeutics based on allogeneic cells and tissues will be defined and regulated. Certainly, the legal framework does not have to be as comprehensive as for human CBMP; a leaner solution is conceivable, similar to the special provisions for advanced-therapy medicinal products laid down in the German Medicines Act.

  3. Feeding cells induced by phytoparasitic nematodes require γ-tubulin ring complex for microtubule reorganization.

    Directory of Open Access Journals (Sweden)

    Mohamed Youssef Banora

    2011-12-01

    Full Text Available Reorganization of the microtubule network is important for the fast isodiametric expansion of giant-feeding cells induced by root-knot nematodes. The efficiency of microtubule reorganization depends on the nucleation of new microtubules, their elongation rate and activity of microtubule severing factors. New microtubules in plants are nucleated by cytoplasmic or microtubule-bound γ-tubulin ring complexes. Here we investigate the requirement of γ-tubulin complexes for giant feeding cells development using the interaction between Arabidopsis and Meloidogyne spp. as a model system. Immunocytochemical analyses demonstrate that γ-tubulin localizes to both cortical cytoplasm and mitotic microtubule arrays of the giant cells where it can associate with microtubules. The transcripts of two Arabidopsis γ-tubulin (TUBG1 and TUBG2 and two γ-tubulin complex proteins genes (GCP3 and GCP4 are upregulated in galls. Electron microscopy demonstrates association of GCP3 and γ-tubulin as part of a complex in the cytoplasm of giant cells. Knockout of either or both γ-tubulin genes results in the gene dose-dependent alteration of the morphology of feeding site and failure of nematode life cycle completion. We conclude that the γ-tubulin complex is essential for the control of microtubular network remodelling in the course of initiation and development of giant-feeding cells, and for the successful reproduction of nematodes in their plant hosts.

  4. Rapid CD4+ T-cell responses to bacterial flagellin require dendritic cell expression of Syk and CARD9.

    Science.gov (United States)

    Atif, Shaikh M; Lee, Seung-Joo; Li, Lin-Xi; Uematsu, Satoshi; Akira, Shizuo; Gorjestani, Sara; Lin, Xin; Schweighoffer, Edina; Tybulewicz, Victor L J; McSorley, Stephen J

    2015-02-01

    Toll-like receptors (TLRs) can recognize microbial patterns and utilize adaptor molecules, such as-MyD88 or (TRIF TIR-domain-containing adapter-inducing interferon-β), to initiate downstream signaling that ultimately affects the initiation of adaptive immunity. In addition to this inflammatory role, TLR5 expression on dendritic cells can favor antigen presentation of flagellin peptides and thus increase the sensitivity of flagellin-specific T-cell responses in vitro and in vivo. Here, we examined the role of alternative signaling pathways that might regulate flagellin antigen presentation in addition to MyD88. These studies suggest a requirement for spleen tyrosine kinase, a noncanonical TLR-signaling adaptor molecule, and its downstream molecule CARD9 in regulating the sensitivity of flagellin-specific CD4(+) T-cell responses in vitro and in vivo. Thus, a previously unappreciated signaling pathway plays an important role in regulating the dominance of flagellin-specific T-cell responses. PMID:25430631

  5. Efficient retina formation requires suppression of both Activin and BMP signaling pathways in pluripotent cells

    Directory of Open Access Journals (Sweden)

    Kimberly A. Wong

    2015-03-01

    Full Text Available Retina formation requires the correct spatiotemporal patterning of key regulatory factors. While it is known that repression of several signaling pathways lead to specification of retinal fates, addition of only Noggin, a known BMP antagonist, can convert pluripotent Xenopus laevis animal cap cells to functional retinal cells. The aim of this study is to determine the intracellular molecular events that occur during this conversion. Surprisingly, blocking BMP signaling alone failed to mimic Noggin treatment. Overexpressing Noggin in pluripotent cells resulted in a concentration-dependent suppression of both Smad1 and Smad2 phosphorylation, which act downstream of BMP and Activin signaling, respectively. This caused a decrease in downstream targets: endothelial marker, xk81, and mesodermal marker, xbra. We treated pluripotent cells with dominant-negative receptors or the chemical inhibitors, dorsomorphin and SB431542, which each target either the BMP or Activin signaling pathway. We determined the effect of these treatments on retina formation using the Animal Cap Transplant (ACT assay; in which treated pluripotent cells were transplanted into the eye field of host embryos. We found that inhibition of Activin signaling, in the presence of BMP signaling inhibition, promotes efficient retinal specification in Xenopus tissue, mimicking the affect of adding Noggin alone. In whole embryos, we found that the eye field marker, rax, expanded when adding both dominant-negative Smad1 and Smad2, as did treating the cells with both dorsomorphin and SB431542. Future studies could translate these findings to a mammalian culture assay, in order to more efficiently produce retinal cells in culture.

  6. Sp110 transcription is induced and required by Anaplasma phagocytophilum for infection of human promyelocytic cells

    Directory of Open Access Journals (Sweden)

    Naranjo Victoria

    2007-09-01

    Full Text Available Abstract Background The tick-borne intracellular pathogen, Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae causes human granulocytic anaplasmosis after infection of polymorphonuclear leucocytes. The human Sp110 gene is a member of the nuclear body (NB components that functions as a nuclear hormone receptor transcriptional coactivator and plays an important role in immunoprotective mechanisms against pathogens in humans. In this research, we hypothesized that Sp110 may be involved in the infection of human promyelocytic HL-60 cells with A. phagocytophilum. Methods The human Sp110 and A. phagocytophilum msp4 mRNA levels were evaluated by real-time RT-PCR in infected human HL-60 cells sampled at 0, 12, 24, 48, 72 and 96 hours post-infection. The effect of Sp110 expression on A. phagocytophilum infection was determined by RNA interference (RNAi. The expression of Sp110 was silenced in HL-60 cells by RNAi using pre-designed siRNAs using the Nucleofector 96-well shuttle system (Amaxa Biosystems, Gaithersburg, MD, USA. The A. phagocytophilum infection levels were evaluated in HL-60 cells after RNAi by real-time PCR of msp4 and normalizing against human Alu sequences. Results While Sp110 mRNA levels increased concurrently with A. phagocytophilum infections in HL-60 cells, the silencing of Sp110 expression by RNA interference resulted in decreased infection levels. Conclusion These results demonstrated that Sp110 expression is required for A. phagocytophilum infection and multiplication in HL-60 cells, and suggest a previously undescribed mechanism by which A. phagocytophilum modulates Sp110 mRNA levels to facilitate establishment of infection of human HL-60 cells.

  7. Matrix metalloproteinase-10 is required for lung cancer stem cell maintenance, tumor initiation and metastatic potential.

    Directory of Open Access Journals (Sweden)

    Verline Justilien

    Full Text Available Matrix metalloproteinases (Mmps stimulate tumor invasion and metastasis by degrading the extracellular matrix. Here we reveal an unexpected role for Mmp10 (stromelysin 2 in the maintenance and tumorigenicity of mouse lung cancer stem-like cells (CSC. Mmp10 is highly expressed in oncosphere cultures enriched in CSCs and RNAi-mediated knockdown of Mmp10 leads to a loss of stem cell marker gene expression and inhibition of oncosphere growth, clonal expansion, and transformed growth in vitro. Interestingly, clonal expansion of Mmp10 deficient oncospheres can be restored by addition of exogenous Mmp10 protein to the culture medium, demonstrating a direct role for Mmp10 in the proliferation of these cells. Oncospheres exhibit enhanced tumor-initiating and metastatic activity when injected orthotopically into syngeneic mice, whereas Mmp10-deficient cultures show a severe defect in tumor initiation. Conversely, oncospheres implanted into syngeneic non-transgenic or Mmp10(-/- mice show no significant difference in tumor initiation, growth or metastasis, demonstrating the importance of Mmp10 produced by cancer cells rather than the tumor microenvironment in lung tumor initiation and maintenance. Analysis of gene expression data from human cancers reveals a strong positive correlation between tumor Mmp10 expression and metastatic behavior in many human tumor types. Thus, Mmp10 is required for maintenance of a highly tumorigenic, cancer-initiating, metastatic stem-like cell population in lung cancer. Our data demonstrate for the first time that Mmp10 is a critical lung cancer stem cell gene and novel therapeutic target for lung cancer stem cells.

  8. Nrf2 is required to maintain the self-renewal of glioma stem cells

    International Nuclear Information System (INIS)

    Glioblastomas are deadly cancers that display a functional cellular hierarchy maintained by self-renewing glioma stem cells (GSCs). Self-renewal is a complex biological process necessary for maintaining the glioma stem cells. Nuclear factor rythroid 2-related factor 2(Nrf2) plays a significant role in protecting cells from endogenous and exogenous stresses. Nrf2 is a key nuclear transcription factor that regulates antioxidant response element (ARE)-containing genes. Previous studies have demonstrated the significant role of Nrf2 in the proliferation of glioblastoma, and in their resistance to radioactive therapies. We examined the effect of knocking down Nrf2 in GSCs. Nrf2 expression was down-regulated by shRNA transinfected with lentivirus. Expression levels of Nestin, Nrf2, BMI-1, Sox2 and Cyclin E were assessed by western blotting, quantitative polymerase chain reaction (qPCR) and immunohistochemistry analysis. The capacity for self-renewal in vitro was assessed by genesis of colonies. The capacity for self-renewal in vivo was analyzed by tumor genesis of xenografts in nude mice. Knockdown of Nrf2 inhibited the proliferation of GSCs, and significantly reduced the expression of BMI-1, Sox2 and CyclinE. Knocking down of Nrf2 changed the cell cycle distribution of GSCs by causing an uncharacteristic increase in the proportion of cells in the G2 phase and a decrease in the proportion of cells in the S phase of the cell cycle. Nrf2 is required to maintain the self-renewal of GSCs, and its down-regulation can attenuate the self-renewal of GSCs significantly

  9. No requirement of HCV 5'NCR for HCV-like particles assembly in insect cells

    Institute of Scientific and Technical Information of China (English)

    Wei Zhao; Guo-Yang Liao; Yah-Jun Jiang; Shu-De Jiang

    2003-01-01

    AIM: To express all three HCV structural proteins in the presence or absence of HCV 5'NCR to investigate the requirement of 5'NCR for the assembly of HCV-like particles in insect cells.METHODS: HCV structural protein encoding sequences CE1E2 and 5'NCR-CE1E2 were amplified with PCR.Recombinant baculovirus were constructed with recombinant DNA techniques. HCV structural proteins expressed in insect cells were analyzed by immunofluorescence and SDS-PAGE.Immunoprecipitation experiment of insect cell lysates with anti-E2 monodonal antibody (Mab) was carried out and the immunoprecipitated proteins were subjected to SDS-PAGE and immunoblotting with anti-C, anti-E2 Mabs and HCV positive serum. The virus-like particles in insect cells were visualized by electron microscopy (EM). The HCV-like particles were purified by sucrose gradient centrifugation and identified by EM and immune aggregation EM.RESULTS: The recombinant baculovirus reBV/CE1E2containing HCV C, E1, E2 genes and reBV/CS containing the same structural protein genes plus 5'NCR were constructed. The insect cells infected with either reBV/CE1E2or reBV/CS expressed HCV C, E1 and E2 proteins with a molecular weight of 20 kD, 35 kD and 66 kD respectively.The results of immunoprecipitation and the immunoblotting revealed the coimmunoprecipitation of C, E1, and E2proteins, indicating the interaction of HCV structural proteins expressed in insect cells. Electron microscopy of insect cells infected with reBV/CE1E2 or reBV/CS demonstrated spherical particles (40 to 60 nm in diameter)similar to the HCV virions from sera or hepatic tissues of HCV infected humans. The HCV-like particles were partially purified by sucrose gradient centrifugation, and the purified VLPs showed immuno-reactivity with anti-HCV antibodies.CONCLUSION: HCV 5'NCR is not required for the assembly of HCV-like particles in insect cells, HCV core and envelope proteins are sufficient for viral particle formation.

  10. AKT induces erythroid-cell maturation of JAK2-deficient fetal liver progenitor cells and is required for Epo regulation of erythroid-cell differentiation.

    Science.gov (United States)

    Ghaffari, Saghi; Kitidis, Claire; Zhao, Wei; Marinkovic, Dragan; Fleming, Mark D; Luo, Biao; Marszalek, Joseph; Lodish, Harvey F

    2006-03-01

    AKT serine threonine kinase of the protein kinase B (PKB) family plays essential roles in cell survival, growth, metabolism, and differentiation. In the erythroid system, AKT is known to be rapidly phosphorylated and activated in response to erythropoietin (Epo) engagement of Epo receptor (EpoR) and to sustain survival signals in cultured erythroid cells. Here we demonstrate that activated AKT complements EpoR signaling and supports erythroid-cell differentiation in wild-type and JAK2-deficient fetal liver cells. We show that erythroid maturation of AKT-transduced cells is not solely dependent on AKT-induced cell survival or proliferation signals, suggesting that AKT transduces also a differentiation-specific signal downstream of EpoR in erythroid cells. Down-regulation of expression of AKT kinase by RNA interference, or AKT activity by expression of dominant negative forms, inhibits significantly fetal liver-derived erythroid-cell colony formation and gene expression, demonstrating that AKT is required for Epo regulation of erythroid-cell maturation.

  11. Cutting edge: Human regulatory T cells require IL-35 to mediate suppression and infectious tolerance.

    Science.gov (United States)

    Chaturvedi, Vandana; Collison, Lauren W; Guy, Clifford S; Workman, Creg J; Vignali, Dario A A

    2011-06-15

    Human regulatory T cells (T(reg)) are essential for the maintenance of immune tolerance. However, the mechanisms they use to mediate suppression remain controversial. Although IL-35 has been shown to play an important role in T(reg)-mediated suppression in mice, recent studies have questioned its relevance in human T(reg). In this study, we show that human T(reg) express and require IL-35 for maximal suppressive capacity. Substantial upregulation of EBI3 and IL12A, but not IL10 and TGFB, was observed in activated human T(reg) compared with conventional T cells (T(conv)). Contact-independent T(reg)-mediated suppression was IL-35 dependent and did not require IL-10 or TGF-β. Lastly, human T(reg)-mediated suppression led to the conversion of the suppressed T(conv) into iTr35 cells, an IL-35-induced T(reg) population, in an IL-35-dependent manner. Thus, IL-35 contributes to human T(reg)-mediated suppression, and its conversion of suppressed target T(conv) into IL-35-induced T(reg) may contribute to infectious tolerance.

  12. The α isoform of topoisomerase II is required for hypercompaction of mitotic chromosomes in human cells.

    Science.gov (United States)

    Farr, Christine J; Antoniou-Kourounioti, Melissa; Mimmack, Michael L; Volkov, Arsen; Porter, Andrew C G

    2014-04-01

    As proliferating cells transit from interphase into M-phase, chromatin undergoes extensive reorganization, and topoisomerase (topo) IIα, the major isoform of this enzyme present in cycling vertebrate cells, plays a key role in this process. In this study, a human cell line conditional null mutant for topo IIα and a derivative expressing an auxin-inducible degron (AID)-tagged version of the protein have been used to distinguish real mitotic chromosome functions of topo IIα from its more general role in DNA metabolism and to investigate whether topo IIβ makes any contribution to mitotic chromosome formation. We show that topo IIβ does contribute, with endogenous levels being sufficient for the initial stages of axial shortening. However, a significant effect of topo IIα depletion, seen with or without the co-depletion of topo IIβ, is the failure of chromosomes to hypercompact when delayed in M-phase. This requires much higher levels of topo II protein and is impaired by drugs or mutations that affect enzyme activity. A prolonged delay at the G2/M border results in hyperefficient axial shortening, a process that is topo IIα-dependent. Rapid depletion of topo IIα has allowed us to show that its function during late G2 and M-phase is truly required for shaping mitotic chromosomes.

  13. Endogenous laminin is required for human airway smooth muscle cell maturation

    Directory of Open Access Journals (Sweden)

    Tran Thai

    2006-09-01

    Full Text Available Abstract Background Airway smooth muscle (ASM contraction underlies acute bronchospasm in asthma. ASM cells can switch between a synthetic-proliferative phenotype and a contractile phenotype. While the effects of extracellular matrix (ECM components on modulation of ASM cells to a synthetic phenotype have been reported, the role of ECM components on maturation of ASM cells to a contractile phenotype in adult lung is unclear. As both changes in ECM components and accumulation of contractile ASM are features of airway wall remodelling in asthma, we examined the role of the ECM protein, laminin, in the maturation of contractile phenotype in human ASM cells. Methods Human ASM cells were made senescence-resistant by stable expression of human telomerase reverse transcriptase. Maturation to a contractile phenotype was induced by 7-day serum deprivation, as assessed by immunoblotting for desmin and calponin. The role of laminin on ASM maturation was investigated by comparing the effects of exogenous laminin coated on culture plates, and of soluble laminin peptide competitors. Endogenous expression of laminin chains during ASM maturation was also measured. Results Myocyte binding to endogenously expressed laminin was required for ASM phenotype maturation, as laminin competing peptides (YIGSR or GRGDSP significantly reduced desmin and calponin protein accumulation that otherwise occurs with prolonged serum deprivation. Coating of plastic cell culture dishes with different purified laminin preparations was not sufficient to further promote accumulation of desmin or calponin during 7-day serum deprivation. Expression of α2, β1 and γ1 laminin chains by ASM cells was specifically up-regulated during myocyte maturation, suggesting a key role for laminin-2 in the development of the contractile phenotype. Conclusion While earlier reports suggest exogenously applied laminin slows the spontaneous modulation of ASM to a synthetic phenotype, we show for the

  14. HCN channels are not required for mechanotransduction in sensory hair cells of the mouse inner ear.

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    Geoffrey C Horwitz

    Full Text Available The molecular composition of the hair cell transduction channel has not been identified. Here we explore the novel hypothesis that hair cell transduction channels include HCN subunits. The HCN family of ion channels includes four members, HCN1-4. They were originally identified as the molecular correlates of the hyperpolarization-activated, cyclic nucleotide gated ion channels that carry currents known as If, IQ or Ih. However, based on recent evidence it has been suggested that HCN subunits may also be components of the elusive hair cell transduction channel. To investigate this hypothesis we examined expression of mRNA that encodes HCN1-4 in sensory epithelia of the mouse inner ear, immunolocalization of HCN subunits 1, 2 and 4, uptake of the transduction channel permeable dye, FM1-43 and electrophysiological measurement of mechanotransduction current. Dye uptake and transduction current were assayed in cochlear and vestibular hair cells of wildtype mice exposed to HCN channel blockers or a dominant-negative form of HCN2 that contained a pore mutation and in mutant mice that lacked HCN1, HCN2 or both. We found robust expression of HCNs 1, 2 and 4 but little evidence that localized HCN subunits in hair bundles, the site of mechanotransduction. Although high concentrations of the HCN antagonist, ZD7288, blocked 50-70% of the transduction current, we found no reduction of transduction current in either cochlear or vestibular hair cells of HCN1- or HCN2- deficient mice relative to wild-type mice. Furthermore, mice that lacked both HCN1 and HCN2 also had normal transduction currents. Lastly, we found that mice exposed to the dominant-negative mutant form of HCN2 had normal transduction currents as well. Taken together, the evidence suggests that HCN subunits are not required for mechanotransduction in hair cells of the mouse inner ear.

  15. DNA adenine methylation is required to replicate both Vibrio cholerae chromosomes once per cell cycle.

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    Gaëlle Demarre

    2010-05-01

    Full Text Available DNA adenine methylation is widely used to control many DNA transactions, including replication. In Escherichia coli, methylation serves to silence newly synthesized (hemimethylated sister origins. SeqA, a protein that binds to hemimethylated DNA, mediates the silencing, and this is necessary to restrict replication to once per cell cycle. The methylation, however, is not essential for replication initiation per se but appeared so when the origins (oriI and oriII of the two Vibrio cholerae chromosomes were used to drive plasmid replication in E. coli. Here we show that, as in the case of E. coli, methylation is not essential for oriI when it drives chromosomal replication and is needed for once-per-cell-cycle replication in a SeqA-dependent fashion. We found that oriII also needs SeqA for once-per-cell-cycle replication and, additionally, full methylation for efficient initiator binding. The requirement for initiator binding might suffice to make methylation an essential function in V. cholerae. The structure of oriII suggests that it originated from a plasmid, but unlike plasmids, oriII makes use of methylation for once-per-cell-cycle replication, the norm for chromosomal but not plasmid replication.

  16. PHGDH Expression Is Required for Mitochondrial Redox Homeostasis, Breast Cancer Stem Cell Maintenance, and Lung Metastasis.

    Science.gov (United States)

    Samanta, Debangshu; Park, Youngrok; Andrabi, Shaida A; Shelton, Laura M; Gilkes, Daniele M; Semenza, Gregg L

    2016-08-01

    Intratumoral hypoxia stimulates enrichment of breast cancer stem cells (BCSC), which are critical for metastasis and patient mortality. Here we report a metabolic adaptation that is required for hypoxia-induced BCSC enrichment and metastasis. Hypoxia-inducible factors coordinately regulate expression of genes encoding phosphoglycerate dehydrogenase (PHGDH) and five downstream enzymes in the serine synthesis pathway and mitochondrial one-carbon (folate) cycle. RNAi-mediated silencing of PHGDH expression in both estrogen receptor-positive and negative breast cancer cells led to decreased NADPH levels, disturbed mitochondrial redox homeostasis, and increased apoptosis, which abrogated BCSC enrichment under hypoxic conditions. PHGDH-deficient cells exhibited increased oxidant levels and apoptosis, as well as loss of BCSC enrichment, in response to treatment with carboplatin or doxorubicin. PHGDH-deficient cells were relatively weakly tumorigenic and tumors that did form were deficient in BCSCs, abolishing metastatic capacity. Our findings highlight a role for PHGDH in the formation of secondary (recurrent or metastatic) tumors, with potential implications for therapeutic targeting of advanced cancers. Cancer Res; 76(15); 4430-42. ©2016 AACR. PMID:27280394

  17. The necessary length of carbon nanotubes required to optimize solar cells

    Directory of Open Access Journals (Sweden)

    Barghi Tirdad

    2007-10-01

    Full Text Available Abstract Background In recent years scientists have been trying both to increase the efficiency of solar cells, whilst at the same time reducing dimensions and costs. Increases in efficiency have been brought about by implanting carbon nanotubes onto the surface of solar cells in order to reduce the reflection of sunrays, as well as through the insertion of polymeric arrays into the intrinsic layer for charge separation. Results The experimental results show power rising linearly for intrinsic layer thicknesses between 0–50 nm. Wider thicknesses increase the possibility of recombination of electrons and holes, leading to perturbation of the linear behaviour of output power. This effect is studied and formulated as a function of thickness. Recognition of the critical intrinsic layer thickness can permit one to determine the length of carbon nanotube necessary for optimizing solar cells. Conclusion In this study the behaviour of output power as a function of intrinsic layer thicknesses has been described physically and also simulated. In addition, the implantation of carbon nanotubes into the intrinsic layer and the necessary nanotube length required to optimize solar cells have been suggested.

  18. Recurrent invasive squamous cell carcinoma of the ocular surface requiring penetrating therapeutic sclerokeratoplasty

    Directory of Open Access Journals (Sweden)

    Mark J. Mannis

    2012-12-01

    Full Text Available Purpose: We review a case of invasive squamous cell carcinoma invading the cornea to discuss optimal management. Methods:  Observational case report with histopathologic analysis. Results: Histopathology demonstrates corneal invasion by the tumor that appears to have been completely excised with a large therapeutic keratoplasty and adjuvant cryotherapy. Conclusions: Successful management of ocular surface squamous neoplasia (OSSN requires removal of identifiably abnormal tissue without disruption of normal protective architecture, careful histopathologic analysis, and the employment of adjuvant therapy at the time of or subsequent to surgical excision.

  19. Genes of pyelonephritogenic E. coli required for digalactoside-specific agglutination of human cells.

    OpenAIRE

    Lindberg, F P; Lund, B; Normark, S

    1984-01-01

    Most pyelonephritic Escherichia coli strains bind to digalactoside-containing glycolipids on uroepithelial cells. Purified Pap pili (pili associated with pyelonephritis) show the same binding specificity. A non-polar mutation early in the papA pilin gene abolishes formation of Pap pili but does not affect the degree of digalactoside-specific hemagglutination. Three novel pap genes, papE , papF and papG are defined in this report. The papF and papG gene products are both required for digalacto...

  20. The cell-cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair

    DEFF Research Database (Denmark)

    Sørensen, Claus Storgaard; Hansen, Lasse Tengbjerg; Dziegielewski, Jaroslaw;

    2005-01-01

    The essential checkpoint kinase Chk1 is required for cell-cycle delays after DNA damage or blocked DNA replication. However, it is unclear whether Chk1 is involved in the repair of damaged DNA. Here we establish that Chk1 is a key regulator of genome maintenance by the homologous recombination......, the essential recombination repair protein RAD51 is recruited to DNA repair foci performing a vital role in correct HRR. We demonstrate that Chk1 interacts with RAD51, and that RAD51 is phosphorylated on Thr 309 in a Chk1-dependent manner. Consistent with a functional interplay between Chk1 and RAD51...

  1. Mortalin antibody-conjugated quantum dot transfer from human mesenchymal stromal cells to breast cancer cells requires cell–cell interaction

    International Nuclear Information System (INIS)

    The role of tumor stroma in regulation of breast cancer growth has been widely studied. However, the details on the type of heterocellular cross-talk between stromal and breast cancer cells (BCCs) are still poorly known. In the present study, in order to investigate the intercellular communication between human mesenchymal stromal cells (hMSCs) and breast cancer cells (BCCs, MDA-MB-231), we recruited cell-internalizing quantum dots (i-QD) generated by conjugation of cell-internalizing anti-mortalin antibody and quantum dots (QD). Co-culture of illuminated and color-coded hMSCs (QD655) and BCCs (QD585) revealed the intercellular transfer of QD655 signal from hMSCs to BCCs. The amount of QD double positive BCCs increased gradually within 48 h of co-culture. We found prominent intercellular transfer of QD655 in hanging drop co-culture system and it was non-existent when hMSCs and BBCs cells were co-cultured in trans-well system lacking imminent cell–cell contact. Fluorescent and electron microscope analyses also supported that the direct cell-to-cell interactions may be required for the intercellular transfer of QD655 from hMSCs to BCCs. To the best of our knowledge, the study provides a first demonstration of transcellular crosstalk between stromal cells and BCCs that involve direct contact and may also include a transfer of mortalin, an anti-apoptotic and growth-promoting factor enriched in cancer cells

  2. Mortalin antibody-conjugated quantum dot transfer from human mesenchymal stromal cells to breast cancer cells requires cell–cell interaction

    Energy Technology Data Exchange (ETDEWEB)

    Pietilä, Mika [National Institute of Advanced industrial Sciences and Technology, Tsukuba, Ibaraki 305 8562 (Japan); Lehenkari, Petri [Institute of Biomedicine, Department of Anatomy and Cell Biology, University of Oulu, Aapistie 7, P.O. Box 5000, FIN-90014 (Finland); Institute of Clinical Medicine, Division of Surgery, University of Oulu and Clinical Research Centre, Department of Surgery and Intensive Care, Oulu University Hospital, Aapistie 5a, P.O. Box 5000, FIN-90014 (Finland); Kuvaja, Paula [Institute of Biomedicine, Department of Anatomy and Cell Biology, University of Oulu, Aapistie 7, P.O. Box 5000, FIN-90014 (Finland); Department of Pathology, Oulu University Hospital, P.O. Box 50, FIN-90029 OYS, Oulu (Finland); Kaakinen, Mika [Biocenter Oulu, University of Oulu, P.O. Box 5000, FI-90014 (Finland); Kaul, Sunil C.; Wadhwa, Renu [National Institute of Advanced industrial Sciences and Technology, Tsukuba, Ibaraki 305 8562 (Japan); Uemura, Toshimasa, E-mail: t.uemura@aist.go.jp [National Institute of Advanced industrial Sciences and Technology, Tsukuba, Ibaraki 305 8562 (Japan)

    2013-11-01

    The role of tumor stroma in regulation of breast cancer growth has been widely studied. However, the details on the type of heterocellular cross-talk between stromal and breast cancer cells (BCCs) are still poorly known. In the present study, in order to investigate the intercellular communication between human mesenchymal stromal cells (hMSCs) and breast cancer cells (BCCs, MDA-MB-231), we recruited cell-internalizing quantum dots (i-QD) generated by conjugation of cell-internalizing anti-mortalin antibody and quantum dots (QD). Co-culture of illuminated and color-coded hMSCs (QD655) and BCCs (QD585) revealed the intercellular transfer of QD655 signal from hMSCs to BCCs. The amount of QD double positive BCCs increased gradually within 48 h of co-culture. We found prominent intercellular transfer of QD655 in hanging drop co-culture system and it was non-existent when hMSCs and BBCs cells were co-cultured in trans-well system lacking imminent cell–cell contact. Fluorescent and electron microscope analyses also supported that the direct cell-to-cell interactions may be required for the intercellular transfer of QD655 from hMSCs to BCCs. To the best of our knowledge, the study provides a first demonstration of transcellular crosstalk between stromal cells and BCCs that involve direct contact and may also include a transfer of mortalin, an anti-apoptotic and growth-promoting factor enriched in cancer cells.

  3. Tubule-guided cell-to-cell movement of a plant virus requires class XI myosin motors.

    Directory of Open Access Journals (Sweden)

    Khalid Amari

    2011-10-01

    Full Text Available Cell-to-cell movement of plant viruses occurs via plasmodesmata (PD, organelles that evolved to facilitate intercellular communications. Viral movement proteins (MP modify PD to allow passage of the virus particles or nucleoproteins. This passage occurs via several distinct mechanisms one of which is MP-dependent formation of the tubules that traverse PD and provide a conduit for virion translocation. The MP of tubule-forming viruses including Grapevine fanleaf virus (GFLV recruit the plant PD receptors called Plasmodesmata Located Proteins (PDLP to mediate tubule assembly and virus movement. Here we show that PDLP1 is transported to PD through a specific route within the secretory pathway in a myosin-dependent manner. This transport relies primarily on the class XI myosins XI-K and XI-2. Inactivation of these myosins using dominant negative inhibition results in mislocalization of PDLP and MP and suppression of GFLV movement. We also found that the proper targeting of specific markers of the Golgi apparatus, the plasma membrane, PD, lipid raft subdomains within the plasma membrane, and the tonoplast was not affected by myosin XI-K inhibition. However, the normal tonoplast dynamics required myosin XI-K activity. These results reveal a new pathway of the myosin-dependent protein trafficking to PD that is hijacked by GFLV to promote tubule-guided transport of this virus between plant cells.

  4. The histone variant His2Av is required for adult stem cell maintenance in the Drosophila testis.

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    Jose Rafael Morillo Prado

    2013-11-01

    Full Text Available Many tissues are sustained by adult stem cells, which replace lost cells by differentiation and maintain their own population through self-renewal. The mechanisms through which adult stem cells maintain their identity are thus important for tissue homeostasis and repair throughout life. Here, we show that a histone variant, His2Av, is required cell autonomously for maintenance of germline and cyst stem cells in the Drosophila testis. The ATP-dependent chromatin-remodeling factor Domino is also required in this tissue for adult stem cell maintenance possibly by regulating the incorporation of His2Av into chromatin. Interestingly, although expression of His2Av was ubiquitous, its function was dispensable for germline and cyst cell differentiation, suggesting a specific role for this non-canonical histone in maintaining the stem cell state in these lineages.

  5. Differential requirement for the CD45 splicing regulator hnRNPLL for accumulation of NKT and conventional T cells.

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    Mehmet Yabas

    Full Text Available Natural killer T (NKT cells represent an important regulatory T cell subset that develops in the thymus and contains immature (NK1.1(lo and mature (NK1.1(hi cell subsets. Here we show in mice that an inherited mutation in heterogeneous ribonucleoprotein L-like protein (hnRNPLL(thunder, that shortens the survival of conventional T cells, has no discernible effect on NKT cell development, homeostasis or effector function. Thus, Hnrpll deficiency effectively increases the NKT∶T cell ratio in the periphery. However, Hnrpll mutation disrupts CD45RA, RB and RC exon silencing of the Ptprc mRNA in both NKT and conventional T cells, and leads to a comparably dramatic shift to high molecular weight CD45 isoforms. In addition, Hnrpll mutation has a cell intrinsic effect on the expression of the developmentally regulated cell surface marker NK1.1 on NKT cells in the thymus and periphery but does not affect cell numbers. Therefore our results highlight both overlapping and divergent roles for hnRNPLL between conventional T cells and NKT cells. In both cell subsets it is required as a trans-acting factor to regulate alternative splicing of the Ptprc mRNA, but it is only required for survival of conventional T cells.

  6. Glucose Uptake Is Limiting in T Cell Activation and Requires CD28-Mediated Akt-Dependent and Independent Pathways1

    OpenAIRE

    Jacobs, Sarah R.; Herman, Catherine E.; MacIver, Nancie J.; Wofford, Jessica A.; Wieman, Heather L.; Hammen, Jeremy J.; Rathmell, Jeffrey C.

    2008-01-01

    T cell activation potently stimulates cellular metabolism to support the elevated energetic and biosynthetic demands of growth, proliferation, and effector function. We show that glucose uptake is limiting in T cell activation and that CD28 costimulation is required to allow maximal glucose uptake following TCR stimulation by up-regulating expression and promoting the cell surface trafficking of the glucose transporter Glut1. Regulation of T cell glucose uptake and Glut1 was critical, as low ...

  7. Jun is required in Isl1-expressing progenitor cells for cardiovascular development.

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    Tao Zhang

    Full Text Available Jun is a highly conserved member of the multimeric activator protein 1 transcription factor complex and plays an important role in human cancer where it is known to be critical for proliferation, cell cycle regulation, differentiation, and cell death. All of these biological functions are also crucial for embryonic development. Although all Jun null mouse embryos die at mid-gestation with persistent truncus arteriosus, a severe cardiac outflow tract defect also seen in human congenital heart disease, the developmental mechanisms are poorly understood. Here we show that murine Jun is expressed in a restricted pattern in several cell populations important for cardiovascular development, including the second heart field, pharyngeal endoderm, outflow tract and atrioventricular endocardial cushions and post-migratory neural crest derivatives. Several genes, including Isl1, molecularly mark the second heart field. Isl1 lineages include myocardium, smooth muscle, neural crest, endocardium, and endothelium. We demonstrate that conditional knockout mouse embryos lacking Jun in Isl1-expressing progenitors display ventricular septal defects, double outlet right ventricle, semilunar valve hyperplasia and aortic arch artery patterning defects. In contrast, we show that conditional deletion of Jun in Tie2-expressing endothelial and endocardial precursors does not result in aortic arch artery patterning defects or embryonic death, but does result in ventricular septal defects and a low incidence of semilunar valve defects, atrioventricular valve defects and double outlet right ventricle. Our results demonstrate that Jun is required in Isl1-expressing progenitors and, to a lesser extent, in endothelial cells and endothelial-derived endocardium for cardiovascular development but is dispensable in both cell types for embryonic survival. These data provide a cellular framework for understanding the role of Jun in the pathogenesis of congenital heart disease.

  8. Adaptation of Cholesterol Requiring NS0 Cells to Serum Free Culture Conditions

    Directory of Open Access Journals (Sweden)

    Junaid Muneer Raja

    2011-12-01

    Full Text Available Normal 0 false false false EN-US X-NONE X-NONE Colorectal cancer is the third most common form of cancer and the second leading cause of cancer-related death in the Western world. The answers to such life threatening diseases and cancers are monoclonal antibodies (MAb's which are widely used as therapeutic agents. World demand for currently approved MAb's is on the order of a few kilograms per year. However, new therapeutic MAb's are under development and require doses of several hundred milligrams to a gram over the course of therapy. Very often to cater for the special requirements for the growth of mammalian cells, serum is added to the cell culture medium. However, removal of serum from the cell culture medium is often carried out, especially if the end product is to be used for human consumption, in order to eliminate various disadvantages such as high physiological variability, high batch to batch variability, risk of contamination and high cost, and challenges posed in the downstream processing of the product. In this paper, the adaptation of cholesterol requiring NS0 cells to commercially available serum free media is presented. ABSTRAK: Kanser kolorektum merupakan kanser ketiga paling umum dan kini berada di tempat kedua penyebab kematian berkaitan kanser di negara Barat. Jawapan kepada penyakit yang mengancam nyawa dan penyakit kanser adalah antibodi monoklon (monoclonal antibodies ((MAb's yang digunakan sebagai agen terapeutik. Permintaan dunia terhadap MAb's yang diluluskan adalah dalam bilangan beberapa kilogram setahun. Namun, terapeutik MAb's yang baru adalah di bawah penyelidikan dan memerlukan beberapa ratus dos milligram hingga satu gram dalam satu peringkat terapi. Sering kali untuk memenuhi permintaan terhadap tumbesaran sel mamalia, serum dicampurkan dengan sel kultur perantara. Walaupun begitu, pemindahan serum dari sel kultur perantara sering dilakukan, terutamanya jika produk akhir digunakan untuk kegunaan manusia; untuk

  9. Mind bomb-1 in dendritic cells is specifically required for Notch-mediated T helper type 2 differentiation.

    Directory of Open Access Journals (Sweden)

    Hyun-Woo Jeong

    Full Text Available In dendritic cell (DC-CD4(+ T cell interaction, Notch signaling has been implicated in the CD4(+ T cell activation, proliferation, and subset differentiation. However, there has been a lot of debate on the exact role of Notch signaling. Here, we observed that expression of Mind bomb-1 (Mib1, a critical regulator of Notch ligands for the activation of Notch signaling, increases gradually as precursor cells differentiate into DCs in mice. To clarify the role of Mib1 in DC-CD4(+ T cell interactions, we generated Mib1-null bone marrow-derived DCs. These cells readily expressed Notch ligands but failed to initiate Notch activation in the adjacent cells. Nevertheless, Mib1-null DCs were able to prime the activation and proliferation of CD4(+ T cells, suggesting that Notch activation in CD4(+ T cells is not required for these processes. Intriguingly, stimulation of CD4(+ T cells with Mib1-null DCs resulted in dramatically diminished Th2 cell populations, while preserving Th1 cell populations, both in vitro and in vivo. Our results demonstrate that Mib1 in DCs is critical for the activation of Notch signaling in CD4(+ T cells, and Notch signaling reinforces Th2 differentiation, but is not required for the activation or proliferation of the CD4(+ T cells.

  10. PDGF is required for remyelination-promoting IgM stimulation of oligodendrocyte progenitor cell proliferation.

    Directory of Open Access Journals (Sweden)

    Jens O Watzlawik

    Full Text Available BACKGROUND: Promotion of remyelination is a major goal in treating demyelinating diseases such as multiple sclerosis (MS. The recombinant human monoclonal IgM, rHIgM22, targets myelin and oligodendrocytes (OLs and promotes remyelination in animal models of MS. It is unclear whether rHIgM22-mediated stimulation of lesion repair is due to promotion of oligodendrocyte progenitor cell (OPC proliferation and survival, OPC differentiation into myelinating OLs or protection of mature OLs. It is also unknown whether astrocytes or microglia play a functional role in IgM-mediated lesion repair. METHODS: We assessed the effect of rHIgM22 on cell proliferation in mixed CNS glial and OPC cultures by tritiated-thymidine uptake and by double-label immunocytochemistry using the proliferation marker, Ki-67. Antibody-mediated signaling events, OPC differentiation and OPC survival were investigated and quantified by Western blots. RESULTS: rHIgM22 stimulates OPC proliferation in mixed glial cultures but not in purified OPCs. There is no proliferative response in astrocytes or microglia. rHIgM22 activates PDGFαR in OPCs in mixed glial cultures. Blocking PDGFR-kinase inhibits rHIgM22-mediated OPC proliferation in mixed glia. We confirm in isolated OPCs that rHIgM22-mediated anti-apoptotic signaling and inhibition of OPC differentiation requires PDGF and FGF-2. We observed no IgM-mediated effect in mature OLs in the absence of PDGF and FGF-2. CONCLUSION: Stimulation of OPC proliferation by rHIgM22 depends on co-stimulatory astrocytic and/or microglial factors. We demonstrate that rHIgM22-mediated activation of PDGFαR is required for stimulation of OPC proliferation. We propose that rHIgM22 lowers the PDGF threshold required for OPC proliferation and protection, which can result in remyelination of CNS lesions.

  11. Human Cells Require Non-stop Ribosome Rescue Activity in Mitochondria.

    Directory of Open Access Journals (Sweden)

    Heather A Feaga

    2016-03-01

    Full Text Available Bacteria use trans-translation and the alternative rescue factors ArfA (P36675 and ArfB (Q9A8Y3 to hydrolyze peptidyl-tRNA on ribosomes that stall near the 3' end of an mRNA during protein synthesis. The eukaryotic protein ICT1 (Q14197 is homologous to ArfB. In vitro ribosome rescue assays of human ICT1 and Caulobacter crescentus ArfB showed that these proteins have the same activity and substrate specificity. Both ArfB and ICT1 hydrolyze peptidyl-tRNA on nonstop ribosomes or ribosomes stalled with ≤6 nucleotides extending past the A site, but are unable to hydrolyze peptidyl-tRNA when the mRNA extends ≥14 nucleotides past the A site. ICT1 provided sufficient ribosome rescue activity to support viability in C. crescentus cells that lacked both trans-translation and ArfB. Likewise, expression of ArfB protected human cells from death when ICT1 was silenced with siRNA. These data indicate that ArfB and ICT1 are functionally interchangeable, and demonstrate that ICT1 is a ribosome rescue factor. Because ICT1 is essential in human cells, these results suggest that ribosome rescue activity in mitochondria is required in humans.

  12. Cathepsin L is required for endothelial progenitor cell-induced neovascularization

    Energy Technology Data Exchange (ETDEWEB)

    Urbich, Carmen; Heeschen, Christopher; Aicher, Alexandra; Sasaki, Ken-ichiro; Bruhl, Thomas; Hofmann, Wolf K.; Peters, Christoph; Reinheckel, Thomas; Pennacchio, Len A.; Abolmaali, Nasreddin D.; Chavakis, Emmanouil; Zeiher, Andreas M.; Dimmeler, Stefanie

    2004-01-15

    Infusion of endothelial progenitor cells (EPCs), but not of mature endothelial cells (ECs), promotes neovascularization after ischemia. We performed a gene expression profiling of EPCs and ECs to identify genes, which might be important for the neovascularization capacity of EPCs. Intriguingly, the protease cathepsin L (CathL) was highly expressed in EPCs as opposed to ECs and is essential for matrix degradation and invasion by EPCs in vitro. CathL deficient mice showed impaired functional recovery after hind limb ischemia supporting the concept for an important role of CathL in postnatal neovascularization. Infused CathL deficient progenitor cells failed to home to sites of ischemia and to augment neovascularization. In contrast, over expression of CathL in mature ECs significantly enhanced their invasive activity and induced their neovascularization capacity in vivo. Taken together, CathL plays a crucial role for the integration of circulating EPCs into the ischemic tissue and is required for neovascularization mediated by EPCs.

  13. Autophagy-associated alpha-arrestin signaling is required for conidiogenous cell development in Magnaporthe oryzae

    Science.gov (United States)

    Dong, Bo; Xu, Xiaojin; Chen, Guoqing; Zhang, Dandan; Tang, Mingzhi; Xu, Fei; Liu, Xiaohong; Wang, Hua; Zhou, Bo

    2016-01-01

    Conidiation patterning is evolutionarily complex and mechanism concerning conidiogenous cell differentiation remains largely unknown. Magnaporthe oryzae conidiates in a sympodial way and uses its conidia to infect host and disseminate blast disease. Arrestins are multifunctional proteins that modulate receptor down-regulation and scaffold components of intracellular trafficking routes. We here report an alpha-arrestin that regulates patterns of conidiation and contributes to pathogenicity in M. oryzae. We show that disruption of ARRDC1 generates mutants which produce conidia in an acropetal array and ARRDC1 significantly affects expression profile of CCA1, a virulence-related transcription factor required for conidiogenous cell differentiation. Although germ tubes normally develop appressoria, penetration peg formation is dramatically impaired and Δarrdc1 mutants are mostly nonpathogenic. Fluorescent analysis indicates that EGFP-ARRDC1 puncta are well colocalized with DsRed2-Atg8, and this distribution profile could not be altered in Δatg9 mutants, suggesting ARRDC1 enters into autophagic flux before autophagosome maturation. We propose that M. oryzae employs ARRDC1 to regulate specific receptors in response to conidiation-related signals for conidiogenous cell differentiation and utilize autophagosomes for desensitization of conidiogenous receptor, which transmits extracellular signal to the downstream elements of transcription factors. Our investigation extends novel significance of autophagy-associated alpha-arrestin signaling to fungal parasites. PMID:27498554

  14. ATRX is required for maintenance of the neuroprogenitor cell pool in the embryonic mouse brain

    Directory of Open Access Journals (Sweden)

    Kieran Ritchie

    2014-11-01

    Full Text Available Mutations in the alpha-thalassemia mental retardation X-linked (ATRX gene cause a spectrum of abnormalities including intellectual disability, developmental delay, seizures, and microcephaly. The ATRX protein is highly enriched at heterochromatic repetitive sequences adjacent to the centromere, and ATRX depletion results in chromosome congression, segregation, and cohesion defects. Here, we show that Cre-mediated inactivation of Atrx in the embryonic mouse (Mus musculus brain results in expansion of cerebral cortical layer VI, and a concurrent thinning of layers II–IV. We observed increased cell cycle exit during early-mid neurogenesis, and a depletion of apical progenitors by late neurogenesis in the Atrx-null neocortex, explaining the disproportionate layering. Premature differentiation was associated with an increased generation of outer radial glia (oRG and TBR2-expressing basal progenitors, as well as increased generation of early-born post-mitotic projection neurons. Atrx deletion also reduced the fidelity of mitotic spindle orientation in apical progenitors, where mutant cells were often oriented at non-parallel angles of division relative to the ventricular surface. We conclude that ATRX is required for correct lamination of the mouse neocortex by regulating the timing of neuroprogenitor cell differentiation.

  15. A Bruno-like gene is required for stem cell maintenance in planarians.

    Science.gov (United States)

    Guo, Tingxia; Peters, Antoine H F M; Newmark, Phillip A

    2006-08-01

    The regenerative abilities of freshwater planarians are based on neoblasts, stem cells maintained throughout the animal's life. We show that a member of the Bruno-like family of RNA binding proteins is critical for regulating neoblasts in the planarian Schmidtea mediterranea. Smed-bruno-like (bruli) mRNA and protein are expressed in neoblasts and the central nervous system. Following bruli RNAi, which eliminates detectable Bruli protein, planarians initiate the proliferative response to amputation and form small blastemas but then undergo tissue regression and lysis. We characterize the neoblast population by using antibodies recognizing SMEDWI-1 and Histone H4 (monomethyl-K20) and cell-cycle markers to label subsets of neoblasts and their progeny. bruli knockdown results in a dramatic reduction/elimination of neoblasts. Our analyses indicate that neoblasts lacking Bruli can respond to wound stimuli and generate progeny that can form blastemas and differentiate; yet, they are unable to self-renew. These results suggest that Bruli is required for stem cell maintenance. PMID:16890156

  16. Zebrafish vasa is required for germ-cell differentiation and maintenance.

    Science.gov (United States)

    Hartung, Odelya; Forbes, Meredyth M; Marlow, Florence L

    2014-10-01

    Vasa is a universal marker of the germ line in animals, yet mutations disrupting vasa cause sexually dimorphic infertility, with impaired development of the ovary in some animals and the testis in others. The basis for this sexually dimorphic requirement for Vasa is not clear; in most animals examined, both the male and female gonad express vasa throughout the life of the germ line. Here we characterized a loss-of-function mutation disrupting zebrafish vasa. We show that maternally provided Vasa is stable through the first ten days of development in zebrafish, and thus likely fulfills any early roles for Vasa during germ-line specification, migration, survival, and maintenance. Although zygotic Vasa is not essential for the development of juvenile gonads, vasa mutants develop exclusively as sterile males. Furthermore, phenotypes of vasa;p53 compound mutants are indistinguishable from those of vasa mutants, therefore the failure of vasa mutants to differentiate as females and to support germ-cell development in the testis is not due to p53-mediated apoptosis. Instead, we found that failure to progress beyond the pachytene stage of meiosis causes the loss of germ-line stem cells, leaving empty somatic tubules. Our studies provide insight into the function of zebrafish vasa during female meiosis, differentiation, and maintenance of germ-line stem cells.

  17. The Protrusive Phase and Full Development of Integrin-Dependent Adhesions in Colon Epithelial Cells Require FAK- and ERKMediated Actin Spike Formation: Deregulation in Cancer Cells

    Directory of Open Access Journals (Sweden)

    Valerie G. Brunton

    2001-01-01

    Full Text Available Integrins play an important role in tumour progression by influencing cellular responses and matrix-dependent adhesion. However, the regulation of matrix-dependent adhesion assembly in epithelial cells is poorly understood. We have investigated the integrin and signalling requirements of cell-matrix adhesion assembly in colon carcinoma cells after plating on fibronectin. Adhesion assembly in these, and in the adenoma cells from which they were derived, was largely dependent on αvβ6 integrin and required phosphorylation of FAK on tyrosine-397. The rate of fibronectin-induced adhesion assembly and the expression of both αvβ6 integrin and FAK were increased during the adenoma-to-carcinoma transition. The matrix-dependent adhesion assembly process, particularly the final stages of complex protrusion that is required for optimal cell spreading, required the activity of extracellular signal-regulated kinase (ERK. Furthermore, phosphorylated ERK was targeted to newly forming cell-matrix adhesions in the carcinoma cells but not the adenoma cells, and inhibition of FAK-tyrosine-397 phosphorylation or MEK suppressed the appearance of phosphorylated ERK at peripheral sites. In addition, inhibition of MEK-ERK activation blocked the formation of peripheral actin microspikes that were necessary for the protrusive phase of cell-matrix adhesion assembly. Thus, MEK-ERK-dependent peripheral actin re-organization is required for the full development of integrin-induced adhesions and this pathway is stimulated in an in vitro model of colon cancer progression.

  18. Schistosoma mansoni-mediated suppression of allergic airway inflammation requires patency and Foxp3+ Treg cells.

    Directory of Open Access Journals (Sweden)

    Laura E Layland

    Full Text Available The continual rise of asthma in industrialised countries stands in strong contrast to the situation in developing lands. According to the modified Hygiene Hypothesis, helminths play a major role in suppressing bystander immune responses to allergens, and both epidemiological and experimental studies suggest that the tropical parasitic trematode Schistosoma mansoni elicits such effects. The focus of this study was to investigate which developmental stages of schistosome infection confer suppression of allergic airway inflammation (AAI using ovalbumin (OVA as a model allergen. Moreover, we assessed the functional role and localization of infection-induced CD4(+Foxp3(+ regulatory T cells (Treg in mediating such suppressive effects. Therefore, AAI was elicited using OVA/adjuvant sensitizations with subsequent OVA aerosolic challenge and was induced during various stages of infection, as well as after successful anti-helminthic treatment with praziquantel. The role of Treg was determined by specifically depleting Treg in a genetically modified mouse model (DEREG during schistosome infection. Alterations in AAI were determined by cell infiltration levels into the bronchial system, OVA-specific IgE and Th2 type responses, airway hyper-sensitivity and lung pathology. Our results demonstrate that schistosome infection leads to a suppression of OVA-induced AAI when mice are challenged during the patent phase of infection: production of eggs by fecund female worms. Moreover, this ameliorating effect does not persist after anti-helminthic treatment, and depletion of Treg reverts suppression, resulting in aggravated AAI responses. This is most likely due to a delayed reconstitution of Treg in infected-depleted animals which have strong ongoing immune responses. In summary, we conclude that schistosome-mediated suppression of AAI requires the presence of viable eggs and infection-driven Treg cells. These data provide evidence that helminth derived products

  19. Sox9 is required for precursor cell expansion and extracellular matrix organization during mouse heart valve development

    OpenAIRE

    Lincoln, Joy; Kist, Ralf; Scherer, Gerd; Yutzey, Katherine E.

    2007-01-01

    Heart valve structures derived from mesenchymal cells of the endocardial cushions (EC) are composed of highly organized cell lineages and extracellular matrix. Sox9 is a transcription factor required for both early and late stages of cartilage formation that is also expressed in the developing valves of the heart. The requirements for Sox9 function during valvulogenesis and adult valve homeostasis in mice were examined by conditional inactivation of Sox9 using Tie2-cre and Col2a1-cre transgen...

  20. Asymmetric Receptor Contact is Required for Tyrosine Autophosphorylation of Fibroblast Growth Factor Receptor in Living Cells

    Energy Technology Data Exchange (ETDEWEB)

    Bae, J.; Boggon, T; Tomé, F; Mandiyan, V; Lax, I; Schlessinge, J

    2010-01-01

    Tyrosine autophosphorylation of receptor tyrosine kinases plays a critical role in regulation of kinase activity and in recruitment and activation of intracellular signaling pathways. Autophosphorylation is mediated by a sequential and precisely ordered intermolecular (trans) reaction. In this report we present structural and biochemical experiments demonstrating that formation of an asymmetric dimer between activated FGFR1 kinase domains is required for transphosphorylation of FGFR1 in FGF-stimulated cells. Transphosphorylation is mediated by specific asymmetric contacts between the N-lobe of one kinase molecule, which serves as an active enzyme, and specific docking sites on the C-lobe of a second kinase molecule, which serves a substrate. Pathological loss-of-function mutations or oncogenic activating mutations in this interface may hinder or facilitate asymmetric dimer formation and transphosphorylation, respectively. The experiments presented in this report provide the molecular basis underlying the control of transphosphorylation of FGF receptors and other receptor tyrosine kinases.

  1. Industry requirements for introduction of alternative energies with emphasis on hydrogen fuel cells

    Energy Technology Data Exchange (ETDEWEB)

    Delabbio, F. [Rio Tinto, Canadian Exploration Ltd., Toronto, ON (Canada); Starbuck, D. [Newmont Mining Corp., Denver, CO (United States); Akerman, A. [CVRD-Inco, Toronto, ON (Canada); Betournay, M.C. [Natural Resources Canada, Ottawa, ON (Canada). CANMET Mining and Mineral Sciences Laboratories

    2007-07-01

    This paper discussed issues related to the use of alternate sources of energy in underground mining applications. Hydrogen power systems were examined in relation to operational drivers, available commercial supplies, site supplies, health and safety issues, capital and operating costs, mine production, and the role of government. Hydrogen power systems are being considered for mining applications in an effort to reduce greenhouse gas (GHG) emissions and reduce cooling and ventilation requirements. This article examined a range of issues that must be addressed before alternate energy systems such as hydrogen fuel cell technology can be used in larger-scale underground mining applications. The mining industry supports the development of new technologies. However, the introduction of alternate energy technologies must proceed in steps which include proof of concept testing, the development of generic infrastructure, power systems and regulations, and whole operating system studies. 13 refs., 1 fig.

  2. Pigment cell movement is not required for generation of Turing patterns in zebrafish skin

    Science.gov (United States)

    Bullara, D.; De Decker, Y.

    2015-01-01

    The zebrafish is a model organism for pattern formation in vertebrates. Understanding what drives the formation of its coloured skin motifs could reveal pivotal to comprehend the mechanisms behind morphogenesis. The motifs look and behave like reaction–diffusion Turing patterns, but the nature of the underlying physico-chemical processes is very different, and the origin of the patterns is still unclear. Here we propose a minimal model for such pattern formation based on a regulatory mechanism deduced from experimental observations. This model is able to produce patterns with intrinsic wavelength, closely resembling the experimental ones. We mathematically prove that their origin is a Turing bifurcation occurring despite the absence of cell motion, through an effect that we call differential growth. This mechanism is qualitatively different from the reaction–diffusion originally proposed by Turing, although they both generate the short-range activation and the long-range inhibition required to form Turing patterns. PMID:25959141

  3. Long-term T cell memory requires the surface expression of self-peptide/major histocompatibility complex molecules

    OpenAIRE

    Markiewicz, Mary A.; Girao, Cristina; Opferman, Joseph T.; Sun, Jiling; Hu, Qinghui; Agulnik, Alexander A.; Bishop, Colin E.; Thompson, Craig B.; Ashton-Rickardt, Philip G.

    1998-01-01

    How memory T cells are maintained in vivo is poorly understood. To address this problem, a male-specific peptide (H-Y) was identified and used to activate female anti-H-Y T cells in vitro. Anti-H-Y T cells survived in vivo for at least 70 days in the absence of antigen. This persistence was not because of the intrinsic ability of memory T cells to survive in vivo. Instead, the survival and function of adoptively transferred memory cells was found to require transporter of antigen protein 1-de...

  4. beta-Catenin signaling is required for TGF-beta(1)-induced extracellular matrix production by airway smooth muscle cells

    NARCIS (Netherlands)

    Baarsma, Hoeke A.; Menzen, Mark H.; Halayko, Andrew J.; Meurs, Herman; Kerstjens, Huib A. M.; Gosens, Reinoud

    2011-01-01

    Baarsma HA, Menzen MH, Halayko AJ, Meurs H, Kerstjens HA, Gosens R. beta-Catenin signaling is required for TGF-beta(1)-induced extracellular matrix production by airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 301: L956-L965, 2011. First published September 9, 2011; doi: 10.1152/ajplu

  5. MRG-1 is required for genomic integrity in Caenorhabditis elegans germ cells

    Institute of Scientific and Technical Information of China (English)

    Jing Xu; Xiaojuan Sun; Yudong Jing; Mo Wang; Kai Liu; Youli Jian; Mei Yang; Zhukuan Cheng; Chonglin Yang

    2012-01-01

    During meiotic cell division,proper chromosome synapsis and accurate repair of DNA double strand breaks (DSBs) are required to maintain genomic integrity,loss of which leads to apoptosis or meiotic defects.The mechanisms underlying meiotic chromosome synapsis,DSB repair and apoptosis are not fully understood.Here,we report that the chromodomain-containiug protein MRG-1 is an important factor for genomic integrity in meiosis in Caenorhabditis elegans.Loss ofmrg-1 function resulted in a significant increase in germ cell apoptosis that was partially inhibited by mutations affecting DNA damage checkpoint genes.Consistently,mrg-1 mutant germ lines exhibited SPO-11-generated DSBs and elevated exogenous DNA damage-induced chromosome fragmentation at diakinesis.In addition,the excessive apoptosis in mrg-1 mutants was partially suppressed by loss of the synapsis checkpoint gene pch-2,and a significant number of meiotic nuclei accumulated at the leptotene/zygotene stages with an elevated level of H3K9me2 on the chromatin,which was similarly observed in mutants deficient in the synaptonemal complex,suggesting that the proper progression of chromosome synapsis is likely impaired in the absence of mrg-1.Altogether,these findings suggest that MRG-1 is critical for genomic integrity by promoting meiotic DSB repair and synapsis progression in meiosis.

  6. Membrane repair of human skeletal muscle cells requires Annexin-A5.

    Science.gov (United States)

    Carmeille, Romain; Bouvet, Flora; Tan, Sisareuth; Croissant, Coralie; Gounou, Céline; Mamchaoui, Kamel; Mouly, Vincent; Brisson, Alain R; Bouter, Anthony

    2016-09-01

    Defect in membrane repair contributes to the development of limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. In healthy skeletal muscle, unraveling membrane repair mechanisms requires to establish an exhaustive list of the components of the resealing machinery. Here we show that human myotubes rendered deficient for Annexin-A5 (AnxA5) suffer from a severe defect in membrane resealing. This defect is rescued by the addition of recombinant AnxA5 while an AnxA5 mutant, which is unable to form 2D protein arrays, has no effect. Using correlative light and electron microscopy, we show that AnxA5 binds to the edges of the torn membrane, as early as a few seconds after sarcolemma injury, where it probably self-assembles into 2D arrays. In addition, we observed that membrane resealing is associated with the presence of a cluster of lipid vesicles at the wounded site. AnxA5 is present at the surface of these vesicles and may thus participate in plugging the cell membrane disruption. Finally, we show that AnxA5 behaves similarly in myotubes from a muscle cell line established from a patient suffering from LGMD2B, a myopathy due to dysferlin mutations, which indicates that trafficking of AnxA5 during sarcolemma damage is independent of the presence of dysferlin. PMID:27286750

  7. Lipopolysaccharide (LPS-Induced Biliary Epithelial Cell NRas Activation Requires Epidermal Growth Factor Receptor (EGFR.

    Directory of Open Access Journals (Sweden)

    Christy E Trussoni

    Full Text Available Cholangiocytes (biliary epithelial cells actively participate in microbe-induced proinflammatory responses in the liver and contribute to inflammatory and infectious cholangiopathies. We previously demonstrated that cholangiocyte TLR-dependent NRas activation contributes to proinflammatory/ proliferative responses. We test the hypothesis that LPS-induced activation of NRas requires the EGFR. SV40-transformed human cholangiocytes (H69 cells, or low passage normal human cholangiocytes (NHC, were treated with LPS in the presence or absence of EGFR or ADAM metallopeptidase domain 17 (TACE inhibitors. Ras activation assays, quantitative RT-PCR, and proliferation assays were performed in cells cultured with or without inhibitors or an siRNA to Grb2. Immunofluorescence for phospho-EGFR was performed on LPS-treated mouse samples and specimens from patients with primary sclerosing cholangitis, primary biliary cirrhosis, hepatitis C, and normal livers. LPS-treatment induced an association between the TLR/MyD88 and EGFR/Grb2 signaling apparatus, NRas activation, and EGFR phosphorylation. NRas activation was sensitive to EGFR and TACE inhibitors and correlated with EGFR phosphorylation. The TACE inhibitor and Grb2 depletion prevented LPS-induced IL6 expression (p<0.05 and proliferation (p<0.01. Additionally, cholangiocytes from LPS-treated mouse livers and human primary sclerosing cholangitis (PSC livers exhibited increased phospho-EGFR (p<0.01. Moreover, LPS-induced mouse cholangiocyte proliferation was inhibited by concurrent treatment with the EGFR inhibitor, Erlotinib. Our results suggest that EGFR is essential for LPS-induced, TLR4/MyD88-mediated NRas activation and induction of a robust proinflammatory cholangiocyte response. These findings have implications not only for revealing the signaling potential of TLRs, but also implicate EGFR as an integral component of cholangiocyte TLR-induced proinflammatory processes.

  8. Lipopolysaccharide (LPS)-Induced Biliary Epithelial Cell NRas Activation Requires Epidermal Growth Factor Receptor (EGFR).

    Science.gov (United States)

    Trussoni, Christy E; Tabibian, James H; Splinter, Patrick L; O'Hara, Steven P

    2015-01-01

    Cholangiocytes (biliary epithelial cells) actively participate in microbe-induced proinflammatory responses in the liver and contribute to inflammatory and infectious cholangiopathies. We previously demonstrated that cholangiocyte TLR-dependent NRas activation contributes to proinflammatory/ proliferative responses. We test the hypothesis that LPS-induced activation of NRas requires the EGFR. SV40-transformed human cholangiocytes (H69 cells), or low passage normal human cholangiocytes (NHC), were treated with LPS in the presence or absence of EGFR or ADAM metallopeptidase domain 17 (TACE) inhibitors. Ras activation assays, quantitative RT-PCR, and proliferation assays were performed in cells cultured with or without inhibitors or an siRNA to Grb2. Immunofluorescence for phospho-EGFR was performed on LPS-treated mouse samples and specimens from patients with primary sclerosing cholangitis, primary biliary cirrhosis, hepatitis C, and normal livers. LPS-treatment induced an association between the TLR/MyD88 and EGFR/Grb2 signaling apparatus, NRas activation, and EGFR phosphorylation. NRas activation was sensitive to EGFR and TACE inhibitors and correlated with EGFR phosphorylation. The TACE inhibitor and Grb2 depletion prevented LPS-induced IL6 expression (pphospho-EGFR (p<0.01). Moreover, LPS-induced mouse cholangiocyte proliferation was inhibited by concurrent treatment with the EGFR inhibitor, Erlotinib. Our results suggest that EGFR is essential for LPS-induced, TLR4/MyD88-mediated NRas activation and induction of a robust proinflammatory cholangiocyte response. These findings have implications not only for revealing the signaling potential of TLRs, but also implicate EGFR as an integral component of cholangiocyte TLR-induced proinflammatory processes.

  9. 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

  10. Stem-loop binding protein is required for retinal cell proliferation, neurogenesis, and intraretinal axon pathfinding in zebrafish.

    Science.gov (United States)

    Imai, Fumiyasu; Yoshizawa, Asuka; Matsuzaki, Ayako; Oguri, Eri; Araragi, Masato; Nishiwaki, Yuko; Masai, Ichiro

    2014-10-01

    In the developing retina, neurogenesis and cell differentiation are coupled with cell proliferation. However, molecular mechanisms that coordinate cell proliferation and differentiation are not fully understood. In this study, we found that retinal neurogenesis is severely delayed in the zebrafish stem-loop binding protein (slbp) mutant. SLBP binds to a stem-loop structure at the 3'-end of histone mRNAs, and regulates a replication-dependent synthesis and degradation of histone proteins. Retinal cell proliferation becomes slower in the slbp1 mutant, resulting in cessation of retinal stem cell proliferation. Although retinal stem cells cease proliferation by 2 days postfertilization (dpf) in the slbp mutant, retinal progenitor cells in the central retina continue to proliferate and generate neurons until at least 5dpf. We found that this progenitor proliferation depends on Notch signaling, suggesting that Notch signaling maintains retinal progenitor proliferation when faced with reduced SLBP activity. Thus, SLBP is required for retinal stem cell maintenance. SLBP and Notch signaling are required for retinal progenitor cell proliferation and subsequent neurogenesis. We also show that SLBP1 is required for intraretinal axon pathfinding, probably through morphogenesis of the optic stalk, which expresses attractant cues. Taken together, these data indicate important roles of SLBP in retinal development.

  11. WOX13-like genes are required for reprogramming of leaf and protoplast cells into stem cells in the moss Physcomitrella patens.

    Science.gov (United States)

    Sakakibara, Keiko; Reisewitz, Pascal; Aoyama, Tsuyoshi; Friedrich, Thomas; Ando, Sayuri; Sato, Yoshikatsu; Tamada, Yosuke; Nishiyama, Tomoaki; Hiwatashi, Yuji; Kurata, Tetsuya; Ishikawa, Masaki; Deguchi, Hironori; Rensing, Stefan A; Werr, Wolfgang; Murata, Takashi; Hasebe, Mitsuyasu; Laux, Thomas

    2014-04-01

    Many differentiated plant cells can dedifferentiate into stem cells, reflecting the remarkable developmental plasticity of plants. In the moss Physcomitrella patens, cells at the wound margin of detached leaves become reprogrammed into stem cells. Here, we report that two paralogous P. patens WUSCHEL-related homeobox 13-like (PpWOX13L) genes, homologs of stem cell regulators in flowering plants, are transiently upregulated and required for the initiation of cell growth during stem cell formation. Concordantly, Δppwox13l deletion mutants fail to upregulate genes encoding homologs of cell wall loosening factors during this process. During the moss life cycle, most of the Δppwox13l mutant zygotes fail to expand and initiate an apical stem cell to form the embryo. Our data show that PpWOX13L genes are required for the initiation of cell growth specifically during stem cell formation, in analogy to WOX stem cell functions in seed plants, but using a different cellular mechanism.

  12. Protein crystals in Adenovirus type 5-infected cells: requirements for intranuclear crystallogenesis, structural and functional analysis.

    Directory of Open Access Journals (Sweden)

    Laure Franqueville

    Full Text Available Intranuclear crystalline inclusions have been observed in the nucleus of epithelial cells infected with Adenovirus serotype 5 (Ad5 at late steps of the virus life cycle. Using immuno-electron microscopy and confocal microscopy of cells infected with various Ad5 recombinants modified in their penton base or fiber domains, we found that these inclusions represented crystals of penton capsomers, the heteromeric capsid protein formed of penton base and fiber subunits. The occurrence of protein crystals within the nucleus of infected cells required the integrity of the fiber knob and part of the shaft domain. In the knob domain, the region overlapping residues 489-492 in the FG loop was found to be essential for crystal formation. In the shaft, a large deletion of repeats 4 to 16 had no detrimental effect on crystal inclusions, whereas deletion of repeats 8 to 21 abolished crystal formation without altering the level of fiber protein expression. This suggested a crucial role of the five penultimate repeats in the crystallisation process. Chimeric pentons made of Ad5 penton base and fiber domains from different serotypes were analyzed with respect to crystal formation. No crystal was found when fiber consisted of shaft (S from Ad5 and knob (K from Ad3 (heterotypic S5-K3 fiber, but occurred with homotypic S3K3 fiber. However, less regular crystals were observed with homotypic S35-K35 fiber. TB5, a monoclonal antibody directed against the Ad5 fiber knob was found by immunofluorescence microscopy to react with high efficiency with the intranuclear protein crystals in situ. Data obtained with Ad fiber mutants indicated that the absence of crystalline inclusions correlated with a lower infectivity and/or lower yields of virus progeny, suggesting that the protein crystals might be involved in virion assembly. Thus, we propose that TB5 staining of Ad-infected 293 cells can be used as a prognostic assay for the viability and productivity of fiber-modified Ad5

  13. The Unfolded Protein Response Is Induced by the Cell Wall Integrity Mitogen-activated Protein Kinase Signaling Cascade and Is Required for Cell Wall Integrity in Saccharomyces cerevisiae

    OpenAIRE

    Scrimale, Thomas; DiDone, Louis; de Mesy Bentley, Karen L.; Krysan, Damian J.

    2009-01-01

    The yeast cell wall is an extracellular structure that is dependent on secretory and membrane proteins for its construction. We investigated the role of protein quality control mechanisms in cell wall integrity and found that the unfolded protein response (UPR) and, to a lesser extent, endoplasmic reticulum (ER)-associated degradation (ERAD) pathways are required for proper cell wall construction. Null mutation of IRE1, double mutation of ERAD components (hrd1Δ and ubc7Δ) and ire1Δ, or expres...

  14. Co-stimulation by anti-immunoglobulin is required for B cell activation by CD40Llow T cells

    DEFF Research Database (Denmark)

    Poudrier, J; Owens, T

    1994-01-01

    strongly. Low buoyant density B cells also responded to CD40Llow Th cells. There was no B cell response to resting Th cells. mAb against CD54/intercellular adhesion molecule-1 or major histocompatibility complex (MHC) class II completely inhibited B cell responses to CD40Llow Th1 cells, equivalent...... cell Ag specificity by using anti-CD3/T cell receptor (TcR) monoclonal antibodies (mAb) to activate T cells. To study the role of sIg engagement in the responsiveness of B cells to T help, we pre-treated small resting B cells with soluble anti-kappa mAb prior to contact with an activated Th1 clone....... By reducing the concentration of anti-TcR mAb we obtained low levels of CD40 ligand (CD40Llow) on Th cells, comparable to those expressed by lymph node T cells activated in vitro (ex vivo T cells). In contrast to untreated B cells, which did not respond to CD40Llow Th, anti-Ig-treated B cells responded...

  15. Surrogate light chain is required for central and peripheral B-cell tolerance and inhibits anti-DNA antibody production by marginal zone B cells.

    Science.gov (United States)

    Ren, Weicheng; Grimsholm, Ola; Bernardi, Angelina I; Höök, Nina; Stern, Anna; Cavallini, Nicola; Mårtensson, Inga-Lill

    2015-04-01

    Selection of the primary antibody repertoire takes place in pro-/pre-B cells, and subsequently in immature and transitional B cells. At the first checkpoint, μ heavy (μH) chains assemble with surrogate light (SL) chain into a precursor B-cell receptor. In mice lacking SL chain, μH chain selection is impaired, and serum autoantibody levels are elevated. However, whether the development of autoantibody-producing cells is due to an inability of the resultant B-cell receptors to induce central and/or peripheral B-cell tolerance or other factors is unknown. Here, we show that receptor editing is defective, and that a higher proportion of BM immature B cells are prone to undergoing apoptosis. Furthermore, transitional B cells are also more prone to undergoing apoptosis, with a stronger selection pressure to enter the follicular B-cell pool. Those that enter the marginal zone (MZ) B-cell pool escape selection and survive, possibly due to the B-lymphopenia and elevated levels of B-cell activating factor. Moreover, the MZ B cells are responsible for the elevated IgM anti-dsDNA antibody levels detected in these mice. Thus, the SL chain is required for central and peripheral B-cell tolerance and inhibits anti-DNA antibody production by MZ B cells.

  16. SAP expression in invariant NKT cells is required for cognate help to support B-cell responses.

    Science.gov (United States)

    Detre, Cynthia; Keszei, Marton; Garrido-Mesa, Natividad; Kis-Toth, Katalin; Castro, Wilson; Agyemang, Amma F; Veerapen, Natacha; Besra, Gurdyal S; Carroll, Michael C; Tsokos, George C; Wang, Ninghai; Leadbetter, Elizabeth A; Terhorst, Cox

    2012-07-01

    One of the manifestations of X-linked lymphoproliferative disease (XLP) is progressive agammaglobulinemia, caused by the absence of a functional signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) in T, invariant natural killer T (NKT) cells and NK cells. Here we report that α-galactosylceramide (αGalCer) activated NKT cells positively regulate antibody responses to haptenated protein antigens at multiple checkpoints, including germinal center formation and affinity maturation. Whereas NKT cell-dependent B cell responses were absent in SAP(-/-).B6 mice that completely lack NKT cells, the small number of SAP-deficient NKT cells in SAP(-/-).BALB/c mice adjuvated antibody production, but not the germinal center reaction. To test the hypothesis that SAP-deficient NKT cells can facilitate humoral immunity, SAP was deleted after development in SAP(fl/fl).tgCreERT2.B6 mice. We find that NKT cell intrinsic expression of SAP is dispensable for noncognate helper functions, but is critical for providing cognate help to antigen-specific B cells. These results demonstrate that SLAM-family receptor-regulated cell-cell interactions are not limited to T-B cell conjugates. We conclude that in the absence of SAP, several routes of NKT cell-mediated antibody production are still accessible. The latter suggests that residual NKT cells in XLP patients might contribute to variations in dysgammaglobulinemia.

  17. Notch1 is required in newly postmitotic cells to inhibit the rod photoreceptor fate

    OpenAIRE

    Mizeracka, Karolina; DeMaso, Christina R.; Cepko, Constance L.

    2013-01-01

    Several models of cell fate determination can be invoked to explain how single retinal progenitor cells (RPCs) produce different cell types in a terminal division. To gain insight into this process, the effects of the removal of a cell fate regulator, Notch1, were studied in newly postmitotic cells using a conditional allele of Notch1 (N1-CKO) in mice. Almost all newly postmitotic N1-CKO cells became rod photoreceptors, whereas wild-type (WT) cells achieved a variety of fates. Single cell pro...

  18. Requirements of transcription factor Smad-dependent and -independent TGF-β signaling to control discrete T-cell functions

    OpenAIRE

    Gu, Ai-di; Wang, Yunqi; Lin, Lin; Zhang, Song S.; Wan, Yisong Y.

    2012-01-01

    TGF-β modulates immune response by suppressing non-regulatory T (Treg) function and promoting Treg function. The question of whether TGF-β achieves distinct effects on non-Treg and Treg cells through discrete signaling pathways remains outstanding. In this study, we investigated the requirements of Smad-dependent and -independent TGF-β signaling for T-cell function. Smad2 and Smad3 double deficiency in T cells led to lethal inflammatory disorder in mice. Non-Treg cells were spontaneously acti...

  19. The Nanos3-3'UTR is required for germ cell specific NANOS3 expression in mouse embryos.

    Directory of Open Access Journals (Sweden)

    Hitomi Suzuki

    Full Text Available BACKGROUND: The regulation of gene expression via a 3' untranslated region (UTR plays essential roles in the discrimination of the germ cell lineage from somatic cells during embryogenesis. This is fundamental to the continuation of a species. Mouse NANOS3 is an essential protein required for the germ cell maintenance and is specifically expressed in these cells. However, the regulatory mechanisms that restrict the expression of this gene in the germ cells is largely unknown at present. METHODOLOGY/PRINCIPAL FINDINGS: In our current study, we show that differences in the stability of Nanos3 mRNA between germ cells and somatic cells is brought about in a 3'UTR-dependent manner in mouse embryos. Although Nanos3 is transcribed in both cell lineages, it is efficiently translated only in the germ lineage. We also find that the translational suppression of NANOS3 in somatic cells is caused by a 3'UTR-mediated mRNA destabilizing mechanism. Surprisingly, even when under the control of the CAG promoter which induces strong ubiquitous transcription in both germ cells and somatic cells, the addition of the Nanos3-3'UTR sequence to the coding region of exogenous gene was effective in restricting protein expression in germ cells. CONCLUSIONS/SIGNIFICANCE: Our current study thus suggests that Nanos3-3'UTR has an essential role in translational control in the mouse embryo.

  20. Myc and Fgf Are Required for Zebrafish Neuromast Hair Cell Regeneration.

    Directory of Open Access Journals (Sweden)

    Sang Goo Lee

    Full Text Available Unlike mammals, the non-mammalian vertebrate inner ear can regenerate the sensory cells, hair cells, either spontaneously or through induction after hair cell loss, leading to hearing recovery. The mechanisms underlying the regeneration are poorly understood. By microarray analysis on a chick model, we show that chick hair cell regeneration involves the activation of proliferation genes and downregulation of differentiation genes. Both MYC and FGF are activated in chick hair cell regeneration. Using a zebrafish lateral line neuromast hair cell regeneration model, we show that the specific inhibition of Myc or Fgf suppresses hair cell regeneration, demonstrating that both pathways are essential to the process. Rapid upregulation of Myc and delayed Fgf activation during regeneration suggest a role of Myc in proliferation and Fgf in differentiation. The dorsal-ventral pattern of fgfr1a in the neuromasts overlaps with the distribution of hair cell precursors. By laser ablation, we show that the fgfr1a-positive supporting cells are likely the hair cell precursors that directly give rise to new hair cells; whereas the anterior-posterior fgfr1a-negative supporting cells have heightened proliferation capacity, likely to serve as more primitive progenitor cells to replenish lost precursors after hair cell loss. Thus fgfr1a is likely to mark compartmentalized supporting cell subtypes with different capacities in renewal proliferation and hair cell regeneration. Manipulation of c-MYC and FGF pathways could be explored for mammalian hair cell regeneration.

  1. Myc and Fgf Are Required for Zebrafish Neuromast Hair Cell Regeneration

    Science.gov (United States)

    Obholzer, Nikolaus D.; Sun, Shan; Li, Wenyan; Petrillo, Marco; Dai, Pu; Zhou, Yi; Cotanche, Douglas A.; Megason, Sean G.; Li, Huawei; Chen, Zheng-Yi

    2016-01-01

    Unlike mammals, the non-mammalian vertebrate inner ear can regenerate the sensory cells, hair cells, either spontaneously or through induction after hair cell loss, leading to hearing recovery. The mechanisms underlying the regeneration are poorly understood. By microarray analysis on a chick model, we show that chick hair cell regeneration involves the activation of proliferation genes and downregulation of differentiation genes. Both MYC and FGF are activated in chick hair cell regeneration. Using a zebrafish lateral line neuromast hair cell regeneration model, we show that the specific inhibition of Myc or Fgf suppresses hair cell regeneration, demonstrating that both pathways are essential to the process. Rapid upregulation of Myc and delayed Fgf activation during regeneration suggest a role of Myc in proliferation and Fgf in differentiation. The dorsal-ventral pattern of fgfr1a in the neuromasts overlaps with the distribution of hair cell precursors. By laser ablation, we show that the fgfr1a-positive supporting cells are likely the hair cell precursors that directly give rise to new hair cells; whereas the anterior-posterior fgfr1a-negative supporting cells have heightened proliferation capacity, likely to serve as more primitive progenitor cells to replenish lost precursors after hair cell loss. Thus fgfr1a is likely to mark compartmentalized supporting cell subtypes with different capacities in renewal proliferation and hair cell regeneration. Manipulation of c-MYC and FGF pathways could be explored for mammalian hair cell regeneration. PMID:27351484

  2. Requirement of cAMP signaling for Schwann cell differentiation restricts the onset of myelination.

    Science.gov (United States)

    Bacallao, Ketty; Monje, Paula V

    2015-01-01

    Isolated Schwann cells (SCs) respond to cAMP elevation by adopting a differentiated post-mitotic state that exhibits high levels of Krox-20, a transcriptional enhancer of myelination, and mature SC markers such as the myelin lipid galactocerebroside (O1). To address how cAMP controls myelination, we performed a series of cell culture experiments which compared the differentiating responses of isolated and axon-related SCs to cAMP analogs and ascorbate, a known inducer of axon ensheathment, basal lamina formation and myelination. In axon-related SCs, cAMP induced the expression of Krox-20 and O1 without a concomitant increase in the expression of myelin basic protein (MBP) and without promoting axon ensheathment, collagen synthesis or basal lamina assembly. When cAMP was provided together with ascorbate, a dramatic enhancement of MBP expression occurred, indicating that cAMP primes SCs to form myelin only under conditions supportive of basal lamina formation. Experiments using a combination of cell permeable cAMP analogs and type-selective adenylyl cyclase (AC) agonists and antagonists revealed that selective transmembrane AC (tmAC) activation with forskolin was not sufficient for full SC differentiation and that the attainment of an O1 positive state also relied on the activity of the soluble AC (sAC), a bicarbonate sensor that is insensitive to forskolin and GPCR activation. Pharmacological and immunological evidence indicated that SCs expressed sAC and that sAC activity was required for morphological differentiation and the expression of myelin markers such as O1 and protein zero. To conclude, our data indicates that cAMP did not directly drive myelination but rather the transition into an O1 positive state, which is perhaps the most critical cAMP-dependent rate limiting step for the onset of myelination. The temporally restricted role of cAMP in inducing differentiation independently of basal lamina formation provides a clear example of the uncoupling of signals

  3. Requirement of cAMP signaling for Schwann cell differentiation restricts the onset of myelination.

    Directory of Open Access Journals (Sweden)

    Ketty Bacallao

    Full Text Available Isolated Schwann cells (SCs respond to cAMP elevation by adopting a differentiated post-mitotic state that exhibits high levels of Krox-20, a transcriptional enhancer of myelination, and mature SC markers such as the myelin lipid galactocerebroside (O1. To address how cAMP controls myelination, we performed a series of cell culture experiments which compared the differentiating responses of isolated and axon-related SCs to cAMP analogs and ascorbate, a known inducer of axon ensheathment, basal lamina formation and myelination. In axon-related SCs, cAMP induced the expression of Krox-20 and O1 without a concomitant increase in the expression of myelin basic protein (MBP and without promoting axon ensheathment, collagen synthesis or basal lamina assembly. When cAMP was provided together with ascorbate, a dramatic enhancement of MBP expression occurred, indicating that cAMP primes SCs to form myelin only under conditions supportive of basal lamina formation. Experiments using a combination of cell permeable cAMP analogs and type-selective adenylyl cyclase (AC agonists and antagonists revealed that selective transmembrane AC (tmAC activation with forskolin was not sufficient for full SC differentiation and that the attainment of an O1 positive state also relied on the activity of the soluble AC (sAC, a bicarbonate sensor that is insensitive to forskolin and GPCR activation. Pharmacological and immunological evidence indicated that SCs expressed sAC and that sAC activity was required for morphological differentiation and the expression of myelin markers such as O1 and protein zero. To conclude, our data indicates that cAMP did not directly drive myelination but rather the transition into an O1 positive state, which is perhaps the most critical cAMP-dependent rate limiting step for the onset of myelination. The temporally restricted role of cAMP in inducing differentiation independently of basal lamina formation provides a clear example of the

  4. Requirement of cyclooxygenase-2 expression and prostaglandins for human prostate cancer cell invasion.

    Science.gov (United States)

    Nithipatikom, Kasem; Isbell, Marilyn A; Lindholm, Paul F; Kajdacsy-Balla, Andre; Kaul, Sushma; Campell, William B

    2002-01-01

    The PC-3 Low Invasive cells and the PC-3 High Invasive cells were used to investigate the correlation of the COX-2 expression and its arachidonic acid metabolites, prostaglandins, with their invasiveness through Matrigel using a Boyden chamber assay. The COX-2 expression in PC-3 High Invasive cells was approximately 3-fold higher than in PC-3 Low Invasive cells while the COX-1 expression was similar in both cell sublines. When incubated with arachidonic acid, PGE2 was the major prostaglandin produced by these cells. PC-3 High Invasive cells produced PGE2 approximately 2.5-fold higher than PC-3 Low Invasive cells. PGD2 was the second most abundant prostaglandin produced by these cells. Both indomethacin (a nonspecific COX inhibitor) and NS-398 (a specific COX-2 inhibitor) inhibited the production of prostaglandins and the cell invasion. PGE2 alone did not induce the cell invasion of PC-3 Low Invasive cells. However, PGE2 reversed the inhibition of cell invasion by NS-398 and enhanced the cell invasion of the PC-3 High Invasive cells. In contrast, PGD2 slightly inhibited the cell invasion. These results suggest that in the PC-3 Low Invasive cells, COX-2-derived PGE2 may not be sufficient to induce cell invasion while in the PC-3 High Invasive cells, PGE2 may be sufficient to act as an enhancer for the cell invasion. Further, PGD2 may represent a weak inhibitor and counteracts the effect of PGE2 in the cell invasion. PMID:12498388

  5. 9 CFR 113.51 - Requirements for primary cells used for production of biologics.

    Science.gov (United States)

    2010-01-01

    ... of the final pool of harvested material or samples of each subculture of cells used to prepare the... completed product or samples of the final pool of harvested material or samples of each subculture of cells... cells or each subculture of primary cells used to prepare a biological product shall be shown free...

  6. T Cell Cancer Therapy Requires CD40-CD40L Activation of Tumor Necrosis Factor and Inducible Nitric-Oxide-Synthase-Producing Dendritic Cells.

    Science.gov (United States)

    Marigo, Ilaria; Zilio, Serena; Desantis, Giacomo; Mlecnik, Bernhard; Agnellini, Andrielly H R; Ugel, Stefano; Sasso, Maria Stella; Qualls, Joseph E; Kratochvill, Franz; Zanovello, Paola; Molon, Barbara; Ries, Carola H; Runza, Valeria; Hoves, Sabine; Bilocq, Amélie M; Bindea, Gabriela; Mazza, Emilia M C; Bicciato, Silvio; Galon, Jérôme; Murray, Peter J; Bronte, Vincenzo

    2016-09-12

    Effective cancer immunotherapy requires overcoming immunosuppressive tumor microenvironments. We found that local nitric oxide (NO) production by tumor-infiltrating myeloid cells is important for adoptively transferred CD8(+) cytotoxic T cells to destroy tumors. These myeloid cells are phenotypically similar to inducible nitric oxide synthase (NOS2)- and tumor necrosis factor (TNF)-producing dendritic cells (DC), or Tip-DCs. Depletion of immunosuppressive, colony stimulating factor 1 receptor (CSF-1R)-dependent arginase 1(+) myeloid cells enhanced NO-dependent tumor killing. Tumor elimination via NOS2 required the CD40-CD40L pathway. We also uncovered a strong correlation between survival of colorectal cancer patients and NOS2, CD40, and TNF expression in their tumors. Our results identify a network of pro-tumor factors that can be targeted to boost cancer immunotherapies.

  7. Sorbent Material Property Requirements for On-Board Hydrogen Storage for Automotive Fuel Cell Systems.

    Energy Technology Data Exchange (ETDEWEB)

    Ahluwalia, R. K.; Peng, J-K; Hua, T. Q.

    2015-05-25

    Material properties required for on-board hydrogen storage in cryogenic sorbents for use with automotive polymer electrolyte membrane (PEM) fuel cell systems are discussed. Models are formulated for physical, thermodynamic and transport properties, and for the dynamics of H-2 refueling and discharge from a sorbent bed. A conceptual storage configuration with in-bed heat exchanger tubes, a Type-3 containment vessel, vacuum insulation and requisite balance-of-plant components is developed to determine the peak excess sorption capacity and differential enthalpy of adsorption for 5.5 wt% system gravimetric capacity and 55% well-to-tank (WTT) efficiency. The analysis also determines the bulk density to which the material must be compacted for the storage system to reach 40 g.L-1 volumetric capacity. Thermal transport properties and heat transfer enhancement methods are analyzed to estimate the material thermal conductivity needed to achieve 1.5 kg.min(-1) H-2 refueling rate. Operating temperatures and pressures are determined for 55% WTT efficiency and 95% usable H-2. Needs for further improvements in material properties are analyzed that would allow reduction of storage pressure to 50 bar from 100 bar, elevation of storage temperature to 175-200 K from 150 K, and increase of WTT efficiency to 57.5% or higher.

  8. Cited2 is required for the maintenance of glycolytic metabolism in adult hematopoietic stem cells.

    Science.gov (United States)

    Du, Jinwei; Li, Qiang; Tang, Fangqiang; Puchowitz, Michelle A; Fujioka, Hisashi; Dunwoodie, Sally L; Danielpour, David; Yang, Yu-Chung

    2014-01-15

    Mammalian adult hematopoietic stem cells (HSCs) reside in the hypoxic bone marrow microenvironment and display a distinct metabolic phenotype compared with their progenitors. It has been proposed that HSCs generate energy mainly through anaerobic glycolysis in a pyruvate dehydrogenase kinase (Pdk)-dependent manner. Cited2 is an essential regulator for HSC quiescence, apoptosis, and function. Herein, we show that conditional deletion of Cited2 in murine HSCs results in elevated levels of reactive oxygen species, decreased cellular glutathione content, increased mitochondrial activity, and decreased glycolysis. At the molecular level, Cited2 deficiency significantly reduced the expression of genes involved in metabolism, such as Pdk2, Pdk4, and lactate dehydrogenases B and D (LDHB and LDHD). Cited2-deficient HSCs also exhibited increased Akt signaling, concomitant with elevated mTORC1 activity and phosphorylation of FoxOs. Further, inhibition of PI3/Akt, but not mTORC1, partially rescued the repression of Pdk4 caused by deletion of Cited2. Altogether, our results suggest that Cited2 is required for the maintenance of adult HSC glycolytic metabolism likely through regulating Pdk2, Pdk4, LDHB, LDHD, and Akt activity. PMID:24083546

  9. All Three Rows of Outer Hair Cells Are Required for Cochlear Amplification

    Directory of Open Access Journals (Sweden)

    Michio Murakoshi

    2015-01-01

    Full Text Available In the mammalian auditory system, the three rows of outer hair cells (OHCs located in the cochlea are thought to increase the displacement amplitude of the organ of Corti. This cochlear amplification is thought to contribute to the high sensitivity, wide dynamic range, and sharp frequency selectivity of the hearing system. Recent studies have shown that traumatic stimuli, such as noise exposure and ototoxic acid, cause functional loss of OHCs in one, two, or all three rows. However, the degree of decrease in cochlear amplification caused by such functional losses remains unclear. In the present study, a finite element model of a cross section of the gerbil cochlea was constructed. Then, to determine effects of the functional losses of OHCs on the cochlear amplification, changes in the displacement amplitude of the basilar membrane (BM due to the functional losses of OHCs were calculated. Results showed that the displacement amplitude of the BM decreases significantly when a single row of OHCs lost its function, suggesting that all three rows of OHCs are required for cochlear amplification.

  10. HIV-1 Vpr-induced apoptosis is cell cycle dependent and requires Bax but not ANT.

    Directory of Open Access Journals (Sweden)

    Joshua L Andersen

    2006-12-01

    Full Text Available The HIV-1 accessory protein viral protein R (Vpr causes G2 arrest and apoptosis in infected cells. We previously identified the DNA damage-signaling protein ATR as the cellular factor that mediates Vpr-induced G2 arrest and apoptosis. Here, we examine the mechanism of induction of apoptosis by Vpr and how it relates to induction of G2 arrest. We find that entry into G2 is a requirement for Vpr to induce apoptosis. We investigated the role of the mitochondrial permeability transition pore by knockdown of its essential component, the adenine nucleotide translocator. We found that Vpr-induced apoptosis was unaffected by knockdown of ANT. Instead, apoptosis is triggered through a different mitochondrial pore protein, Bax. In support of the idea that checkpoint activation and apoptosis induction are functionally linked, we show that Bax activation by Vpr was ablated when ATR or GADD45alpha was knocked down. Certain mutants of Vpr, such as R77Q and I74A, identified in long-term nonprogressors, have been proposed to inefficiently induce apoptosis while activating the G2 checkpoint in a normal manner. We tested the in vitro phenotypes of these mutants and found that their abilities to induce apoptosis and G2 arrest are indistinguishable from those of HIV-1NL4-3 vpr, providing additional support to the idea that G2 arrest and apoptosis induction are mechanistically linked.

  11. The SHIP2 interactor Myo1c is required for cell migration in 1321 N1 glioblastoma cells.

    Science.gov (United States)

    Edimo, William's Elong; Ramos, Ana Raquel; Ghosh, Somadri; Vanderwinden, Jean-Marie; Erneux, Christophe

    2016-08-01

    The phosphoinositide 5-phosphatases consist of several enzymes that have been shown to modulate cell migration and invasion. SHIP2, one family member, is known to interact with growth factor receptors and cytoskeletal proteins. In a human model of glioblastoma 1321 N1 cells, we recently identified Myo1c as a new interactor of SHIP2. This was shown in a complex of proteins also containing filamin A. We show here that SHIP2 localization at lamellipodia and ruffles is impaired in Myo1c depleted cells. In the absence of Myo1c, N1 cells tend to associate to form clusters. Cell migration is very much reduced in Myo1c depleted cells, concomitantly with a decrease in FAK Tyr397 phosphorylation, focal adhesion length and PI(4,5)P2 immunostaining. In N1 cells, Myo1c is thus important for lamellipodia formation to assemble a protein complex containing SHIP2 to facilitate cell migration. PMID:27246739

  12. NK cell survival mediated through the regulatory synapse with human DCs requires IL-15Rα

    OpenAIRE

    Brilot, Fabienne; Strowig, Till; Roberts, Susanne M.; Arrey, Frida; Münz, Christian

    2007-01-01

    DCs activate NK cells during innate immune responses to viral infections. However, the composition and kinetics of the immunological synapse mediating this interaction are largely unknown. Here, we report the rapid formation of an immunological synapse between human resting NK cells and mature DCs. Although inhibitory NK cell receptors were polarized to this synapse, where they are known to protect mature DCs from NK cell lysis, the NK cell also received activation signals that induced mobili...

  13. Placental Growth Factor Expression Is Required for Bone Marrow Endothelial Cell Support of Primitive Murine Hematopoietic Cells

    OpenAIRE

    Xiaoying Zhou; Barsky, Lora W.; Adams, Gregor B

    2013-01-01

    Two distinct microenvironmental niches that regulate hematopoietic stem/progenitor cell physiology in the adult bone marrow have been proposed; the endosteal and the vascular niche. While extensive studies have been performed relating to molecular interactions in the endosteal niche, the mechanisms that regulate hematopoietic stem/progenitor cell interaction with bone marrow endothelial cells are less well defined. Here we demonstrate that endothelial cells derived from the bone marrow suppor...

  14. Glucose-dependent anaplerosis in cancer cells is required for cellular redox balance in the absence of glutamine

    Science.gov (United States)

    Cetinbas, Naniye Mallı; Sudderth, Jessica; Harris, Robert C.; Cebeci, Aysun; Negri, Gian L.; Yılmaz, Ömer H.; Deberardinis, Ralph J.; Sorensen, Poul H.

    2016-09-01

    Cancer cells have altered metabolism compared to normal cells, including dependence on glutamine (GLN) for survival, known as GLN addiction. However, some cancer cell lines do not require GLN for survival and the basis for this discrepancy is not well understood. GLN is a precursor for antioxidants such as glutathione (GSH) and NADPH, and GLN deprivation is therefore predicted to deplete antioxidants and increase reactive oxygen species (ROS). Using diverse human cancer cell lines we show that this occurs only in cells that rely on GLN for survival. Thus, the preference for GLN as a dominant antioxidant source defines GLN addiction. We show that despite increased glucose uptake, GLN addicted cells do not metabolize glucose via the TCA cycle when GLN is depleted, as revealed by 13C-glucose labeling. In contrast, GLN independent cells can compensate by diverting glucose-derived pyruvate into the TCA cycle. GLN addicted cells exhibit reduced PDH activity, increased PDK1 expression, and PDK inhibition partially rescues GLN starvation-induced ROS and cell death. Finally, we show that combining GLN starvation with pro-oxidants selectively kills GLN addicted cells. These data highlight a major role for GLN in maintaining redox balance in cancer cells that lack glucose-dependent anaplerosis.

  15. A virtual lymph node model to dissect the requirements for T-cell activation by synapses and kinapses

    Science.gov (United States)

    Moreau, Hélène D; Bogle, Gib; Bousso, Philippe

    2016-01-01

    The initiation of T-cell responses in lymph nodes requires T cells to integrate signals delivered by dendritic cells (DCs) during long-lasting contacts (synapses) or more transient interactions (kinapses). However, it remains extremely challenging to understand how a specific sequence of contacts established by T cells ultimately dictates T-cell fate. Here, we have coupled a computational model of T-cell migration and interactions with DCs with a real-time, flow cytometry-like representation of T-cell activation. In this model, low-affinity peptides trigger T-cell proliferation through kinapses but we show that this process is only effective under conditions of high DC densities and prolonged antigen availability. By contrast, high-affinity peptides favor synapse formation and a vigorous proliferation under a wide range of antigen presentation conditions. In line with the predictions, decreasing the DC density in vivo selectively abolished proliferation induced by the low-affinity peptide. Finally, our results suggest that T cells possess a biochemical memory of previous stimulations of at least 1–2 days. We propose that the stability of T-cell–DC interactions, apart from their signaling potency, profoundly influences the robustness of T-cell activation. By offering the ability to control parameters that are difficult to manipulate experimentally, the virtual lymph node model provides new possibilities to tackle the fundamental mechanisms that regulate T-cell responses elicited by infections or vaccines. PMID:27089942

  16. Production in a factory (the cell) requires high level of organisation : the cell: The plant’s smallest building block

    NARCIS (Netherlands)

    Heuvelink, E.

    2015-01-01

    The cell is the plant’s smallest building block. Many cultivation techniques and climate control measures have an effect at this level. Some knowledge about the functioning of the cell is therefore very useful. Many components of the cell have bizarre names so to understand it all better, for the pu

  17. Timing of Tissue-specific Cell Division Requires a Differential Onset of Zygotic Transcription during Metazoan Embryogenesis.

    Science.gov (United States)

    Wong, Ming-Kin; Guan, Daogang; Ng, Kaoru Hon Chun; Ho, Vincy Wing Sze; An, Xiaomeng; Li, Runsheng; Ren, Xiaoliang; Zhao, Zhongying

    2016-06-10

    Metazoan development demands not only precise cell fate differentiation but also accurate timing of cell division to ensure proper development. How cell divisions are temporally coordinated during development is poorly understood. Caenorhabditis elegans embryogenesis provides an excellent opportunity to study this coordination due to its invariant development and widespread division asynchronies. One of the most pronounced asynchronies is a significant delay of cell division in two endoderm progenitor cells, Ea and Ep, hereafter referred to as E2, relative to its cousins that mainly develop into mesoderm organs and tissues. To unravel the genetic control over the endoderm-specific E2 division timing, a total of 822 essential and conserved genes were knocked down using RNAi followed by quantification of cell cycle lengths using in toto imaging of C. elegans embryogenesis and automated lineage. Intriguingly, knockdown of numerous genes encoding the components of general transcription pathway or its regulatory factors leads to a significant reduction in the E2 cell cycle length but an increase in cell cycle length of the remaining cells, indicating a differential requirement of transcription for division timing between the two. Analysis of lineage-specific RNA-seq data demonstrates an earlier onset of transcription in endoderm than in other germ layers, the timing of which coincides with the birth of E2, supporting the notion that the endoderm-specific delay in E2 division timing demands robust zygotic transcription. The reduction in E2 cell cycle length is frequently associated with cell migration defect and gastrulation failure. The results suggest that a tissue-specific transcriptional activation is required to coordinate fate differentiation, division timing, and cell migration to ensure proper development.

  18. Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

    Science.gov (United States)

    Jorgensen, Niklas Rye; Teilmann, Stefan Cuoni; Henriksen, Zanne; Civitelli, Roberto; Sorensen, Ole Helmer; Steinberg, Thomas H.

    2003-01-01

    The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal of extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx43 (UMR/Cx43) we confirmed that nifedipine sensitivity of ICW required Cx43 expression. In human osteoblastic cells, gap junction-dependent ICW also required activation of L-type calcium channels and influx of extracellular calcium.

  19. Yeast spore germination: a requirement for Ras protein activity during re-entry into the cell cycle.

    OpenAIRE

    Herman, P K; Rine, J.

    1997-01-01

    Saccharomyces cerevisiae spore germination is a process in which quiescent, non-dividing spores become competent for mitotic cell division. Using a novel assay for spore uncoating, we found that spore germination was a multi-step process whose nutritional requirements differed from those for mitotic division. Although both processes were controlled by nutrient availability, efficient spore germination occurred in conditions that did not support cell division. In addition, germination did not ...

  20. Oct4 is required for lineage priming in the developing inner cell mass of the mouse blastocyst

    OpenAIRE

    Le Bin, Gloryn Chia; Muñoz-Descalzo, Silvia; Kurowski, Agata; Leitch, Harry; Lou, Xinghua; Mansfield, William; Etienne-Dumeau, Charles; Grabole, Nils; Mulas, Carla; Niwa, Hitoshi; Hadjantonakis, Anna-Katerina; Nichols, Jennifer

    2014-01-01

    The transcription factor Oct4 is required in vitro for establishment and maintenance of embryonic stem cells and for reprogramming somatic cells to pluripotency. In vivo, it prevents the ectopic differentiation of early embryos into trophoblast. Here, we further explore the role of Oct4 in blastocyst formation and specification of epiblast versus primitive endoderm lineages using conditional genetic deletion. Experiments involving mouse embryos deficient for both maternal and zygotic Oct4 sug...

  1. The Mi-2-like Smed-CHD4 gene is required for stem cell differentiation in the planarian Schmidtea mediterranea

    OpenAIRE

    Scimone, M. Lucila; Meisel, Joshua; Reddien, Peter W.

    2010-01-01

    Freshwater planarians are able to regenerate any missing part of their body and have extensive tissue turnover because of the action of dividing cells called neoblasts. Neoblasts provide an excellent system for in vivo study of adult stem cell biology. We identified the Smed-CHD4 gene, which is predicted to encode a chromatin-remodeling protein similar to CHD4/Mi-2 proteins, as required for planarian regeneration and tissue homeostasis. Following inhibition of Smed-CHD4 with RNA interference ...

  2. The Mi-2-like Smed-CHD4 gene is required for stem cell differentiation in the planarian Schmidtea mediterranea.

    Science.gov (United States)

    Scimone, M Lucila; Meisel, Joshua; Reddien, Peter W

    2010-04-01

    Freshwater planarians are able to regenerate any missing part of their body and have extensive tissue turnover because of the action of dividing cells called neoblasts. Neoblasts provide an excellent system for in vivo study of adult stem cell biology. We identified the Smed-CHD4 gene, which is predicted to encode a chromatin-remodeling protein similar to CHD4/Mi-2 proteins, as required for planarian regeneration and tissue homeostasis. Following inhibition of Smed-CHD4 with RNA interference (RNAi), neoblast numbers were initially normal, despite an inability of the animals to regenerate. However, the proliferative response of neoblasts to amputation or growth stimulation in Smed-CHD4(RNAi) animals was diminished. Smed-CHD4(RNAi) animals displayed a dramatic reduction in the numbers of certain neoblast progeny cells. Smed-CHD4 was required for the formation of these neoblast progeny cells. Together, these results indicate that Smed-CHD4 is required for neoblasts to produce progeny cells committed to differentiation in order to control tissue turnover and regeneration and suggest a crucial role for CHD4 proteins in stem cell differentiation. PMID:20223763

  3. The transcription factor NFAT5 is required for cyclin expression and cell cycle progression in cells exposed to hypertonic stress.

    Directory of Open Access Journals (Sweden)

    Katherine Drews-Elger

    Full Text Available BACKGROUND: Hypertonicity can perturb cellular functions, induce DNA damage-like responses and inhibit proliferation. The transcription factor NFAT5 induces osmoprotective gene products that allow cells to adapt to sustained hypertonic conditions. Although it is known that NFAT5-deficient lymphocytes and renal medullary cells have reduced proliferative capacity and viability under hypertonic stress, less is understood about the contribution of this factor to DNA damage responses and cell cycle regulation. METHODOLOGY/PRINCIPAL FINDINGS: We have generated conditional knockout mice to obtain NFAT5(-/- T lymphocytes, which we used as a model of proliferating cells to study NFAT5-dependent responses. We show that hypertonicity triggered an early, NFAT5-independent, genotoxic stress-like response with induction of p53, p21 and GADD45, downregulation of cyclins, and cell cycle arrest. This was followed by an NFAT5-dependent adaptive phase in wild-type cells, which induced an osmoprotective gene expression program, downregulated stress markers, resumed cyclin expression and proliferation, and displayed enhanced NFAT5 transcriptional activity in S and G2/M. In contrast, NFAT5(-/- cells failed to induce osmoprotective genes and exhibited poorer viability. Although surviving NFAT5(-/- cells downregulated genotoxic stress markers, they underwent cell cycle arrest in G1/S and G2/M, which was associated with reduced expression of cyclins E1, A2 and B1. We also show that pathologic hypertonicity levels, as occurring in plasma of patients and animal models of osmoregulatory disorders, inhibited the induction of cyclins and aurora B kinase in response to T cell receptor stimulation in fresh NFAT5(-/- lymphocytes. CONCLUSIONS/SIGNIFICANCE: We conclude that NFAT5 facilitates cell proliferation under hypertonic conditions by inducing an osmoadaptive response that enables cells to express fundamental regulators needed for cell cycle progression.

  4. Successful Reconstruction of Tooth Germ with Cell Lines Requires Coordinated Gene Expressions from the Initiation Stage

    Directory of Open Access Journals (Sweden)

    Yasuhiro Tomooka

    2012-10-01

    Full Text Available Tooth morphogenesis is carried out by a series of reciprocal interactions between the epithelium and mesenchyme in embryonic germs. Previously clonal dental epithelial cell (epithelium of molar tooth germ (emtg lines were established from an embryonic germ. They were odontogenic when combined with a dental mesenchymal tissue, although the odontogenesis was quantitatively imperfect. To improve the microenvironment in the germs, freshly isolated dental epithelial cells were mixed with cells of lines, and germs were reconstructed in various combinations. The results demonstrated that successful tooth construction depends on the mixing ratio, the age of dental epithelial cells and the combination with cell lines. Analyses of gene expression in these germs suggest that some signal(s from dental epithelial cells makes emtg cells competent to communicate with mesenchymal cells and the epithelial and mesenchymal compartments are able to progress  odontogenesis from the initiation stage.

  5. neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas

    OpenAIRE

    Gradwohl, Gérard; Dierich, Andrée; LeMeur, Marianne; Guillemot, François

    2000-01-01

    In the mammalian pancreas, the endocrine cell types of the islets of Langerhans, including the α-, β-, δ-, and pancreatic polypeptide cells as well as the exocrine cells, derive from foregut endodermal progenitors. Recent genetic studies have identified a network of transcription factors, including Pdx1, Isl1, Pax4, Pax6, NeuroD, Nkx2.2, and Hlxb9, regulating the development of islet cells at different stages, but the molecular mechanisms controlling the specification of pancreatic endocrine ...

  6. Requirement for noncognate interaction with T cells for the activation of B cell immunoglobulin secretion by IL-2

    DEFF Research Database (Denmark)

    Owens, T

    1991-01-01

    23.1+ TH1 clone E9.D4 in F23.1 (anti-T cell receptor V-beta 8)-coated microwells. This induced polyclonal B cell activation to enter cell cycle (thymidine incorporation) at 2 days and to secrete immunoglobulin at 5 days. An anti-IL-2 mAb (S4B6) inhibited antibody production completely. Anti-IL-2 did...... not inhibit either LPS-induced B cell responses, or T cell activation (measured as IL-3 secretion). Anti-IL-2 receptor (anti-Tac) mAbs also inhibited T-dependent B cell responses, without affecting LPS responses. An anti-IFN-gamma mAb partially inhibited Ig secretion, without affecting entry into...

  7. Differential requirements for the induction of interleukin 2 responsiveness in L3T4+ and Lyt-2+ T cell subsets

    OpenAIRE

    1985-01-01

    Minimal requirements for the induction of interleukin 2 (IL-2) responsiveness in purified subsets of murine T lymphocytes have been investigated. Whereas Lyt-2+ cells could be induced to IL-2-dependent growth by lectin, phorbol ester, or calcium ionophore, none of these stimuli was by itself sufficient for L3T4+ cells. The latter cells could, however, be induced to respond to IL-2 by combinations of lectin plus phorbol ester or ionophore plus phorbol ester (but not lectin plus ionophore). Und...

  8. Phoenix is required for mechanosensory hair cell regeneration in the zebrafish lateral line.

    Directory of Open Access Journals (Sweden)

    Martine Behra

    2009-04-01

    Full Text Available In humans, the absence or irreversible loss of hair cells, the sensory mechanoreceptors in the cochlea, accounts for a large majority of acquired and congenital hearing disorders. In the auditory and vestibular neuroepithelia of the inner ear, hair cells are accompanied by another cell type called supporting cells. This second cell population has been described as having stem cell-like properties, allowing efficient hair cell replacement during embryonic and larval/fetal development of all vertebrates. However, mammals lose their regenerative capacity in most inner ear neuroepithelia in postnatal life. Remarkably, reptiles, birds, amphibians, and fish are different in that they can regenerate hair cells throughout their lifespan. The lateral line in amphibians and in fish is an additional sensory organ, which is used to detect water movements and is comprised of neuroepithelial patches, called neuromasts. These are similar in ultra-structure to the inner ear's neuroepithelia and they share the expression of various molecular markers. We examined the regeneration process in hair cells of the lateral line of zebrafish larvae carrying a retroviral integration in a previously uncharacterized gene, phoenix (pho. Phoenix mutant larvae develop normally and display a morphologically intact lateral line. However, after ablation of hair cells with copper or neomycin, their regeneration in pho mutants is severely impaired. We show that proliferation in the supporting cells is strongly decreased after damage to hair cells and correlates with the reduction of newly formed hair cells in the regenerating phoenix mutant neuromasts. The retroviral integration linked to the phenotype is in a novel gene with no known homologs showing high expression in neuromast supporting cells. Whereas its role during early development of the lateral line remains to be addressed, in later larval stages phoenix defines a new class of proteins implicated in hair cell regeneration.

  9. Cell cycle requirements for transduction by foamy virus vectors compared to those of oncovirus and lentivirus vectors.

    Science.gov (United States)

    Trobridge, Grant; Russell, David W

    2004-03-01

    Retroviral vectors based on foamy viruses (FV) are efficient gene delivery vehicles for therapeutic and research applications. While previous studies have shown that FV vectors transduce quiescent cell cultures more efficiently than oncoviral vectors, their specific cell cycle requirements have not been determined. Here we compare the transduction frequencies of FV vectors with those of onco- and lentiviral vectors in nondividing and dividing normal human fibroblasts by several methods. FV vectors transduced serum-deprived fibroblast cultures more efficiently than oncoretroviral vectors and at rates comparable to those of lentiviral vectors. However, in these cultures FV vectors only transduced a subpopulation of proliferating cells, as determined by bromodeoxyuridine staining for DNA synthesis. In contrast to lentiviral vectors, FV vectors were unable to transduce human fibroblasts arrested by aphidicolin (G(1)/S phase) or gamma-irradiation (G(2) phase), and a partial cell cycle that included mitosis but not DNA synthesis was required. We could not determine if mitosis facilitated nuclear entry of FV vectors, since cell-free vector preparations contained long terminal repeat circles, precluding their use as nuclear markers. In contrast to oncoviral vectors, both FV and lentiviral vectors efficiently transduced G(0) fibroblasts that were later stimulated to divide. In the case of FV vectors, this was due to the persistence of a stable transduction intermediate in quiescent cells. Our findings support the use of FV vectors as a safe and effective alternative to lentiviral vectors for ex vivo transduction of stem cells that are quiescent during culture but divide following transplantation.

  10. Repression of the integrated papillomavirus E6/E7 promoter is required for growth suppression of cervical cancer cells.

    Science.gov (United States)

    Francis, D A; Schmid, S I; Howley, P M

    2000-03-01

    The human papillomavirus (HPV) E2 protein is an important regulator of viral E6 and E7 gene expression. E2 can repress the viral promoter for E6 and E7 expression as well as block progression of the cell cycle in cancer cells harboring the DNA of "high-risk" HPV types. Although the phenomenon of E2-mediated growth arrest of HeLa cells and other HPV-positive cancer cells has been well documented, the specific mechanism by which E2 affects cellular proliferation has not yet been elucidated. Here, we show that bovine papillomavirus (BPV) E2-induced growth arrest of HeLa cells requires the repression of the E6 and E7 promoter. This repression is specific for E2TA and not E2TR, a BPV E2 variant that lacks the N-terminal transactivation domain. We demonstrate that expression of HPV16 E6 and E7 from a heterologous promoter that is not regulated by E2 rescues HeLa cells from E2-mediated growth arrest. Our data indicate that the pathway of E2-mediated growth arrest of HeLa cells requires repression of E6 and E7 expression through an activity specified by the transactivation domain of E2TA. PMID:10684283

  11. Insulin-secreting β cells require a post-genomic concept.

    Science.gov (United States)

    Jiang, Fang-Xu; Morahan, Grant

    2016-05-25

    Pancreatic insulin-secreting β cells are essential in maintaining normal glucose homeostasis accomplished by highly specialized transcription of insulin gene, of which occupies up to 40% their transcriptome. Deficiency of these cells causes diabetes mellitus, a global public health problem. Although tremendous endeavors have been made to generate insulin-secreting cells from human pluripotent stem cells (i.e., primitive cells capable of giving rise to all cell types in the body), a regenerative therapy to diabetes has not yet been established. Furthermore, the nomenclature of β cells has become inconsistent, confusing and controversial due to the lack of standardized positive controls of developmental stage-matched in vivo cells. In order to minimize this negative impact and facilitate critical research in this field, a post-genomic concept of pancreatic β cells might be helpful. In this review article, we will briefly describe how β cells were discovered and islet lineage is developed that may help understand the cause of nomenclatural controversy, suggest a post-genomic definition and finally provide a conclusive remark on future research of this pivotal cell. PMID:27226815

  12. Regulatory requirements in the good manufacturing practice production of an epithelial cell graft for ocular surface reconstruction.

    Science.gov (United States)

    Sheth-Shah, Radhika; Vernon, Amanda J; Seetharaman, Shankar; Neale, Michael H; Daniels, Julie T

    2016-04-01

    In the past decade, stem cell therapy has been increasingly employed for the treatment of various diseases. Subsequently, there has been a great interest in the manufacture of stem cells under good manufacturing practice, which is required by law for their use in humans. The cells for sight Stem Cell Therapy Research Unit, based at UCL Institute of Ophthalmology, delivers somatic cell-based and tissue-engineered therapies to patients suffering from blinding eye diseases at Moorfields Eye Hospital (London, UK). The following article is based on our experience in the conception, design, construction, validation and manufacturing within a good manufacturing practice manufacturing facility based in the UK. As such the regulations can be extrapolated to the 28 members stated within the EU. However, the principles may have a broad relevance outside the EU.

  13. Efficient T-cell surveillance of the CNS requires expression of the CXC chemokine receptor 3

    DEFF Research Database (Denmark)

    Christensen, Jeanette Erbo; Nansen, Anneline; Moos, Torben;

    2004-01-01

    T-cells play an important role in controlling viral infections inside the CNS. To study the role of the chemokine receptor CXCR3 in the migration and positioning of virus-specific effector T-cells within the brain, CXCR3-deficient mice were infected intracerebrally with lymphocytic choriomeningitis......-cell-mediated immunopathology. Quantitative analysis of the cellular infiltrate in CSF of infected mice revealed modest, if any, decrease in the number of mononuclear cells recruited to the meninges in the absence of CXCR3. However, immunohistological analysis disclosed a striking impairment of CD8+ T-cells from CXCR3......-deficient mice to migrate from the meninges into the outer layers of the brain parenchyma despite similar localization of virus-infected target cells. Reconstitution of CXCR3-deficient mice with wild-type CD8+ T-cells completely restored susceptibility to LCMV-induced meningitis. Thus, taken together, our...

  14. SH-2-containing protein tyrosine phosphatase 1 is required for IL-4-induced IL-4R expression in spleen cells

    Institute of Scientific and Technical Information of China (English)

    CAO Xin; HUANG Zan; FAN Jingyi

    2005-01-01

    To investigate the role of SH-2-containing protein tyrosine phosphatase 1, SHP-1, in IL-4-induced IL-4 receptor (IL-4R) expression, we examined IL-4 receptor α-chain (IL-4Rα) mRNA expression in Na3VO4-treated wild type (WT) spleen cells and measured IL-4R mRNA in IL-4-stimulated spleen cells of viable motheaten mice (mev/mev). It is found that IL-4-induced IL-4R mRNA expression was impaired in Na3VO4-treated WT spleen cells and IL-4-stimulated mev/mev spleen cells. Here we show that the impaired IL-4-induced IL-4RαmRNA expression was due to reduced expression of IL-4R that led to impaired STAT6 signaling. We further demonstrate that reduction of IL-4Rαprotein expression in mev/mev spleen cells was due to alteration in cell compositions. In mev/mev spleen, the percentages of CD4+, CD8+, and CD19+ cells expressing relatively high levels of IL-4R were reduced dramatically while the percentages of Mac-1+ and Gr-1+ cells with relative low levels of IL-4R increased greatly. Despite the profound effect of reduced expression of IL-4R protein, the IL-4RαmRNA expression was comparable in spleen cells of littermate control mice (+/() and mev/mev mice and no differences were found in B cells, T cells, and macrophages, suggesting cell type-specific downregulation of IL-4R expression in macrophages through a posttranscriptional mechanism. Our study suggests that SHP-1 is required for IL-4-meidated function and indirectly regulates IL-4-meidated function in spleen cells by affecting hematopoiesis.

  15. NOTCH3 expression is induced in mural cells through an autoregulatory loop that requires endothelial-expressed JAGGED1.

    Science.gov (United States)

    Liu, Hua; Kennard, Simone; Lilly, Brenda

    2009-02-27

    Endothelial cells and mural cells (smooth muscle cells, pericytes, or fibroblasts) are known to communicate with one another. Their interactions not only serve to support fully functional blood vessels but also can regulate vessel assembly and differentiation or maturation. In an effort to better understand the molecular components of this heterotypic interaction, we used a 3D model of angiogenesis and screened for genes, which were modulated by coculturing of these 2 different cell types. In doing so, we discovered that NOTCH3 is one gene whose expression is robustly induced in mural cells by coculturing with endothelial cells. Knockdown by small interfering RNA revealed that NOTCH3 is necessary for endothelial-dependent mural cell differentiation, whereas overexpression of NOTCH3 is sufficient to promote smooth muscle gene expression. Moreover, NOTCH3 contributes to the proangiogenic abilities of mural cells cocultured with endothelial cells. Interestingly, we found that the expression of NOTCH3 is dependent on Notch signaling, because the gamma-secretase inhibitor DAPT blocked its upregulation. Furthermore, in mural cells, a dominant-negative Mastermind-like1 construct inhibited NOTCH3 expression, and endothelial-expressed JAGGED1 was required for its induction. Additionally, we demonstrated that NOTCH3 could promote its own expression and that of JAGGED1 in mural cells. Taken together, these data provide a mechanism by which endothelial cells induce the differentiation of mural cells through activation and induction of NOTCH3. These findings also suggest that NOTCH3 has the capacity to maintain a differentiated phenotype through a positive-feedback loop that includes both autoregulation and JAGGED1 expression.

  16. Nuclear removal during terminal lens fiber cell differentiation requires CDK1 activity: appropriating mitosis-related nuclear disassembly.

    Science.gov (United States)

    Chaffee, Blake R; Shang, Fu; Chang, Min-Lee; Clement, Tracy M; Eddy, Edward M; Wagner, Brad D; Nakahara, Masaki; Nagata, Shigekazu; Robinson, Michael L; Taylor, Allen

    2014-09-01

    Lens epithelial cells and early lens fiber cells contain the typical complement of intracellular organelles. However, as lens fiber cells mature they must destroy their organelles, including nuclei, in a process that has remained enigmatic for over a century, but which is crucial for the formation of the organelle-free zone in the center of the lens that assures clarity and function to transmit light. Nuclear degradation in lens fiber cells requires the nuclease DNase IIβ (DLAD) but the mechanism by which DLAD gains access to nuclear DNA remains unknown. In eukaryotic cells, cyclin-dependent kinase 1 (CDK1), in combination with either activator cyclins A or B, stimulates mitotic entry, in part, by phosphorylating the nuclear lamin proteins leading to the disassembly of the nuclear lamina and subsequent nuclear envelope breakdown. Although most post-mitotic cells lack CDK1 and cyclins, lens fiber cells maintain these proteins. Here, we show that loss of CDK1 from the lens inhibited the phosphorylation of nuclear lamins A and C, prevented the entry of DLAD into the nucleus, and resulted in abnormal retention of nuclei. In the presence of CDK1, a single focus of the phosphonuclear mitotic apparatus is observed, but it is not focused in CDK1-deficient lenses. CDK1 deficiency inhibited mitosis, but did not prevent DNA replication, resulting in an overall reduction of lens epithelial cells, with the remaining cells possessing an abnormally large nucleus. These observations suggest that CDK1-dependent phosphorylations required for the initiation of nuclear membrane disassembly during mitosis are adapted for removal of nuclei during fiber cell differentiation.

  17. Profilin is required for viral morphogenesis, syncytium formation, and cell-specific stress fiber induction by respiratory syncytial virus

    Directory of Open Access Journals (Sweden)

    Barik Sailen

    2003-05-01

    Full Text Available Abstract Background Actin is required for the gene expression and morphogenesis of respiratory syncytial virus (RSV, a clinically important Pneumovirus of the Paramyxoviridae family. In HEp-2 cells, RSV infection also induces actin stress fibers, which may be important in the immunopathology of the RSV disease. Profilin, a major regulator of actin polymerization, stimulates viral transcription in vitro. Thus, we tested the role of profilin in RSV growth and RSV-actin interactions in cultured cells (ex vivo. Results We tested three cell lines: HEp-2 (human, A549 (human, and L2 (rat. In all three, RSV grew well and produced fused cells (syncytium, and two RSV proteins, namely, the phosphoprotein P and the nucleocapsid protein N, associated with profilin. In contrast, induction of actin stress fibers by RSV occurred in HEp-2 and L2 cells, but not in A549. Knockdown of profilin by RNA interference had a small effect on viral macromolecule synthesis but strongly inhibited maturation of progeny virions, cell fusion, and induction of stress fibers. Conclusions Profilin plays a cardinal role in RSV-mediated cell fusion and viral maturation. In contrast, interaction of profilin with the viral transcriptional proteins P and N may only nominally activate viral RNA-dependent RNA polymerase. Stress fiber formation is a cell-specific response to infection, requiring profilin and perhaps other signaling molecules that are absent in certain cell lines. Stress fibers per se play no role in RSV replication in cell culture. Clearly, the cellular architecture controls multiple steps of host-RSV interaction, some of which are regulated by profilin.

  18. The Wnt receptor, Lrp5, is expressed by mouse mammary stem cells and is required to maintain the basal lineage.

    Directory of Open Access Journals (Sweden)

    Nisha M Badders

    Full Text Available BACKGROUND: Ectopic Wnt signaling induces increased stem/progenitor cell activity in the mouse mammary gland, followed by tumor development. The Wnt signaling receptors, Lrp5/6, are uniquely required for canonical Wnt activity. Previous data has shown that the absence of Lrp5 confers resistance to Wnt1-induced tumor development. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that all basal mammary cells express Lrp5, and co-express Lrp6 in a similar fashion. Though Wnt dependent transcription of key target genes is relatively unchanged in mammary epithelial cell cultures, the absence of Lrp5 specifically depletes adult regenerative stem cell activity (to less than 1%. Stem cell activity can be enriched by >200 fold (over 80% of activity, based on high Lrp5 expression alone. Though Lrp5 null glands have apparent normal function, the basal lineage is relatively reduced (from 42% basal/total epithelial cells to 22% and Lrp5-/- mammary epithelial cells show enhanced expression of senescence-associated markers in vitro, as measured by expression of p16(Ink4a and TA-p63. CONCLUSIONS/SIGNIFICANCE: This is the first single biomarker that has been demonstrated to be functionally involved in stem cell maintenance. Together, these results demonstrate that Wnt signaling through Lrp5 is an important component of normal mammary stem cell function.

  19. The Proteomic Landscape of Human Ex Vivo Regulatory and Conventional T Cells Reveals Specific Metabolic Requirements

    Science.gov (United States)

    Procaccini, Claudio; Carbone, Fortunata; Di Silvestre, Dario; Brambilla, Francesca; De Rosa, Veronica; Galgani, Mario; Faicchia, Deriggio; Marone, Gianni; Tramontano, Donatella; Corona, Marco; Alviggi, Carlo; Porcellini, Antonio; La Cava, Antonio; Mauri, Pierluigi; Matarese, Giuseppe

    2016-01-01

    Summary Human CD4+CD25hiFoxp3+CD127− Treg and CD4+CD25−Foxp3− Tconv cell functions are governed by their metabolic requirements. Here we report a comprehensive comparative analysis between ex vivo human Treg and Tconv cells that comprises analyses of the proteomic networks in subcellular compartments. We identified a dominant proteomic signature at the metabolic level that primarily impacted the highly-tuned balance between glucose and fatty-acid oxidation in the two cell types. Ex vivo Treg cells were highly glycolytic while Tconv cells used predominantly fatty-acid oxidation (FAO). When cultured in vitro, Treg cells engaged both glycolysis and FAO to proliferate, while Tconv cell proliferation mainly relied on glucose metabolism. Our unbiased proteomic analysis provides a molecular picture of the impact of metabolism on ex vivo human Treg versus Tconv cell functions that might be relevant for therapeutic manipulations of these cells. PMID:26885861

  20. Primary human cervical carcinoma cells require human papillomavirus E6 and E7 expression for ongoing proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Magaldi, Thomas G.; Almstead, Laura L. [Department of Genetics, Yale School of Medicine, P.O. Box 208005, New Haven, CT 06520-8005 (United States); Bellone, Stefania [Department of Obstetrics and Gynecology and Reproductive Sciences, Yale School of Medicine, P.O. Box 208063, New Haven, CT 06520-8063 (United States); Prevatt, Edward G. [Department of Genetics, Yale School of Medicine, P.O. Box 208005, New Haven, CT 06520-8005 (United States); Santin, Alessandro D. [Department of Obstetrics and Gynecology and Reproductive Sciences, Yale School of Medicine, P.O. Box 208063, New Haven, CT 06520-8063 (United States); Yale Comprehensive Cancer Center, P.O. Box 208028, New Haven, CT 06520-8028 (United States); DiMaio, Daniel, E-mail: daniel.dimaio@yale.edu [Department of Genetics, Yale School of Medicine, P.O. Box 208005, New Haven, CT 06520-8005 (United States); Department of Therapeutic Radiology, Yale School of Medicine, P.O. Box 208040, New Haven, CT 06520-8040 (United States); Department of Molecular Biophysics and Biochemistry, Yale School of Medicine, P.O. Box 208024 (United States); Yale Comprehensive Cancer Center, P.O. Box 208028, New Haven, CT 06520-8028 (United States)

    2012-01-05

    Repression of human papillomavirus (HPV) E6 and E7 oncogenes in established cervical carcinoma cell lines causes senescence due to reactivation of cellular tumor suppressor pathways. Here, we determined whether ongoing expression of HPV16 or HPV18 oncogenes is required for the proliferation of primary human cervical carcinoma cells in serum-free conditions at low passage number after isolation from patients. We used an SV40 viral vector expressing the bovine papillomavirus E2 protein to repress E6 and E7 in these cells. To enable efficient SV40 infection and E2 gene delivery, we first incubated the primary cervical cancer cells with the ganglioside GM1, a cell-surface receptor for SV40 that is limiting in these cells. Repression of HPV in primary cervical carcinoma cells caused them to undergo senescence, but the E2 protein had little effect on HPV-negative primary cells. These data suggest that E6 and E7 dependence is an inherent property of human cervical cancer cells.

  1. N-Cadherin Induction by ECM Stiffness and FAK Overrides the Spreading Requirement for Proliferation of Vascular Smooth Muscle Cells

    Directory of Open Access Journals (Sweden)

    Keeley L. Mui

    2015-03-01

    Full Text Available In contrast to the accepted pro-proliferative effect of cell-matrix adhesion, the proliferative effect of cadherin-mediated cell-cell adhesion remains unresolved. Here, we studied the effect of N-cadherin on cell proliferation in the vasculature. We show that N-cadherin is induced in smooth muscle cells (SMCs in response to vascular injury, an in vivo model of tissue stiffening and proliferation. Complementary experiments performed with deformable substrata demonstrated that stiffness-mediated activation of a focal adhesion kinase (FAK-p130Cas-Rac signaling pathway induces N-cadherin. Additionally, by culturing paired and unpaired SMCs on microfabricated adhesive islands of different areas, we found that N-cadherin relaxes the spreading requirement for SMC proliferation. In vivo SMC deletion of N-cadherin strongly reduced injury-induced cycling. Finally, SMC-specific deletion of FAK inhibited proliferation after vascular injury, and this was accompanied by reduced induction of N-cadherin. Thus, a stiffness- and FAK-dependent induction of N-cadherin connects cell-matrix to cell-cell adhesion and regulates the degree of cell spreading needed for cycling.

  2. Chromatin remodeling enzyme Brg1 is required for mouse lens fiber cell terminal differentiation and its denucleation

    Directory of Open Access Journals (Sweden)

    He Shuying

    2010-11-01

    Full Text Available Abstract Background Brahma-related gene 1 (Brg1, also known as Smarca4 and Snf2β encodes an adenosine-5'-triphosphate (ATP-dependent catalytical subunit of the (switch/sucrose nonfermentable (SWI/SNF chromatin remodeling complexes. SWI/SNF complexes are recruited to chromatin through multiple mechanisms, including specific DNA-binding factors (for example, heat shock transcription factor 4 (Hsf4 and paired box gene 6 (Pax6, chromatin structural proteins (for example, high-mobility group A1 (HMGA1 and/or acetylated core histones. Previous studies have shown that a single amino acid substitution (K798R in the Brg1 ATPase domain acts via a dominant-negative (dn mechanism. Genetic studies have demonstrated that Brg1 is an essential gene for early (that is, prior implantation mouse embryonic development. Brg1 also controls neural stem cell maintenance, terminal differentiation of multiple cell lineages and organs including the T-cells, glial cells and limbs. Results To examine the roles of Brg1 in mouse lens development, a dnBrg1 transgenic construct was expressed using the lens-specific αA-crystallin promoter in postmitotic lens fiber cells. Morphological studies revealed abnormal lens fiber cell differentiation in transgenic lenses resulting in cataract. Electron microscopic studies showed abnormal lens suture formation and incomplete karyolysis (that is, denucleation of lens fiber cells. To identify genes regulated by Brg1, RNA expression profiling was performed in embryonic day 15.5 (E15.5 wild-type and dnBrg1 transgenic lenses. In addition, comparisons between differentially expressed genes in dnBrg1 transgenic, Pax6 heterozygous and Hsf4 homozygous lenses identified multiple genes coregulated by Brg1, Hsf4 and Pax6. DNase IIβ, a key enzyme required for lens fiber cell denucleation, was found to be downregulated in each of the Pax6, Brg1 and Hsf4 model systems. Lens-specific deletion of Brg1 using conditional gene targeting demonstrated that

  3. Increased T follicular helper cells and germinal center B cells are required for cGVHD and bronchiolitis obliterans

    OpenAIRE

    Flynn, Ryan; Du, Jing; Veenstra, Rachelle G.; Reichenbach, Dawn K.; Panoskaltsis-Mortari, Angela; Taylor, Patricia A.; Freeman, Gordon J.; Serody, Jonathan S.; Murphy, William J.; Munn, David H.; Sarantopoulos, Stefanie; Luznik, Leo; Maillard, Ivan; Koreth, John; Cutler, Corey

    2014-01-01

    T follicular helper cells and germinal center B cells are increased and strongly correlate with the development of cGVHD in a murine model.Blocking mAbs for IL-21, ICOS, and CD40L are potential novel therapeutics for cGVHD.

  4. Requirement for CD40 ligand, CD4(+) T cells, and B cells in an infectious mononucleosis-like syndrome

    DEFF Research Database (Denmark)

    Brooks, J W; Hamilton-Easton, A M; Christensen, J P;

    1999-01-01

    Respiratory challenge with the murine gammaherpesvirus 68 (gammaHV-68) results in productive infection of the lung, the establishment of latency in B lymphocytes and other cell types, transient splenomegaly, and prolonged clonal expansion of activated CD8(+) CD62L(lo) T cells, particularly a Vbet...

  5. Active Stat3 is required for survival of human squamous cell carcinoma cells in serum-free conditions

    Directory of Open Access Journals (Sweden)

    DiGiovanni John

    2006-04-01

    Full Text Available Abstract Background Squamous cell carcinoma (SCC of the skin is the most aggressive form of non-melanoma skin cancer (NMSC, and is the single most commonly diagnosed cancer in the U.S., with over one million new cases reported each year. Recent studies have revealed an oncogenic role of activated signal transducer and activator of transcription 3 (Stat3 in many human tumors, especially in those of epithelial origin, including skin SCC. Stat3 is a mediator of numerous growth factor and cytokine signaling pathways, all of which activate it through phosphorylation of tyrosine 705. Results To further address the role of Stat3 in skin SCC tumorigenesis, we have analyzed a panel of human skin-derived cell lines ranging from normal human epidermal keratinocytes (NHEK, to non-tumorigenic transformed skin cells (HaCaT, to highly tumorigenic cells (SRB1-m7 and SRB12-p9 and observed a positive correlation between Stat3 phosphorylation and SCC malignancy. We next determined the role of Stat3 activity in cell proliferation and viability under serum-free culture conditions. This was accomplished by suppressing Stat3 activity in the SRB12-p9 cells through stable expression of a dominant negative acting form of Stat3β, which contains a tyrosine 705 to phenylalanine mutation (S3DN. The S3DN cells behaved similar to parental SRB12-p9 cells when cultured in optimal growth conditions, in the presence of 10% fetal calf serum. However, unlike the SRB12-p9 cells, S3DN cells underwent apoptotic cell death when cultured in serum-free medium (SFM. This was evidenced by multiple criteria, including accumulation of sub-G1 particles, induced PARP cleavage, and acquisition of the characteristic morphological changes associated with apoptosis. Conclusion This study provides direct evidence for a role for Stat3 in maintaining cell survival in the conditions of exogenous growth factor deprivation produced by culture in SFM. We also propose that delivery of the S3DN gene or

  6. Neuronal cell fate decisions:  O2 and CO2 sensing neurons require egl-13/Sox5

    DEFF Research Database (Denmark)

    Gramstrup Petersen, Jakob; Pocock, Roger David John

    2013-01-01

    We recently conducted a study that aimed to describe the differentiation mechanisms used to generate O2 and CO2 sensing neurons in C. elegans. We identified egl-13/Sox5 to be required for the differentiation of both O2 and CO2 sensing neurons. We found that egl-13 functions cell autonomously...

  7. Vangl2-regulated polarisation of second heart field-derived cells is required for outflow tract lengthening during cardiac development.

    Directory of Open Access Journals (Sweden)

    Simon A Ramsbottom

    2014-12-01

    Full Text Available Planar cell polarity (PCP is the mechanism by which cells orient themselves in the plane of an epithelium or during directed cell migration, and is regulated by a highly conserved signalling pathway. Mutations in the PCP gene Vangl2, as well as in other key components of the pathway, cause a spectrum of cardiac outflow tract defects. However, it is unclear why cells within the mesodermal heart tissue require PCP signalling. Using a new conditionally floxed allele we show that Vangl2 is required solely within the second heart field (SHF to direct normal outflow tract lengthening, a process that is required for septation and normal alignment of the aorta and pulmonary trunk with the ventricular chambers. Analysis of a range of markers of polarised epithelial tissues showed that in the normal heart, undifferentiated SHF cells move from the dorsal pericardial wall into the distal outflow tract where they acquire an epithelial phenotype, before moving proximally where they differentiate into cardiomyocytes. Thus there is a transition zone in the distal outflow tract where SHF cells become more polarised, turn off progenitor markers and start to differentiate to cardiomyocytes. Membrane-bound Vangl2 marks the proximal extent of this transition zone and in the absence of Vangl2, the SHF-derived cells are abnormally polarised and disorganised. The consequent thickening, rather than lengthening, of the outflow wall leads to a shortened outflow tract. Premature down regulation of the SHF-progenitor marker Isl1 in the mutants, and accompanied premature differentiation to cardiomyocytes, suggests that the organisation of the cells within the transition zone is important for maintaining the undifferentiated phenotype. Thus, Vangl2-regulated polarisation and subsequent acquisition of an epithelial phenotype is essential to lengthen the tubular outflow vessel, a process that is essential for on-going cardiac morphogenesis.

  8. β-catenin is selectively required for the expansion and regeneration of mature pancreatic acinar cells in mice

    Directory of Open Access Journals (Sweden)

    Matthew D. Keefe

    2012-07-01

    The size of the pancreas is determined by intrinsic factors, such as the number of progenitor cells, and by extrinsic signals that control the fate and proliferation of those progenitors. Both the exocrine and endocrine compartments of the pancreas undergo dramatic expansion after birth and are capable of at least partial regeneration following injury. Whether the expansion of these lineages relies on similar mechanisms is unknown. Although we have shown that the Wnt signaling component β-catenin is selectively required in mouse embryos for the generation of exocrine acinar cells, this protein has been ascribed various functions in the postnatal pancreas, including proliferation and regeneration of islet as well as acinar cells. To address whether β-catenin remains important for the maintenance and expansion of mature acinar cells, we have established a system to follow the behavior and fate of β-catenin-deficient cells during postnatal growth and regeneration in mice. We find that β-catenin is continuously required for the establishment and maintenance of acinar cell mass, extending from embryonic specification through juvenile and adult self-renewal and regeneration. This requirement is not shared with islet cells, which proliferate and function normally in the absence of β-catenin. These results make distinct predictions for the relative role of Wnt–β-catenin signaling in the etiology of human endocrine and exocrine disease. We suggest that loss of Wnt–β-catenin activity is unlikely to drive islet dysfunction, as occurs in type 2 diabetes, but that β-catenin is likely to promote human acinar cell proliferation following injury, and might therefore contribute to the resolution of acute or chronic pancreatitis.

  9. echinus, required for interommatidial cell sorting and cell death in the Drosophila pupal retina, encodes a protein with homology to ubiquitin-specific proteases

    Directory of Open Access Journals (Sweden)

    Gorski Sharon M

    2007-07-01

    Full Text Available Abstract Background Programmed cell death is used to remove excess cells between ommatidia in the Drosophila pupal retina. This death is required to establish the crystalline, hexagonal packing of ommatidia that characterizes the adult fly eye. In previously described echinus mutants, interommatidial cell sorting, which precedes cell death, occurred relatively normally. Interommatidial cell death was partially suppressed, resulting in adult eyes that contained excess pigment cells, and in which ommatidia were mildly disordered. These results have suggested that echinus functions in the pupal retina primarily to promote interommatidial cell death. Results We generated a number of new echinus alleles, some likely null mutants. Analysis of these alleles provides evidence that echinus has roles in cell sorting as well as cell death. echinus encodes a protein with homology to ubiquitin-specific proteases. These proteins cleave ubiquitin-conjugated proteins at the ubiquitin C-terminus. The echinus locus encodes multiple splice forms, including two proteins that lack residues thought to be critical for deubiquitination activity. Surprisingly, ubiquitous expression in the eye of versions of Echinus that lack residues critical for ubiquitin specific protease activity, as well as a version predicted to be functional, rescue the echinus loss-of-function phenotype. Finally, genetic interactions were not detected between echinus loss and gain-of-function and a number of known apoptotic regulators. These include Notch, EGFR, the caspases Dronc, Drice, Dcp-1, Dream, the caspase activators, Rpr, Hid, and Grim, the caspase inhibitor DIAP1, and Lozenge or Klumpfuss. Conclusion The echinus locus encodes multiple splice forms of a protein with homology to ubiquitin-specific proteases, but protease activity is unlikely to be required for echinus function, at least when echinus is overexpressed. Characterization of likely echinus null alleles and genetic interactions

  10. Six1 is required for mouse dental follicle cell and human periodontal ligament-derived cell proliferation.

    Science.gov (United States)

    Kawasaki, Tatsuki; Takahashi, Masanori; Yajima, Hiroshi; Mori, Yoshiyuki; Kawakami, Kiyoshi

    2016-08-01

    The periodontal ligament (PDL) is a connective tissue that attaches the tooth cementum to the alveolar bone and is derived from dental follicle cells (DFCs). The DFCs form fibroblasts, osteoblasts, cementoblasts, and PDL stem cells (PDLSCs). We previously reported homeobox transcription factor Six1 expression in mouse DFCs. However, the role of Six1 in periodontal tissue development is largely unknown. In this study, we analyzed SIX1 expression in mouse periodontal tissue cells during postnatal development and adulthood. We also addressed the role of SIX1 in mouse periodontium development and in human cultured PDL-derived cells (PDLCs). In mouse development, SIX1 production was abundant in DFCs and PDL cells by 2 weeks, but it was greatly diminished in the PDL at 4 weeks and in adults. Although the SIX1-positive cell distribution was sparse in the adult PDL, SIX1-positive cells were observed with low expression levels. We used 5-ethynyl-2'-deoxyuridine (EdU) for cell labeling to reveal numerous EdU/SIX1-double positive cells at 2 weeks; however, a few EdU-positive cells remained at 4 weeks. The proportion of DFCs that incorporated EdU was significantly lower in Six1-deficient mice compared with wild-type mice at E18.5. In human PDLCs, SIX1 was intensely expressed, and SIX1-knockdown using siRNA reduced proliferating PDLCs. Our results suggest that SIX1 is a key proliferation regulator in mouse DFCs and human PDLCs, which provides novel insight into Six family gene function in mammals. PMID:27241908

  11. The zinc finger transcription factor Gfi1, implicated in lymphomagenesis, is required for inner ear hair cell differentiation and survival

    Science.gov (United States)

    Wallis, Deeann; Hamblen, Melanie; Zhou, Yi; Venken, Koen J T.; Schumacher, Armin; Grimes, H. Leighton; Zoghbi, Huda Y.; Orkin, Stuart H.; Bellen, Hugo J.

    2003-01-01

    Gfi1 was first identified as causing interleukin 2-independent growth in T cells and lymphomagenesis in mice. Much work has shown that Gfi1 and Gfi1b, a second mouse homolog, play pivotal roles in blood cell lineage differentiation. However, neither Gfi1 nor Gfi1b has been implicated in nervous system development, even though their invertebrate homologues, senseless in Drosophila and pag-3 in C. elegans are expressed and required in the nervous system. We show that Gfi1 mRNA is expressed in many areas that give rise to neuronal cells during embryonic development in mouse, and that Gfi1 protein has a more restricted expression pattern. By E12.5 Gfi1 mRNA is expressed in both the CNS and PNS as well as in many sensory epithelia including the developing inner ear epithelia. At later developmental stages, Gfi1 expression in the ear is refined to the hair cells and neurons throughout the inner ear. Gfi1 protein is expressed in a more restricted pattern in specialized sensory cells of the PNS, including the eye, presumptive Merkel cells, the lung and hair cells of the inner ear. Gfi1 mutant mice display behavioral defects that are consistent with inner ear anomalies, as they are ataxic, circle, display head tilting behavior and do not respond to noise. They have a unique inner ear phenotype in that the vestibular and cochlear hair cells are differentially affected. Although Gfi1-deficient mice initially specify inner ear hair cells, these hair cells are disorganized in both the vestibule and cochlea. The outer hair cells of the cochlea are improperly innervated and express neuronal markers that are not normally expressed in these cells. Furthermore, Gfi1 mutant mice lose all cochlear hair cells just prior to and soon after birth through apoptosis. Finally, by five months of age there is also a dramatic reduction in the number of cochlear neurons. Hence, Gfi1 is expressed in the developing nervous system, is required for inner ear hair cell differentiation, and its loss

  12. Protein SUMOylation Is Required for Regulatory T Cell Expansion and Function.

    Science.gov (United States)

    Ding, Xiao; Wang, Aibo; Ma, Xiaopeng; Demarque, Maud; Jin, Wei; Xin, Huawei; Dejean, Anne; Dong, Chen

    2016-07-26

    Foxp3-expressing regulatory T (Treg) cells are essential for immune tolerance; however, the molecular mechanisms underlying Treg cell expansion and function are still not well understood. SUMOylation is a protein post-translational modification characterized by covalent attachment of SUMO moieties to lysines. UBC9 is the only E2 conjugating enzyme involved in this process, and loss of UBC9 completely abolishes the SUMOylation pathway. Here, we report that selective deletion of Ubc9 within the Treg lineage results in fatal early-onset autoimmunity similar to Foxp3 mutant mice. Ubc9-deficient Treg cells exhibit severe defects in TCR-driven homeostatic proliferation, accompanied by impaired activation and compromised suppressor function. Importantly, TCR ligation enhanced SUMOylation of IRF4, a critical regulator of Treg cell function downstream of TCR signals, which regulates its stability in Treg cells. Our data thus have demonstrated an essential role of SUMOylation in the expansion and function of Treg cells. PMID:27425617

  13. Division genes in Escherichia coli are expressed coordinately to cell septum requirements by gearbox promoters.

    Science.gov (United States)

    Aldea, M; Garrido, T; Pla, J; Vicente, M

    1990-11-01

    The cell division ftsQAZ cluster and the ftsZ-dependent bolA morphogene of Escherichia coli are found to be driven by gearboxes, a distinct class of promoters characterized by showing an activity that is inversely dependent on growth rate. These promoters contain specific sequences upstream from the mRNA start point, and their -10 region is essential for the inverse growth rate dependence. Gearbox promoters are essential for driving ftsQAZ and bolA gene expression so that the encoded products are synthesized at constant amounts per cell independently of cell size. This mode of regulation would be expected for the expression of proteins that either play a regulatory role in cell division or form a stoichiometric component of the septum, a structure that, independently of cell size and growth rate, is produced once per cell cycle.

  14. Structural characteristics of an antigen required for its interaction with Ia and recognition by T cells

    DEFF Research Database (Denmark)

    Sette, A; Buus, S; Colon, S;

    1987-01-01

    A detailed analysis of the residues within an immunogenic peptide that endow it with the capacity to interact with Ia and to be recognized by T cells is presented. Ia interacts with only a few of the peptide residues and overall exhibits a very broad specificity. Some residues appear to interact...... both with Ia and with T cells, leading to a model in which a peptide antigen is 'sandwiched' between Ia and the T-cell receptor....

  15. Successful Reconstruction of Tooth Germ with Cell Lines Requires Coordinated Gene Expressions from the Initiation Stage

    OpenAIRE

    Yasuhiro Tomooka; Akihiko Komine

    2012-01-01

    Tooth morphogenesis is carried out by a series of reciprocal interactions between the epithelium and mesenchyme in embryonic germs. Previously clonal dental epithelial cell (epithelium of molar tooth germ (emtg)) lines were established from an embryonic germ. They were odontogenic when combined with a dental mesenchymal tissue, although the odontogenesis was quantitatively imperfect. To improve the microenvironment in the germs, freshly isolated dental epithelial cells were mixed with cells o...

  16. Androgen receptor signaling is required for androgen-sensitive human prostate cancer cell proliferation and survival

    Directory of Open Access Journals (Sweden)

    Day Wanda V

    2005-04-01

    Full Text Available Abstract Background Androgens and androgen receptors (AR regulate normal prostate development and growth. They also are involved in pathological development of prostatic diseases, including benign prostatic hyperplasia (BPH and prostate cancer (PCa. Antiandrogen therapy for PCa, in conjunction with chemical or surgical castration, offers initial positive responses and leads to massive prostate cell death. However, cancer cells later appear as androgen-independent PCa. To investigate the role of AR in prostate cell proliferation and survival, we introduced a vector-based small interfering RNA (siRNA. This siRNA targeted 5'-untranslated region of AR mRNA for extended suppression of AR expression in androgen-sensitive human prostate LNCaP cells. Results The siRNA design successfully suppressed endogenous AR expression, as revealed by western blotting and immunofluorescence staining in LNCaP cells. LNCaP cells did not proliferate in the absence of AR and underwent apoptosis, based on elevated phospho-Histone H2B expression and higher number of apoptotic body as compared to control cells. Conclusion We demonstrated that AR is vital for prostate cell proliferation and survival in this androgen-sensitive prostate cell line. These results further strengthen the hypothesis that AR can be a therapeutic target for treating androgen-sensitive stages of PCa. Unlike antiandorgens, however, siRNA targeting AR provides a direct inactivation of AR function through the suppression of AR protein expression.

  17. Evidence That Mast Cells Are Not Required for Healing of Splinted Cutaneous Excisional Wounds in Mice

    OpenAIRE

    Nauta, Allison C.; Grova, Monica; Montoro, Daniel T.; Zimmermann, Andrew; Tsai, Mindy; Geoffrey C Gurtner; Galli, Stephen J.; Longaker, Michael T.

    2013-01-01

    Wound healing is a complex biological process involving the interaction of many cell types to replace lost or damaged tissue. Although the biology of wound healing has been extensively investigated, few studies have focused on the role of mast cells. In this study, we investigated the possible role of mast cells in wound healing by analyzing aspects of cutaneous excisional wound healing in three types of genetically mast cell-deficient mice. We found that C57BL/6-KitW-sh/W-sh , WBB6F1-KitW/W-...

  18. Monocytes are required to prime peripheral blood T cells to undergo apoptosis.

    OpenAIRE

    Wu, M. X.; Daley, J F; Rasmussen, R A; Schlossman, S F

    1995-01-01

    Freshly isolated, human peripheral blood T (PBT) cells are largely resistant to the apoptotic effects of anti-CD3 monoclonal antibody, ionomycin, or phorbol 12-myristate 13-acetate (PMA). We demonstrate here, however, that PBT cells, including both CD4+ and CD8+ cell populations, can be readily induced to undergo apoptosis when cocultured with either autologous or allogeneic monocytes (Mo) in PMA-containing medium. Incubation of PBT cells with Mo at a ratio of 1:1 for 18 hr resulted in maxima...

  19. Recruitment of Mediator Complex by Cell Type and Stage-Specific Factors Required for Tissue-Specific TAF Dependent Gene Activation in an Adult Stem Cell Lineage.

    Science.gov (United States)

    Lu, Chenggang; Fuller, Margaret T

    2015-12-01

    Onset of terminal differentiation in adult stem cell lineages is commonly marked by robust activation of new transcriptional programs required to make the appropriate differentiated cell type(s). In the Drosophila male germ line stem cell lineage, the switch from proliferating spermatogonia to spermatocyte is accompanied by one of the most dramatic transcriptional changes in the fly, as over 1000 new transcripts turn on in preparation for meiosis and spermatid differentiation. Here we show that function of the coactivator complex Mediator is required for activation of hundreds of new transcripts in the spermatocyte program. Mediator appears to act in a sequential hierarchy, with the testis activating Complex (tMAC), a cell type specific form of the Mip/dREAM general repressor, required to recruit Mediator subunits to the chromatin, and Mediator function required to recruit the testis TAFs (tTAFs), spermatocyte specific homologs of subunits of TFIID. Mediator, tMAC and the tTAFs co-regulate expression of a major set of spermatid differentiation genes. The Mediator subunit Med22 binds the tMAC component Topi when the two are coexpressed in S2 cells, suggesting direct recruitment. Loss of Med22 function in spermatocytes causes meiosis I maturation arrest male infertility, similar to loss of function of the tMAC subunits or the tTAFs. Our results illuminate how cell type specific versions of the Mip/dREAM complex and the general transcription machinery cooperate to drive selective gene activation during differentiation in stem cell lineages. PMID:26624996

  20. Sinorhizobium meliloti Cells Require Biotin and either Cobalt or Methionine for Growth

    OpenAIRE

    Watson, Robert J.; Heys, Roselyn; Martin, Teresa; Savard, Marc

    2001-01-01

    Sinorhizobium meliloti is usually cultured in rich media containing yeast extract. It has been suggested that some components of yeast extract are also required for growth in minimal medium. We tested 27 strains of this bacterium and found that none were able to grow in minimal medium when methods to limit carryover of yeast extract were used during inoculation. By fractionation of yeast extract, two required growth factors were identified. Biotin was found to be absolutely required for growt...

  1. Slow cluster formation of purified human or rhesus T cells requires protein kinase C and LFA-1.

    Science.gov (United States)

    Eylar, E H; Molina, C; Báez, I; Kessler, M

    1996-03-01

    Homotropic T cell adhesion, as generally studied, consists of a rapid, transient binding process that is measured over a 15-120 min. period. Here we report a slow type of adhesion process occurring with human or rhesus T cells, purified from peripheral blood, that manifests itself by the formation of rounded, multi-layer clusters which may contain hundreds of cells. The maximal number and size of the clusters peak 1-2 days after the addition of phorbol ester, an absolute requirement. The number of clusters formed is proportional to phorbol ester concentration up to 1.25 ng/mL. Phorbol esters such as phorbol myristate acetate (PMA), phorbol dibutyrate (PDB), and 7-octylindolactam (OIL) induced optimal cluster formation at 1-13 ng/mL, levels slightly higher than that required to induce mitogenesis of purified T cells. Phorbol itself and the alpha-form of the ester were inactive. Both cluster formation and mitogenesis (stimulated by Con A or anti-CD3) are completely inhibited by staurosporin at 12.5 ng/mL. Even at 2.5 ng/mL, 74% of cluster formation was inhibited, which strongly implies a crucial role for protein kinase C. In the presence of accessory cells, T cell clusters were suppressed. Monoclonal Ab such as anti-CD3, mouse anti-CD3 followed by anti-mouse IgG, anti-CD4, anti-CD4A, anti-CD2, anti-CD8, and anti-CD45 did not induce cluster formation. None were inhibitory or stimulatory in the presence of PMA, except for anti-CD3 which enhanced cluster formation by 26%. However, anti-LFA-1 beta-chain (mouse monoclonal) completely blocked cluster formation over the range studied (63-1000 ng/mL) for both human and rhesus cells; rat anti-LFA-1 only blocked human cell adhesion. Anti LFA-1 only partially inhibited T cell mitogenesis. These results show that slow cluster formation shares the LFA-1 and phorbol ester requirements of the rapid adhesion of T cells requiring LFA-1 and ICAM-1. However, cluster occurs at a very low phorbol ester concentration, appears more

  2. Oct4 is required for lineage priming in the developing inner cell mass of the mouse blastocyst.

    Science.gov (United States)

    Le Bin, Gloryn Chia; Muñoz-Descalzo, Silvia; Kurowski, Agata; Leitch, Harry; Lou, Xinghua; Mansfield, William; Etienne-Dumeau, Charles; Grabole, Nils; Mulas, Carla; Niwa, Hitoshi; Hadjantonakis, Anna-Katerina; Nichols, Jennifer

    2014-03-01

    The transcription factor Oct4 is required in vitro for establishment and maintenance of embryonic stem cells and for reprogramming somatic cells to pluripotency. In vivo, it prevents the ectopic differentiation of early embryos into trophoblast. Here, we further explore the role of Oct4 in blastocyst formation and specification of epiblast versus primitive endoderm lineages using conditional genetic deletion. Experiments involving mouse embryos deficient for both maternal and zygotic Oct4 suggest that it is dispensable for zygote formation, early cleavage and activation of Nanog expression. Nanog protein is significantly elevated in the presumptive inner cell mass of Oct4 null embryos, suggesting an unexpected role for Oct4 in attenuating the level of Nanog, which might be significant for priming differentiation during epiblast maturation. Induced deletion of Oct4 during the morula to blastocyst transition disrupts the ability of inner cell mass cells to adopt lineage-specific identity and acquire the molecular profile characteristic of either epiblast or primitive endoderm. Sox17, a marker of primitive endoderm, is not detected following prolonged culture of such embryos, but can be rescued by provision of exogenous FGF4. Interestingly, functional primitive endoderm can be rescued in Oct4-deficient embryos in embryonic stem cell complementation assays, but only if the host embryos are at the pre-blastocyst stage. We conclude that cell fate decisions within the inner cell mass are dependent upon Oct4 and that Oct4 is not cell-autonomously required for the differentiation of primitive endoderm derivatives, as long as an appropriate developmental environment is established. PMID:24504341

  3. Scleraxis is required for cell lineage differentiation and extracellular matrix remodeling during murine heart valve formation in vivo.

    Science.gov (United States)

    Levay, Agata K; Peacock, Jacqueline D; Lu, Yinhui; Koch, Manuel; Hinton, Robert B; Kadler, Karl E; Lincoln, Joy

    2008-10-24

    Heart valve structures, derived from mesenchyme precursor cells, are composed of differentiated cell types and extracellular matrix arranged to facilitate valve function. Scleraxis (scx) is a transcription factor required for tendon cell differentiation and matrix organization. This study identified high levels of scx expression in remodeling heart valve structures at embryonic day 15.5 through postnatal stages using scx-GFP reporter mice and determined the in vivo function using mice null for scx. Scx(-/-) mice display significantly thickened heart valve structures from embryonic day 17.5, and valves from mutant mice show alterations in valve precursor cell differentiation and matrix organization. This is indicated by decreased expression of the tendon-related collagen type XIV, increased expression of cartilage-associated genes including sox9, as well as persistent expression of mesenchyme cell markers including msx1 and snai1. In addition, ultrastructure analysis reveals disarray of extracellular matrix and collagen fiber organization within the valve leaflet. Thickened valve structures and increased expression of matrix remodeling genes characteristic of human heart valve disease are observed in juvenile scx(-/-) mice. In addition, excessive collagen deposition in annular structures within the atrioventricular junction is observed. Collectively, our studies have identified an in vivo requirement for scx during valvulogenesis and demonstrate its role in cell lineage differentiation and matrix distribution in remodeling valve structures.

  4. LKB1 Is Required for the Development and Maintenance of Stereocilia in Inner Ear Hair Cells in Mice.

    Directory of Open Access Journals (Sweden)

    Yuqin Men

    Full Text Available The LKB1 gene, which encodes a serine/threonine kinase, was discovered to play crucial roles in cell differentiation, proliferation, and the establishment of cell polarity. In our study, LKB1 conditional knockout mice (Atoh1-LKB1-/- mice were generated to investigate LKB1 function in the inner ear. Tests of auditory brainstem response and distortion product otoacoustic emissions revealed significant decreases in the hearing sensitivities of the Atoh1-LKB1-/- mice. In Atoh1-LKB1-/- mice, malformations of hair cell stereocilliary bundles were present as early as postnatal day 1 (P1, a time long before the maturation of the hair cell bundles. In addition, we also observed outer hair cell (OHC loss starting at P14. The impaired stereocilliary bundles occurred long before the presence of hair cell loss. Stereociliary cytoskeletal structure depends on the core actin-based cytoskeleton and several actin-binding proteins. By Western blot, we examined actin-binding proteins, specifically ERM (ezrin/radixin/moesin proteins involved in the regulation of the actin cytoskeleton of hair cell stereocilia. Our results revealed that the phosphorylation of ERM proteins (pERM was significantly decreased in mutant mice. Thus, we propose that the decreased pERM may be a key factor for the impaired stereocillia function, and the damaged stereocillia may induce hair cell loss and hearing impairments. Taken together, our data indicates that LKB1 is required for the development and maintenance of stereocilia in the inner ear.

  5. Energy-requiring uptake of prostasomes and PC3 cell-derived exosomes into non-malignant and malignant cells

    OpenAIRE

    Panaretakis, Theocharis; Ronquist, Karl Göran; Sanchez, Claire; Dubois, Louise; Chioureas, Dimitris; Fonseca, Pedro; Larsson, Anders; Ullén, Anders; Yachnin, Jeffrey; Ronquist, Gunnar

    2016-01-01

    Epithelial cells lining the prostate acini release, in a regulated manner (exocytosis), nanosized vesicles called prostasomes that belong to the exosome family. Prostate cancer cells have preserved this ability to generate and export exosomes to the extracellular space. We previously demonstrated that human prostasomes have an ATP-forming capacity. In this study, we compared the capacity of extracellular vesicles (EVs) to generate ATP between normal seminal prostasomes and exosomes secreted b...

  6. Rapid CD4+ T-cell responses to bacterial flagellin require dendritic cell expression of Syk and CARD9

    OpenAIRE

    Atif, Shaikh M.; Lee, Seung-Joo; Li, Lin-Xi; Uematsu, Satoshi; Akira, Shizuo; Gorjestani, Sara; Lin, Xin; Schweighoffer, Edina; Tybulewicz, Victor L.J.; McSorley, Stephen J.

    2014-01-01

    Toll-like receptors (TLRs) can recognize microbial patterns and utilize adaptor molecules, such as-MyD88 or (TRIF TIR-domain-containing adapter-inducing interferon-β), to initiate downstream signaling that ultimately affects the initiation of adaptive immunity. In addition to this inflammatory role, TLR5 expression on dendritic cells can favor antigen presentation of flagellin peptides and thus increase the sensitivity of flagellin-specific T-cell responses in vitro and in vivo. Here, we exam...

  7. The MP65 gene is required for cell wall integrity, adherence to epithelial cells and biofilm formation in Candida albicans

    Directory of Open Access Journals (Sweden)

    Girolamo Antonietta

    2011-05-01

    Full Text Available Abstract Background The MP65 gene of Candida albicans (orf19.1779 encodes a putative β-glucanase mannoprotein of 65 kDa, which plays a main role in a host-fungus relationship, morphogenesis and pathogenicity. In this study, we performed an extensive analysis of a mp65Δ mutant to assess the role of this protein in cell wall integrity, adherence to epithelial cells and biofilm formation. Results The mp65Δ mutant showed a high sensitivity to a range of cell wall-perturbing and degrading agents, especially Congo red, which induced morphological changes such as swelling, clumping and formation of hyphae. The mp65Δ mutant showed an activation of two MAPKs (Mkc1p and Cek1p, a high level of expression of two stress-related genes (DDR48 and SOD5, and a modulated expression of β-glucan epitopes, but no gross changes in cell wall polysaccharide composition. Interestingly, the mp65Δ mutant displayed a marked reduction in adhesion to BEC and Caco-2 cells and severe defects in biofilm formation when compared to the wild type. All of the mentioned properties were totally or partially recovered in a revertant strain, demonstrating the specificity of gene deletion. Conclusions We demonstrate that the MP65 gene of Candida albicans plays a significant role in maintaining cell wall integrity, as well as in adherence to epithelia and biofilm formation, which are major virulence attributes of this fungus.

  8. Mechanotransduction in mouse inner ear hair cells requires transmembrane channel-like genes

    NARCIS (Netherlands)

    Kawashima, Yoshiyuki; Geleoc, Gwenaelle S. G.; Kurima, Kiyoto; Labay, Valentina; Lelli, Andrea; Asai, Yukako; Makishima, Tomoko; Wu, Doris K.; Della Santina, Charles C.; Holt, Jeffrey R.; Griffith, Andrew J.

    2011-01-01

    Inner ear hair cells convert the mechanical stimuli of sound, gravity, and head movement into electrical signals. This mechanotransduction process is initiated by opening of cation channels near the tips of hair cell stereocilia. Since the identity of these ion channels is unknown, and mutations in

  9. Arabidopsis R-SNARE proteins VAMP721 and VAMP722 are required for cell plate formation.

    Directory of Open Access Journals (Sweden)

    Liang Zhang

    Full Text Available BACKGROUND: Cell plate formation during plant cytokinesis is facilitated by SNARE complex-mediated vesicle fusion at the cell-division plane. However, our knowledge regarding R-SNARE components of membrane fusion machinery for cell plate formation remains quite limited. METHODOLOGY/PRINCIPAL FINDINGS: We report the in vivo function of Arabidopsis VAMP721 and VAMP722, two closely sequence-related R-SNAREs, in cell plate formation. Double homozygous vamp721vamp722 mutant seedlings showed lethal dwarf phenotypes and were characterized by rudimentary roots, cotyledons and hypocotyls. Furthermore, cell wall stubs and incomplete cytokinesis were frequently observed in vamp721vamp722 seedlings. Confocal images revealed that green fluorescent protein-tagged VAMP721 and VAMP722 were preferentially localized to the expanding cell plates in dividing cells. Drug treatments and co-localization analyses demonstrated that punctuate organelles labeled with VAMP721 and VAMP722 represented early endosomes overlapped with VHA-a1-labeled TGN, which were distinct from Golgi stacks and prevacuolar compartments. In addition, protein traffic to the plasma membrane, but not to the vacuole, was severely disrupted in vamp721vamp722 seedlings by subcellular localization of marker proteins. CONCLUSION/SIGNIFICANCE: These observations suggest that VAMP721 and VAMP722 are involved in secretory trafficking to the plasma membrane via TGN/early endosomal compartment, which contributes substantially to cell plate formation during plant cytokinesis.

  10. N-cadherin is required for the polarized cell behaviors that drive neurulation in the zebrafish.

    Science.gov (United States)

    Hong, Elim; Brewster, Rachel

    2006-10-01

    Through the direct analysis of cell behaviors, we address the mechanisms underlying anterior neural tube morphogenesis in the zebrafish and the role of the cell adhesion molecule N-cadherin (N-cad) in this process. We demonstrate that although the mode of neurulation differs at the morphological level between amphibians and teleosts, the underlying cellular mechanisms are conserved. Contrary to previous reports, the zebrafish neural plate is a multi-layered structure, composed of deep and superficial cells that converge medially while undergoing radial intercalation, to form a single cell-layered neural tube. Time-lapse recording of individual cell behaviors reveals that cells are polarized along the mediolateral axis and exhibit protrusive activity. In N-cad mutants, both convergence and intercalation are blocked. Moreover, although N-cad-depleted cells are not defective in their ability to form protrusions, they are unable to maintain them stably. Taken together, these studies uncover key cellular mechanisms underlying neural tube morphogenesis in teleosts, and reveal a role for cadherins in promoting the polarized cell behaviors that underlie cellular rearrangements and shape the vertebrate embryo.

  11. Disparate Requirement for T Cells in Resistance to Mucosal and Acute Systemic Candidiasis

    OpenAIRE

    Jones-Carson, Jessica; Vazquez-Torres, Andres; Warner, Thomas; Balish, Edward

    2000-01-01

    Although highly susceptible to orogastric candidiasis, T-cell receptor δ- and α-chain knockout mice, deficient in γδ and αβ T cells, respectively, were found to be resistant to disseminated candidiasis of endogenous origin and to acute systemic candidiasis (resulting from intravenous injection).

  12. Codes and Standards Requirements for Deployment of Emerging Fuel Cell Technologies

    Energy Technology Data Exchange (ETDEWEB)

    Burgess, R.; Buttner, W.; Riykin, C.

    2011-12-01

    The objective of this NREL report is to provide information on codes and standards (of two emerging hydrogen power fuel cell technology markets; forklift trucks and backup power units), that would ease the implementation of emerging fuel cell technologies. This information should help project developers, project engineers, code officials and other interested parties in developing and reviewing permit applications for regulatory compliance.

  13. Rab5 is required in metastatic cancer cells for Caveolin-1-enhanced Rac1 activation, migration and invasion.

    Science.gov (United States)

    Díaz, Jorge; Mendoza, Pablo; Ortiz, Rina; Díaz, Natalia; Leyton, Lisette; Stupack, Dwayne; Quest, Andrew F G; Torres, Vicente A

    2014-06-01

    Rab5 is a small GTPase that regulates early endosome trafficking and other cellular processes, including cell adhesion and migration. Specifically, Rab5 promotes Rac1 activation and cancer cell migration, but little is known about the upstream regulators of Rab5. We have previously shown that the scaffolding protein Caveolin-1 (CAV1) promotes Rac1 activation and migration of cancer cells. Here, we hypothesized that CAV1 stimulates Rab5 activation, leading to increased Rac1 activity and cell migration. Expression of CAV1 in B16-F10 mouse melanoma and HT-29(US) human colon adenocarcinoma cells increased the GTP loading of Rab5, whereas shRNA-mediated targeting of endogenous CAV1 in MDA-MB-231 breast cancer cells decreased Rab5-GTP levels. Accordingly, shRNA-mediated downregulation of Rab5 decreased CAV1-mediated Rac1 activation, cell migration and invasion in B16-F10 and HT-29(US) cells. Expression of CAV1 was accompanied by increased recruitment of Tiam1, a Rac1 guanine nucleotide exchange factor (GEF), to Rab5-positive early endosomes. Using the inhibitor NSC23766, Tiam1 was shown to be required for Rac1 activation and cell migration induced by CAV1 and Rab5. Mechanistically, we provide evidence implicating p85α (also known as PIK3R1), a Rab5 GTPase-activating protein (GAP), in CAV1-dependent effects, by showing that CAV1 recruits p85α, precluding p85α-mediated Rab5 inactivation and increasing cell migration. In summary, these studies identify a novel CAV1-Rab5-Rac1 signaling axis, whereby CAV1 prevents Rab5 inactivation, leading to increased Rac1 activity and enhanced tumor cell migration and invasion.

  14. Bcl6 Is Required for Somatic Hypermutation and Gene Conversion in Chicken DT40 Cells.

    Directory of Open Access Journals (Sweden)

    Alan M Williams

    Full Text Available The activation induced cytosine deaminase (AID mediates diversification of B cell immunoglobulin genes by the three distinct yet related processes of somatic hypermutation (SHM, class switch recombination (CSR, and gene conversion (GCV. SHM occurs in germinal center B cells, and the transcription factor Bcl6 is a key regulator of the germinal center B cell gene expression program, including expression of AID. To test the hypothesis that Bcl6 function is important for the process of SHM, we compared WT chicken DT40 B cells, which constitutively perform SHM/GCV, to their Bcl6-deficient counterparts. We found that Bcl6-deficient DT40 cells were unable to perform SHM and GCV despite enforced high level expression of AID and substantial levels of AID in the nucleus of the cells. To gain mechanistic insight into the GCV/SHM dependency on Bcl6, transcriptional features of a highly expressed SHM target gene were analyzed in Bcl6-sufficient and -deficient DT40 cells. No defect was observed in the accumulation of single stranded DNA in the target gene as a result of Bcl6 deficiency. In contrast, association of Spt5, an RNA polymerase II (Pol II and AID binding factor, was strongly reduced at the target gene body relative to the transcription start site in Bcl6-deficient cells as compared to WT cells. However, partial reconstitution of Bcl6 function substantially reconstituted Spt5 association with the target gene body but did not restore detectable SHM. Our observations suggest that in the absence of Bcl6, Spt5 fails to associate efficiently with Pol II at SHM targets, perhaps precluding robust AID action on the SHM target DNA. Our data also suggest, however, that Spt5 binding is not sufficient for SHM of a target gene even in DT40 cells with strong expression of AID.

  15. Bcl6 Is Required for Somatic Hypermutation and Gene Conversion in Chicken DT40 Cells

    Science.gov (United States)

    Williams, Alan M.; Maman, Yaakov; Alinikula, Jukka; Schatz, David G.

    2016-01-01

    The activation induced cytosine deaminase (AID) mediates diversification of B cell immunoglobulin genes by the three distinct yet related processes of somatic hypermutation (SHM), class switch recombination (CSR), and gene conversion (GCV). SHM occurs in germinal center B cells, and the transcription factor Bcl6 is a key regulator of the germinal center B cell gene expression program, including expression of AID. To test the hypothesis that Bcl6 function is important for the process of SHM, we compared WT chicken DT40 B cells, which constitutively perform SHM/GCV, to their Bcl6-deficient counterparts. We found that Bcl6-deficient DT40 cells were unable to perform SHM and GCV despite enforced high level expression of AID and substantial levels of AID in the nucleus of the cells. To gain mechanistic insight into the GCV/SHM dependency on Bcl6, transcriptional features of a highly expressed SHM target gene were analyzed in Bcl6-sufficient and -deficient DT40 cells. No defect was observed in the accumulation of single stranded DNA in the target gene as a result of Bcl6 deficiency. In contrast, association of Spt5, an RNA polymerase II (Pol II) and AID binding factor, was strongly reduced at the target gene body relative to the transcription start site in Bcl6-deficient cells as compared to WT cells. However, partial reconstitution of Bcl6 function substantially reconstituted Spt5 association with the target gene body but did not restore detectable SHM. Our observations suggest that in the absence of Bcl6, Spt5 fails to associate efficiently with Pol II at SHM targets, perhaps precluding robust AID action on the SHM target DNA. Our data also suggest, however, that Spt5 binding is not sufficient for SHM of a target gene even in DT40 cells with strong expression of AID. PMID:26900682

  16. Osteocytes, not Osteoblasts or Lining Cells, are the Main Source of the RANKL Required for Osteoclast Formation in Remodeling Bone.

    Directory of Open Access Journals (Sweden)

    Jinhu Xiong

    Full Text Available The cytokine receptor activator of nuclear factor kappa B ligand (RANKL, encoded by the Tnfsf11 gene, is essential for osteoclastogenesis and previous studies have shown that deletion of the Tnfsf11 gene using a Dmp1-Cre transgene reduces osteoclast formation in cancellous bone by more than 70%. However, the Dmp1-Cre transgene used in those studies leads to recombination in osteocytes, osteoblasts, and lining cells making it unclear whether one or more of these cell types produce the RANKL required for osteoclast formation in cancellous bone. Because osteoblasts, osteocytes, and lining cells have distinct locations and functions, distinguishing which of these cell types are sources of RANKL is essential for understanding the orchestration of bone remodeling. To distinguish between these possibilities, we have now created transgenic mice expressing the Cre recombinase under the control of regulatory elements of the Sost gene, which is expressed in osteocytes but not osteoblasts or lining cells in murine bone. Activity of the Sost-Cre transgene in osteocytes, but not osteoblast or lining cells, was confirmed by crossing Sost-Cre transgenic mice with tdTomato and R26R Cre-reporter mice, which express tdTomato fluorescent protein or LacZ, respectively, only in cells expressing the Cre recombinase or their descendants. Deletion of the Tnfsf11 gene in Sost-Cre mice led to a threefold decrease in osteoclast number in cancellous bone and increased cancellous bone mass, mimicking the skeletal phenotype of mice in which the Tnfsf11 gene was deleted using the Dmp1-Cre transgene. These results demonstrate that osteocytes, not osteoblasts or lining cells, are the main source of the RANKL required for osteoclast formation in remodeling cancellous bone.

  17. Transmembrane Protein 184A Is a Receptor Required for Vascular Smooth Muscle Cell Responses to Heparin.

    Science.gov (United States)

    Pugh, Raymond J; Slee, Joshua B; Farwell, Sara Lynn N; Li, Yaqiu; Barthol, Trista; Patton, Walter A; Lowe-Krentz, Linda J

    2016-03-01

    Vascular cell responses to exogenous heparin have been documented to include decreased vascular smooth muscle cell proliferation following decreased ERK pathway signaling. However, the molecular mechanism(s) by which heparin interacts with cells to induce those responses has remained unclear. Previously characterized monoclonal antibodies that block heparin binding to vascular cells have been found to mimic heparin effects. In this study, those antibodies were employed to isolate a heparin binding protein. MALDI mass spectrometry data provide evidence that the protein isolated is transmembrane protein 184A (TMEM184A). Commercial antibodies against three separate regions of the TMEM184A human protein were used to identify the TMEM184A protein in vascular smooth muscle cells and endothelial cells. A GFP-TMEM184A construct was employed to determine colocalization with heparin after endocytosis. Knockdown of TMEM184A eliminated the physiological responses to heparin, including effects on ERK pathway activity and BrdU incorporation. Isolated GFP-TMEM184A binds heparin, and overexpression results in additional heparin uptake. Together, these data support the identification of TMEM184A as a heparin receptor in vascular cells.

  18. Downregulation of the transcription factor KLF4 is required for the lineage commitment of T cells

    Institute of Scientific and Technical Information of China (English)

    Xiaomin Wen; Haifeng Liu; Gang Xiao; Xiaolong Liu

    2011-01-01

    The roles of the reprogramming factors Oct4,Sox2,c-Myc and Klf4 in early T cell development are incompletely defined.Here,we show that Klf4 is the only reprogramming factor whose expression is downregulated when early thymic progenitors (ETPs) differentiate into T cells.Enforced expression of Klf4 in uncommitted progenitors severely impaired T cell development mainly at the DN2-to-DN3 transition when T cell lineage commitment occurs and affected the transcription of a variety of genes with crucial functions in early T cell development,including genes involved in microenvironmental signaling (IL-7Rα),Notch target genes (Deltexl),and essential T cell lineage regulatory or inhibitory genes (Bcllla,SpiB,and ldl).The survival of thymocytes and the rearrangement at the Tcrb locus were impaired in the presence of enforced Klf4 expression.The defects in the DN1-to-DN2 and DN2-to-DN3 transitions in Klf4 transgenic mice could not be rescued by the introduction of a TCR transgene,but was partially rescued by restoring the expression of IL-7Rα.Thus,our data indicate that the downregulation of Klf4 is a prerequisite for T cell lineage commitment.

  19. CLCA2 Interactor EVA1 Is Required for Mammary Epithelial Cell Differentiation.

    Directory of Open Access Journals (Sweden)

    Grace Ramena

    Full Text Available CLCA2 is a p53-, p63-inducible transmembrane protein that is frequently downregulated in breast cancer. It is induced during differentiation of human mammary epithelial cells, and its knockdown causes epithelial-to-mesenchymal transition (EMT. To determine how CLCA2 promotes epithelial differentiation, we searched for interactors using membrane dihybrid screening. We discovered a strong interaction with the cell junctional protein EVA1 (Epithelial V-like Antigen 1 and confirmed it by co-immunoprecipitation. Like CLCA2, EVA1 is a type I transmembrane protein that is regulated by p53 and p63. It is thought to mediate homophilic cell-cell adhesion in diverse epithelial tissues. We found that EVA1 is frequently downregulated in breast tumors and breast cancer cell lines, especially those of mesenchymal phenotype. Moreover, knockdown of EVA1 in immortalized human mammary epithelial cells (HMEC caused EMT, implying that EVA1 is essential for epithelial differentiation. Both EVA1 and CLCA2 co-localized with E-cadherin at cell-cell junctions. The interacting domains were delimited by deletion analysis, revealing the site of interaction to be the transmembrane segment (TMS. The primary sequence of the CLCA2 TMS was found to be conserved in CLCA2 orthologs throughout mammals, suggesting that its interaction with EVA1 co-evolved with the mammary gland. A screen for other junctional interactors revealed that CLCA2 was involved in two different complexes, one with EVA1 and ZO-1, the other with beta catenin. Overexpression of CLCA2 caused downregulation of beta catenin and beta catenin-activated genes. Thus, CLCA2 links a junctional adhesion molecule to cytosolic signaling proteins that modulate proliferation and differentiation. These results may explain how attenuation of CLCA2 causes EMT and why CLCA2 and EVA1 are frequently downregulated in metastatic breast cancer cell lines.

  20. Interactions of efficiency and material requirements for terrestrial silicon solar cells

    Science.gov (United States)

    Bowler, D. L.; Wolf, M.

    1980-01-01

    The transport velocity transformation method was used to analyze solar cell designs to determine optimum cell structures. It was found that low resistivity materials should be used up to the onset of Auger recombination; a properly designed three-layer structure permits base region approaching an ideal device in performance; and that higher resistivity front regions will need more sophisticated grid metallization structures than those used now. It was concluded that new features will provide idealized silicon cell structures yielding airmass 1 efficiencies in the 24-26.5% range, with real efficiencies near 22%.

  1. Donor Requirements for Regulatory T Cell Suppression of Murine Graft Versus Host Disease

    OpenAIRE

    Pierini, Antonio; Colonna, Lucrezia; Alvarez, Maite; Schneidawind, Dominik; Nishikii, Hidekazu; Baker, Jeanette; Pan, Yuqiong; Florek, Mareike; Kim, Byung-Su; Negrin, Robert S.

    2015-01-01

    Adoptive transfer of freshly isolated natural occurring CD4+CD25+FoxP3+ regulatory T cells (Treg) prevents graft versus host disease (GvHD) in several animal models and following hematopoietic cell transplantation (HCT) in clinical trials. Donor derived Treg have been mainly used as they share the same MHC with conventional CD4+ and CD8+ T cells (Tcon) that are primarily responsible for GvHD. Third-party derived Treg are a promising alternative for cellular therapy as they can be prepared in ...

  2. Dual Requirement of Cytokine and Activation Receptor Triggering for Cytotoxic Control of Murine Cytomegalovirus by NK Cells

    Science.gov (United States)

    Pak-Wittel, Melissa A.; Yang, Liping; Schreiber, Robert D.; Yokoyama, Wayne M.

    2015-01-01

    Natural killer (NK) cells play a critical role in controlling murine cytomegalovirus (MCMV) and can mediate both cytokine production and direct cytotoxicity. The NK cell activation receptor, Ly49H, is responsible for genetic resistance to MCMV in C57BL/6 mice. Recognition of the viral m157 protein by Ly49H is sufficient for effective control of MCMV infection. Additionally, during the host response to infection, distinct immune and non-immune cells elaborate a variety of pleiotropic cytokines which have the potential to impact viral pathogenesis, NK cells, and other immune functions, both directly and indirectly. While the effects of various immune deficiencies have been examined for general antiviral phenotypes, their direct effects on Ly49H-dependent MCMV control are poorly understood. To specifically interrogate Ly49H-dependent functions, herein we employed an in vivo viral competition approach to show Ly49H-dependent MCMV control is specifically mediated through cytotoxicity but not IFNγ production. Whereas m157 induced Ly49H-dependent degranulation, efficient cytotoxicity also required either IL-12 or type I interferon (IFN-I) which acted directly on NK cells to produce granzyme B. These studies demonstrate that both of these distinct NK cell-intrinsic mechanisms are integrated for optimal viral control by NK cells. PMID:26720279

  3. The Microtubule Plus-End Tracking Protein CLASP2 Is Required for Hematopoiesis and Hematopoietic Stem Cell Maintenance

    Directory of Open Access Journals (Sweden)

    Ksenija Drabek

    2012-10-01

    Full Text Available Mammalian CLASPs are microtubule plus-end tracking proteins whose essential function as regulators of microtubule behavior has been studied mainly in cultured cells. We show here that absence of murine CLASP2 in vivo results in thrombocytopenia, progressive anemia, and pancytopenia, due to defects in megakaryopoiesis, in erythropoiesis, and in the maintenance of hematopoietic stem cell activity. Furthermore, microtubule stability and organization are affected upon attachment of Clasp2 knockout hematopoietic stem-cell-enriched populations, and these cells do not home efficiently toward their bone marrow niche. Strikingly, CLASP2-deficient hematopoietic stem cells contain severely reduced mRNA levels of c-Mpl, which encodes the thrombopoietin receptor, an essential factor for megakaryopoiesis and hematopoietic stem cell maintenance. Our data suggest that thrombopoietin signaling is impaired in Clasp2 knockout mice. We propose that the CLASP2-mediated stabilization of microtubules is required for proper attachment, homing, and maintenance of hematopoietic stem cells and that this is necessary to sustain c-Mpl transcription.

  4. TRANSFORMING GROWTH FACTOR-BETA MEDIATED SUPPRESSION OF ANTI-TUMOR T CELLS REQUIRES FOXP1 TRANSCRIPTION FACTOR EXPRESSION

    Science.gov (United States)

    Stephen, Tom L.; Rutkowski, Melanie R.; Allegrezza, Michael J.; Perales-Puchalt, Alfredo; Tesone, Amelia J.; Svoronos, Nikolaos; Nguyen, Jenny M.; Sarmin, Fahmida; Borowsky, Mark E.; Tchou, Julia; Conejo-Garcia, Jose R.

    2014-01-01

    SUMMARY Tumor-reactive T cells become unresponsive in advanced tumors. Here we have characterized a common mechanism of T cell unresponsiveness in cancer driven by the up-regulation of the transcription factor Forkhead box protein P1 (Foxp1), which prevents CD8+ T cells from proliferating and up-regulating Granzyme-B and interferon-γ (IFN-γ) in response to tumor antigens. Accordingly, Foxp1-deficient lymphocytes induced rejection of incurable tumors, and promoted protection against tumor re-challenge. Mechanistically, Foxp1 interacted with the transcription factors Smad2 and Smad3 in pre-activated CD8+ T cells in response to microenvironmental transforming growth factor-β (TGF-β), and was essential for its suppressive activity. Therefore, Smad2 and Smad3-mediated c-Myc repression requires Foxp1 expression in T cells. Furthermore, Foxp1 directly mediated TGF-β-induced c-Jun transcriptional repression, which abrogated T cell activity. Our results unveil a fundamental mechanism of T cell unresponsiveness different from anergy or exhaustion, driven by TGF-β signaling on tumor-associated lymphocytes undergoing Foxp1-dependent transcriptional regulation. PMID:25238097

  5. Discrete levels of Twist activity are required to direct distinct cell functions during gastrulation and somatic myogenesis.

    Directory of Open Access Journals (Sweden)

    Ming-Ching Wong

    Full Text Available Twist (Twi, a conserved basic helix-loop-helix transcriptional regulator, directs the epithelial-to-mesenchymal transition (EMT, and regulates changes in cell fate, cell polarity, cell division and cell migration in organisms from flies to humans. Analogous to its role in EMT, Twist has been implicated in metastasis in numerous cancer types, including breast, pancreatic and prostate. In the Drosophila embryo, Twist is essential for discrete events in gastrulation and mesodermal patterning. In this study, we derive a twi allelic series by examining the various cellular events required for gastrulation in Drosophila. By genetically manipulating the levels of Twi activity during gastrulation, we find that coordination of cell division is the most sensitive cellular event, whereas changes in cell shape are the least sensitive. Strikingly, we show that by increasing levels of Snail expression in a severe twi hypomorphic allelic background, but not a twi null background, we can reconstitute gastrulation and produce viable adult flies. Our results demonstrate that the level of Twi activity determines whether the cellular events of ventral furrow formation, EMT, cell division and mesodermal migration occur.

  6. A synthetic interaction screen identifies factors selectively required for proliferation and TERT transcription in p53-deficient human cancer cells.

    Directory of Open Access Journals (Sweden)

    Li Xie

    Full Text Available Numerous genetic and epigenetic alterations render cancer cells selectively dependent on specific genes and regulatory pathways, and represent potential vulnerabilities that can be therapeutically exploited. Here we describe an RNA interference (RNAi-based synthetic interaction screen to identify genes preferentially required for proliferation of p53-deficient (p53- human cancer cells. We find that compared to p53-competent (p53+ human cancer cell lines, diverse p53- human cancer cell lines are preferentially sensitive to loss of the transcription factor ETV1 and the DNA damage kinase ATR. In p53- cells, RNAi-mediated knockdown of ETV1 or ATR results in decreased expression of the telomerase catalytic subunit TERT leading to growth arrest, which can be reversed by ectopic TERT expression. Chromatin immunoprecipitation analysis reveals that ETV1 binds to a region downstream of the TERT transcriptional start-site in p53- but not p53+ cells. We find that the role of ATR is to phosphorylate and thereby stabilize ETV1. Our collective results identify a regulatory pathway involving ETV1, ATR, and TERT that is preferentially important for proliferation of diverse p53- cancer cells.

  7. MicroRNA-155 is required for effector CD8+ T cell responses to virus infection and cancer

    Science.gov (United States)

    DUDDA, Jan C.; SALAUN, Bruno; JI, Yun; PALMER, Douglas C.; Monnot, Gwennaelle C.; MERCK, Estelle; BOUDOUSQUIE, Caroline; UTZSCHNEIDER, Daniel T.; ESCOBAR, Thelma M.; PERRET, Rachel; MULJO, Stefan A.; HEBEISEN, Michael; RUFER, Nathalie; ZEHN, Dietmar; DONDA, Alena; RESTIFO, Nicholas P.; HELD, Werner; GATTINONI, Luca; ROMERO, Pedro

    2013-01-01

    SUMMARY MicroRNAs regulate the function of several immune cells but their role in promoting CD8+ T-cell immunity remains unknown. Here we report that miR-155 is required for CD8+ T-cell responses to both virus and cancer. In the absence of miR-155, accumulation of effector CD8+ T cells was severely reduced during acute and chronic viral infections and control of virus replication was impaired. Similarly, Mir155-/- CD8+ T cells were in effective at controlling tumor growth, whereas miR-155 overexpression enhanced the antitumor response. miR-155 deficiency resulted in accumulation of SOCS-1 causing defective cytokine signaling through STAT5. Consistently, enforced expression of SOCS-1 in CD8+ T cells phenocopied the miR-155 deficiency, whereas SOCS-1 silencing augmented tumor destruction. These findings identify miR-155 and its target SOCS-1 as key regulators of effector CD8+ T cells that can be modulated to potentiate immunotherapies for infectious diseases and cancer. PMID:23601686

  8. BTG1 is required to maintain the pool of stem and progenitor cells of dentate gyrus and subventricular zone

    Directory of Open Access Journals (Sweden)

    Stefano eFarioli-Vecchioli

    2012-08-01

    Full Text Available Btg1 belongs to a family of cell cycle inhibitory genes. We observed that Btg1 is highly expressed in adult neurogenic niches, i.e., the dentate gyrus and subventricular zone (SVZ. Thus, we generated Btg1 knockout mice to analyze the role of Btg1 in the process of generation of adult new neurons.Ablation of Btg1 causes a transient increase of the proliferating dentate gyrus stem and progenitor cells at post-natal day 7; however, at two months of age the number of these proliferating cells, as well as of mature neurons, greatly decreases compared to wild-type controls. Remarkably, adult dentate gyrus stem and progenitor cells of Btg1-null mice exit cell cycle after completing the S phase, highly express p53 and p21, and within 5 days undergo apoptosis. In SVZ we observed an equivalent decrease, associated to apoptosis, of Btg1-null stem cells, neuroblasts and neurons; furthermore, neurospheres derived from SVZ stem cells showed an age-dependent decrease of the self-renewal and expansion capacity.We conclude that the ablation of Btg1 reduces the pool of dividing adult stem and progenitor cells in dentate gyrus and SVZ by decreasing their proliferative capacity and inducing apoptosis, likely reflecting the impairment of the control of the cell cycle transition from G1 to S phase. As a result, the ability of Btg1-null mice to discriminate among overlapping contextual memories was affected. Thus, Btg1 appears to be required for maintaining adult stem and progenitor cells quiescence and self-renewal.

  9. Dynamin 2 is required for actin assembly in phagocytosis in Sertoli cells

    International Nuclear Information System (INIS)

    Dynamin 2 has been reported to be implicated in phagocytosis. However, the mode of action of dynamin is poorly understood. In this study, we examined whether dynamin 2 participates in actin assembly during phagocytosis in Sertoli cells. In the presence of dynasore, a dynamin inhibitor, phagocytosis was reduced by 60-70% in Sertoli cells and macrophages. Scanning electron microscopy revealed that Sertoli cells treated with dynasore were unable to form phagocytic cups. In addition, dysfunction of dynamin 2 reduced both actin polymerization and recruitment of actin and dynamin 2 to phosphatidylinositol (4,5) bisphosphate [PI(4,5)P2]-containing liposomes. The formation of dynamin 2-positive ruffles of Sertoli cells was decreased by 60-70% by sequestering PI(4,5)P2 either by expression of PH domain of PLCδ or treatment with neomycin. These results strongly suggest that dynamin 2 is involved in actin dynamics and the formation of dynamin 2-positive ruffles during phagocytosis.

  10. Autophagy is required for stem cell mobilization by G-CSF

    DEFF Research Database (Denmark)

    Leveque-El Mouttie, Lucie; Vu, Therese; Lineburg, Katie E.;

    2015-01-01

    Granulocyte colony-stimulating factor (G-CSF) is widely used clinically to prevent neutropenia after cytotoxic chemotherapy and to mobilize hematopoietic stem cells (HSCs) for transplantation. Autophagy, a process of cytoplasmic component recycling, maintains cellular homeostasis and protects...... the cell during periods of metabolic stress or nutrient deprivation. We have observed that G-CSF activates autophagy in neutrophils and HSCs from both mouse and human donors. Furthermore, G-CSF-induced neutrophil and HSC mobilization is impaired in the absence of autophagy. In contrast, autophagy...... is dispensable for direct HSC mobilization in response to the CXCR4 antagonist AMD3100. Altogether, these data demonstrate an important role for G-CSF in invoking autophagy within hematopoietic and myeloid cells and suggest that this pathway is critical for ensuring cell survival in response to clinically...

  11. Ligands, cell-based models, and readouts required for Toll-like receptor action.

    LENUS (Irish Health Repository)

    Dellacasagrande, Jerome

    2012-02-01

    This chapter details the tools that are available to study Toll-like receptor (TLR) biology in vitro. This includes ligands, host cells, and readouts. The use of modified TLRs to circumvent some technical problems is also discussed.

  12. Perlecan is required for FGF-2 signaling in the neural stem cell niche

    Directory of Open Access Journals (Sweden)

    Aurelien Kerever

    2014-03-01

    Full Text Available In the adult subventricular zone (neurogenic niche, neural stem cells double-positive for two markers of subsets of neural stem cells in the adult central nervous system, glial fibrillary acidic protein and CD133, lie in proximity to fractones and to blood vessel basement membranes, which contain the heparan sulfate proteoglycan perlecan. Here, we demonstrate that perlecan deficiency reduces the number of both GFAP/CD133-positive neural stem cells in the subventricular zone and new neurons integrating into the olfactory bulb. We also show that FGF-2 treatment induces the expression of cyclin D2 through the activation of the Akt and Erk1/2 pathways and promotes neurosphere formation in vitro. However, in the absence of perlecan, FGF-2 fails to promote neurosphere formation. These results suggest that perlecan is a component of the neurogenic niche that regulates FGF-2 signaling and acts by promoting neural stem cell self-renewal and neurogenesis.

  13. Identification of a region of simian virus 40 large T antigen required for cell transformation.

    OpenAIRE

    Chen, S.; Paucha, E

    1990-01-01

    A series of replication-competent simian virus 40 (SV40) large T antigens with point and deletion mutations in the amino acid sequence between residues 105 and 115 were examined for the ability to immortalize primary cultures of mouse and rat cells. The results show that certain mutants, including one that deletes the entire region, are able to immortalize. However, consistent with previous data, the immortalized cells are not fully transformed, as judged by doubling time, sensitivity to conc...

  14. Matrix Metalloproteinase-10 Is Required for Lung Cancer Stem Cell Maintenance, Tumor Initiation and Metastatic Potential

    OpenAIRE

    Verline Justilien; Regala, Roderick P.; I-Chu Tseng; Walsh, Michael P.; Jyotica Batra; Radisky, Evette S.; Murray, Nicole R.; Fields, Alan P.

    2012-01-01

    Matrix metalloproteinases (Mmps) stimulate tumor invasion and metastasis by degrading the extracellular matrix. Here we reveal an unexpected role for Mmp10 (stromelysin 2) in the maintenance and tumorigenicity of mouse lung cancer stem-like cells (CSC). Mmp10 is highly expressed in oncosphere cultures enriched in CSCs and RNAi-mediated knockdown of Mmp10 leads to a loss of stem cell marker gene expression and inhibition of oncosphere growth, clonal expansion, and transformed growth in vitro. ...

  15. Autophagy is required for self-renewal and differentiation of adult human stem cells

    Institute of Scientific and Technical Information of China (English)

    Souzan Salemi; Shida Yousefi; Mihai A Constantinescu; Martin F Fey; Hans-Uwe Simon

    2012-01-01

    Dear Editor,Patients receiving anticancer therapy usually suffer from side effects,such as loss of hair,epithelial barrier defects,and myelosuppression,which,however,are usually reversible when the therapy is discontinued.Although this clinical experience suggests that adult stem cells may survive such therapeutic interventions,the underlying molecular regulatory mechanisms are poorly understood.Such regulatory mechanisms also seem to protect adult stem cells from acquiring mutations,which could lead to additional tumorigenesis.

  16. Efficient lymphoreticular prion propagation requires PrP(c) in stromal and hematopoietic cells.

    OpenAIRE

    Kaeser, P S; Klein, M A; Schwarz, P.; Aguzzi, A

    2001-01-01

    In most prion diseases, infectivity accumulates in lymphoreticular organs early after infection. Defects in hematopoietic compartments, such as impaired B-cell maturation, or in stromal compartments, such as abrogation of follicular dendritic cells, can delay or prevent lymphoreticular prion colonization. However, the nature of the compartment in which prion replication takes place is controversial, and it is unclear whether this compartment coincides with that expressing the normal prion pro...

  17. Efficient Lymphoreticular Prion Propagation Requires PrPc in Stromal and Hematopoietic Cells

    OpenAIRE

    Kaeser, Pascal S.; Klein, Michael A.; Schwarz, Petra; Aguzzi, Adriano

    2001-01-01

    In most prion diseases, infectivity accumulates in lymphoreticular organs early after infection. Defects in hematopoietic compartments, such as impaired B-cell maturation, or in stromal compartments, such as abrogation of follicular dendritic cells, can delay or prevent lymphoreticular prion colonization. However, the nature of the compartment in which prion replication takes place is controversial, and it is unclear whether this compartment coincides with that expressing the normal prion pro...

  18. Presenilins are required for maintenance of neural stem cells in the developing brain

    Directory of Open Access Journals (Sweden)

    Kim Woo-Young

    2008-01-01

    Full Text Available Abstract The early embryonic lethality of mutant mice bearing germ-line deletions of both presenilin genes precluded the study of their functions in neural development. We therefore employed the Cre-loxP technology to generate presenilin conditional double knockout (PS cDKO mice, in which expression of both presenilins is inactivated in neural progenitor cells (NPC or neural stem cells and their derivative neurons and glia beginning at embryonic day 11 (E11. In PS cDKO mice, dividing NPCs labeled by BrdU are decreased in number beginning at E13.5. By E15.5, fewer than 20% of NPCs remain in PS cDKO mice. The depletion of NPCs is accompanied by severe morphological defects and hemorrhages in the PS cDKO embryonic brain. Interkinetic nuclear migration of NPCs is also disrupted in PS cDKO embryos, as evidenced by displacement of S-phase and M-phase nuclei in the ventricular zone of the telencephalon. Furthermore, the depletion of neural progenitor cells in PS cDKO embryos is due to NPCs exiting cell cycle and differentiating into neurons rather than reentering cell cycle between E13.5 and E14.5 following PS inactivation in most NPCs. The length of cell cycle, however, is unchanged in PS cDKO embryos. Expression of Notch target genes, Hes1 and Hes5, is significantly decreased in PS cDKO brains, whereas Dll1 expression is up-regulated, indicating that Notch signaling is effectively blocked by PS inactivation. These findings demonstrate that presenilins are essential for neural progenitor cells to re-enter cell cycle and thus ensure proper expansion of neural progenitor pool during embryonic neural development.

  19. APC is required for muscle stem cell proliferation and skeletal muscle tissue repair

    OpenAIRE

    Parisi, Alice; Lacour, Floriane; Giordani, Lorenzo; Colnot, Sabine; Maire, Pascal; Le Grand, Fabien

    2015-01-01

    The tumor suppressor adenomatous polyposis coli (APC) is a crucial regulator of many stem cell types. In constantly cycling stem cells of fast turnover tissues, APC loss results in the constitutive activation of a Wnt target gene program that massively increases proliferation and leads to malignant transformation. However, APC function in skeletal muscle, a tissue with a low turnover rate, has never been investigated. Here we show that conditional genetic disruption of APC in adult muscle ste...

  20. The necessary length of carbon nanotubes required to optimize solar cells

    OpenAIRE

    Barghi Tirdad; Saeedi Mohammad; Vaezzadeh Majid; Sadeghi Mohammad

    2007-01-01

    Abstract Background In recent years scientists have been trying both to increase the efficiency of solar cells, whilst at the same time reducing dimensions and costs. Increases in efficiency have been brought about by implanting carbon nanotubes onto the surface of solar cells in order to reduce the reflection of sunrays, as well as through the insertion of polymeric arrays into the intrinsic layer for charge separation. Results The experimental results show power rising linearly for intrinsi...

  1. CTLA-4 is Required by CD4+CD25+ Treg to Control CD4+ T Cell Lymphopenia-Induced Proliferation

    OpenAIRE

    Sojka, Dorothy K.; Hughson, Angela; Fowell, Deborah J.

    2009-01-01

    CTLA-4 is constitutively expressed by CD4+CD25+Foxp3+ regulatory T cells (Treg) but its precise role in Treg function is not clear. Although blockade of CTLA-4 interferes with Treg function, studies using CTLA-4 deficient Treg have failed to reveal an essential requirement for CTLA-4 in Treg suppression in vivo. Conditional deletion of CTLA-4 in Foxp3+ T cells disrupts immune homeostasis in vivo but the immune processes disrupted by CTLA-4 deletion have not been determined. We demonstrate tha...

  2. A tetanus toxin sensitive protein other than VAMP 2 is required for exocytosis in the pancreatic acinar cell.

    Science.gov (United States)

    Padfield, P J

    2000-11-01

    The neurotoxin sensitivity of regulated exocytosis in the pancreatic acinar cell was investigated using streptolysin-O permeabilized pancreatic acini. Treatment of permeabilized acini with botulinum toxin B (BoNT/B) or botulinum toxin D (BoNT/D) had no detectable effect on Ca(2+)-dependent amylase secretion but did result in the complete cleavage of VAMP 2. In comparison, tetanus toxin (TeTx) treatment both significantly inhibited Ca(2+)-dependent amylase secretion and cleaved VAMP 2. These results indicate that regulated exocytosis in the pancreatic acinar cell requires a tetanus toxin sensitive protein(s) other than VAMP 2.

  3. Homologs of SCAR/WAVE complex components are required for epidermal cell morphogenesis in rice.

    Science.gov (United States)

    Zhou, Wenqi; Wang, Yuchuan; Wu, Zhongliang; Luo, Liang; Liu, Ping; Yan, Longfeng; Hou, Suiwen

    2016-07-01

    Filamentous actins (F-actins) play a vital role in epidermal cell morphogenesis. However, a limited number of studies have examined actin-dependent leaf epidermal cell morphogenesis events in rice. In this study, two recessive mutants were isolated: less pronounced lobe epidermal cell2-1 (lpl2-1) and lpl3-1, whose leaf and stem epidermis developed a smooth surface, with fewer serrated pavement cell (PC) lobes, and decreased papillae. The lpl2-1 also exhibited irregular stomata patterns, reduced plant height, and short panicles and roots. Molecular genetic studies demonstrated that LPL2 and LPL3 encode the PIROGI/Specifically Rac1-associated protein 1 (PIR/SRA1)-like and NCK-associated protein 1 (NAP1)-like proteins, respectively, two components of the suppressor of cAMP receptor/Wiskott-Aldrich syndrome protein-family verprolin-homologous protein (SCAR/WAVE) regulatory complex involved in actin nucleation and function. Epidermal cells exhibited abnormal arrangement of F-actins in both lpl2 and lpl3 expanding leaves. Moreover, the distorted trichomes of Arabidopsis pir could be partially restored by an overexpression of LPL2 A yeast two-hybrid assay revealed that LPL2 can directly interact with LPL3 in vitro Collectively, the results indicate that LPL2 and LPL3 are two functionally conserved homologs of the SCAR/WAVE complex components, and that they play an important role in controlling epidermal cell morphogenesis in rice by organising F-actin.

  4. Device Architecture and Lifetime Requirements for High Efficiency Multicrystalline Silicon Solar Cells

    Energy Technology Data Exchange (ETDEWEB)

    Wagner, H.; Hofstetter, J.; Mitchell, B.; Altermatt, P.; Buonassisi, T.

    2015-03-23

    We present a numerical simulation study of different multicrystalline silicon materials and solar cell architectures to understand today's efficiency limitations and future efficiency possibilities. We compare conventional full-area BSF and PERC solar cells to future cell designs with a gallium phosphide heteroemitter. For all designs, mc-Si materials with different excess carrier lifetime distributions are used as simulation input parameters to capture a broad range of materials. The results show that conventional solar cell designs are sufficient for generalized mean lifetimes between 40 – 90 μs, but do not give a clear advantage in terms of efficiency for higher mean lifetime mc-Si material because they are often limited by recombination in the phosphorus diffused emitter region. Heteroemitter designs instead increase in cell efficiency considerable up to generalized mean lifetimes of 380 μs because they are significantly less limited by recombination in the emitter and the bulk lifetime becomes more important. In conclusion, to benefit from increasing mc-Si lifetime, new cell designs, especially heteroemitter, are desirable.

  5. Rel Is Required for Morphogenesis of Resting Cells in Mycobacterium smegmatis

    Science.gov (United States)

    Wu, Mu-Lu; Chan, Chuu Ling; Dick, Thomas

    2016-01-01

    Recently we showed that upon transfer of growing Mycobacterium smegmatis into saline, the bacilli exited the canonical cell division cycle and formed septated multi-nucleoided cells. Under shock starvation (i.e., in saline without any carbon source), differentiation terminated at this stage with internally remodeled Large Resting Cells (LARCs). Whereas under gentle starvation (i.e., in saline with trace amounts of a carbon source), the septated multi-nucleoided bacilli completed cell division and separated into mono-nucleoided Small Resting Cells (SMRCs). This demonstrated that the non-sporulating mycobacteria are in fact capable of forming morphologically differentiated resting cells when exposed to starvation. Depending on the specific starvation conditions they can form two different resting cell types, LARCs or SMRCs, which share a common cellular differentiation pathway. The mRNA encoding the (p)ppGpp synthetase Rel was found to be transiently upregulated immediately upon starvation under both conditions, suggesting a role for the stringent response factor in both LARC and SMRC development. Here, we disrupted Rel function by generating two types of mutant M. smegmatis strains: a rel nonsense mutant (relE4TAG) in which translation is prematurely terminated at codon 4, and a rel deletion mutant (Δrel) in which the entire coding sequence was deleted. Both mutants showed identical phenotypes: sparse septum formation, less DNA compaction, and failure in formation of both the septated multi-nucleoided LARCs and the small-cell morphotype SMRC under starvation conditions. All phenotypes were rescued through the introduction of a wild-type copy of rel. Therefore, we conclude that loss-of-function mutations in rel block the development of both LARCs and SMRCs by preventing the first morphogenetic step in mycobacterial resting cell development, the formation of septated multi-nucleoided cells. Interestingly, in contrast to Rel’s role in most other bacteria, starvation

  6. Rel is required for morphogenesis of resting cells in Mycobacterium smegmatis

    Directory of Open Access Journals (Sweden)

    Mu-Lu Wu

    2016-08-01

    Full Text Available Recently we showed that upon transfer of growing Mycobacterium smegmatis into saline, the bacilli exited the canonical cell division cycle and formed septated multi-nucleoided cells. Under shock starvation (i.e. in saline without any carbon source, differentiation terminated at this stage with internally remodeled Large Resting Cells, LARCs. Whereas under gentle starvation (i.e. in saline with trace amounts of a carbon source, the septated multi-nucleoided bacilli completed cell division and separated into mono-nucleoided Small Resting Cells, SMRCs. This demonstrated that the non-sporulating mycobacteria are in fact capable of forming morphologically differentiated resting cells when exposed to starvation. Depending on the specific starvation conditions they can form two different resting cell types, LARCs or SMRCs, which share a common cellular differentiation pathway. The mRNA encoding the (pppGpp synthetase Rel was found to be transiently upregulated immediately upon starvation under both conditions, suggesting a role for the stringent response factor in both LARC and SMRC development. Here, we disrupted Rel function by generating two types of mutant M. smegmatis strains: a rel nonsense mutant (relE4TAG in which translation is prematurely terminated at codon 4, and a rel deletion mutant (△rel in which the entire coding sequence was deleted. Both mutants showed identical phenotypes: sparse septum formation, less DNA compaction and failure in formation of both the septated multi-nucleoided LARCs and the small-cell morphotype SMRC under starvation conditions. All phenotypes were rescued through the introduction of a wild-type copy of rel. Therefore, we conclude that loss-of-function mutations in rel block the development of both LARCs and SMRCs by preventing the first morphogenetic step in mycobacterial resting cell development, the formation of septated multi-nucleoided cells. Interestingly, in contrast to Rel’s role in most other bacteria

  7. The RNA recognition motif domains of RBM5 are required for RNA binding and cancer cell proliferation inhibition

    International Nuclear Information System (INIS)

    Highlights: • RNA recognition motif domains of RBM5 are essential for cell proliferation inhibition. • RNA recognition motif domains of RBM5 are essential for apoptosis induction. • RNA recognition motif domains of RBM5 are essential for RNA binding. • RNA recognition motif domains of RBM5 are essential for caspase-2 alternative splicing. - Abstract: RBM5 is a known putative tumor suppressor gene that has been shown to function in cell growth inhibition by modulating apoptosis. RBM5 also plays a critical role in alternative splicing as an RNA binding protein. However, it is still unclear which domains of RBM5 are required for RNA binding and related functional activities. We hypothesized the two putative RNA recognition motif (RRM) domains of RBM5 spanning from amino acids 98–178 and 231–315 are essential for RBM5-mediated cell growth inhibition, apoptosis regulation, and RNA binding. To investigate this hypothesis, we evaluated the activities of the wide-type and mutant RBM5 gene transfer in low-RBM5 expressing A549 cells. We found that, unlike wild-type RBM5 (RBM5-wt), a RBM5 mutant lacking the two RRM domains (RBM5-ΔRRM), is unable to bind RNA, has compromised caspase-2 alternative splicing activity, lacks cell proliferation inhibition and apoptosis induction function in A549 cells. These data provide direct evidence that the two RRM domains of RBM5 are required for RNA binding and the RNA binding activity of RBM5 contributes to its function on apoptosis induction and cell growth inhibition

  8. The RNA recognition motif domains of RBM5 are required for RNA binding and cancer cell proliferation inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Lei [Key Laboratory of Bioresources and Ecoenvironment (Ministry of Education), College of Life Sciences, Sichuan University, Chengdu (China); Zhang, Qing [Jiangsu Key Laboratory of Biological Cancer Therapy, Xuzhou Medical College, Xuzhou 221002 (China); Yang, Yu [Key Laboratory of Bioresources and Ecoenvironment (Ministry of Education), College of Life Sciences, Sichuan University, Chengdu (China); Wu, Chuanfang, E-mail: wuchuanfangsichuan@gmail.com [Key Laboratory of Bioresources and Ecoenvironment (Ministry of Education), College of Life Sciences, Sichuan University, Chengdu (China)

    2014-02-14

    Highlights: • RNA recognition motif domains of RBM5 are essential for cell proliferation inhibition. • RNA recognition motif domains of RBM5 are essential for apoptosis induction. • RNA recognition motif domains of RBM5 are essential for RNA binding. • RNA recognition motif domains of RBM5 are essential for caspase-2 alternative splicing. - Abstract: RBM5 is a known putative tumor suppressor gene that has been shown to function in cell growth inhibition by modulating apoptosis. RBM5 also plays a critical role in alternative splicing as an RNA binding protein. However, it is still unclear which domains of RBM5 are required for RNA binding and related functional activities. We hypothesized the two putative RNA recognition motif (RRM) domains of RBM5 spanning from amino acids 98–178 and 231–315 are essential for RBM5-mediated cell growth inhibition, apoptosis regulation, and RNA binding. To investigate this hypothesis, we evaluated the activities of the wide-type and mutant RBM5 gene transfer in low-RBM5 expressing A549 cells. We found that, unlike wild-type RBM5 (RBM5-wt), a RBM5 mutant lacking the two RRM domains (RBM5-ΔRRM), is unable to bind RNA, has compromised caspase-2 alternative splicing activity, lacks cell proliferation inhibition and apoptosis induction function in A549 cells. These data provide direct evidence that the two RRM domains of RBM5 are required for RNA binding and the RNA binding activity of RBM5 contributes to its function on apoptosis induction and cell growth inhibition.

  9. NMDA receptors on zebrafish Mauthner cells require CaMKII-α for normal development.

    Science.gov (United States)

    Roy, Birbickram; Ferdous, Jannatul; Ali, Declan W

    2015-02-01

    Calcium/calmodulin dependent protein kinase 2 (CaMKII) is a multifunctional protein that is highly enriched in the synapse. It plays important roles in neuronal functions such as synaptic plasticity, synaptogenesis, and neural development. Gene duplication in zebrafish has resulted in the occurrence of seven CaMKII genes (camk2a, camk2b1, camk2b2, camk2g1, camk2g2, camk2d1, and camk2d2) that are developmentally expressed. In this study, we used single cell, real-time quantitative PCR to investigate the expression of CaMKII genes in individual Mauthner cells (M-cells) of 2 days post fertilization (dpf) zebrafish embryos. We found that out of seven different CaMKII genes, only the mRNA for CaMKII-α was expressed in the M-cell at detectable levels, while all other isoforms were undetectable. Morpholino knockdown of CaMKII-α had no significant effect on AMPA synaptic currents (mEPSCs) but decreased the amplitude of NMDA mEPSCs. NMDA events exhibited a biexponential decay with τfast ≈ 30 ms and τslow ≈ 300 ms. Knockdown of CaMKII-α specifically reduced the amplitude of the slow component of the NMDA-mediated currents (mEPSCs), without affecting the fast component, the frequency, or the kinetics of the mEPSCs. Immunolabelling of the M-cell showed increased dendritic arborizations in the morphants compared with controls, and knockdown of CaMKII-α altered locomotor behaviors of touch responses. These results suggest that CaMKII-α is present in embryonic M-cells and that it plays a role in the normal development of excitatory synapses. Our findings pave the way for determining the function of specific CaMKII isoforms during the early stages of M-cell development.

  10. Rational design of medium supplementation strategy for improved influenza viruses production based on analyzing nutritional requirements of MDCK Cells.

    Science.gov (United States)

    Huang, Ding; Xia-Hou, Kang; Liu, Xu-Ping; Zhao, Liang; Fan, Li; Ye, Zhaoyang; Tan, Wen-Song; Luo, Jian; Chen, Ze

    2014-12-12

    Influenza vaccine production using cell culture technology has become popular nowadays. However, to meet the ever increasing demand of influenza vaccine, it is prerequisite to improve the yield of influenza virus in cells. To achieve this, in the present study, the nutritional requirements of MDCK cells in the virus production process were analyzed and a nutrient-feeding strategy was developed accordingly. Based on the consumption rates and corresponding concentration optimization, glucose and fast metabolized amino acids were supplemented into the maintaining medium at the time of infection. Compared with the non-supplemented culture, the average cell specific death rate during 0-48 h post-infection was 0.013 h(-1), which was 40.91% lower in the nutrient-supplemented culture. Total virus titer, HA antigen protein concentration and cell-specific virus yield were (1.88±0.23)×10(3) HA units/50μL, 11.70±0.22 μg/mL and (10.06±1.16)×10(3) virions/cell, respectively, which were 84.04±22.50%, 31.46±2.87% and 86.64±25.81% higher than those in the control, respectively. These data showed that the appropriate supplementation of nutrients during virus production process could reduce cell death, and improve cell-specific virus yield and total influenza virus output. This study laid foundation for the development of cell culture technology for influenza vaccine production.

  11. The NG2 Protein Is Not Required for Glutamatergic Neuron-NG2 Cell Synaptic Signaling.

    Science.gov (United States)

    Passlick, Stefan; Trotter, Jacqueline; Seifert, Gerald; Steinhäuser, Christian; Jabs, Ronald

    2016-01-01

    NG2 glial cells (as from now NG2 cells) are unique in receiving synaptic input from neurons. However, the components regulating formation and maintenance of these neuron-glia synapses remain elusive. The transmembrane protein NG2 has been considered a potential mediator of synapse formation and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) clustering, because it contains 2 extracellular Laminin G/Neurexin/Sex Hormone-Binding Globulin domains, which in neurons are crucial for formation of transsynaptic neuroligin-neurexin complexes. NG2 is connected via Glutamate Receptor-Interacting Protein with GluA2/3-containing AMPARs, thereby possibly mediating receptor clustering in glial postsynaptic density. To elucidate the role of NG2 in neuron-glia communication, we investigated glutamatergic synaptic transmission in juvenile and aged hippocampal NG2 cells of heterozygous and homozygous NG2 knockout mice. Neuron-NG2 cell synapses readily formed in the absence of NG2. Short-term plasticity, synaptic connectivity, postsynaptic AMPAR current kinetics, and density were not affected by NG2 deletion. During development, an NG2-independent acceleration of AMPAR current kinetics and decreased synaptic connectivity were observed. Our results indicate that the lack of NG2 does not interfere with genesis and basic properties of neuron-glia synapses. In addition, we demonstrate frequent expression of neuroligins 1-3 in juvenile and aged NG2 cells, suggesting a role of these molecules in synapse formation between NG2 glia and neurons.

  12. The V protein of canine distemper virus is required for virus replication in human epithelial cells.

    Directory of Open Access Journals (Sweden)

    Noriyuki Otsuki

    Full Text Available Canine distemper virus (CDV becomes able to use human receptors through a single amino acid substitution in the H protein. In addition, CDV strains possessing an intact C protein replicate well in human epithelial H358 cells. The present study showed that CDV strain 007Lm, which was isolated from lymph node tissue of a dog with distemper, failed to replicate in H358 cells, although it possessed an intact C protein. Sequence analyses suggested that a cysteine-to-tyrosine substitution at position 267 of the V protein caused this growth defect. Analyses using H358 cells constitutively expressing the CDV V protein showed that the V protein with a cysteine, but not that with a tyrosine, at this position effectively blocked the interferon-stimulated signal transduction pathway, and supported virus replication of 007Lm in H358 cells. Thus, the V protein as well as the C protein appears to be functional and essential for CDV replication in human epithelial cells.

  13. Requirement of peroxiredoxin on the stationary phase of yeast cell growth.

    Science.gov (United States)

    Watanabe, Toshihiko; Irokawa, Hayato; Ogasawara, Ayako; Iwai, Kenta; Kuge, Shusuke

    2014-02-01

    Toxic chemicals often induce reactive oxygen species (ROS). Although one of the most abundant ROS-sensitive proteins is in the peroxiredoxin (Prx) family, the function of Prx proteins is poorly understood because they are inactivated under high concentrations of hydrogen peroxide. Like mammalian cells, the model eukaryote Saccharomyces cerevisiae possesses multiple Prx proteins. Among the five Prx family proteins, Tsa1 and Ahp1 have the highest and second-highest expression levels, respectively. Here, we focused on a previously uncharacterized phenotype resulting from Tsa1 loss: impaired growth during the late exponential phase. We overexpressed catalase (CTT1) and Ahp1 in cells with disruptions in TSA1 and its homologue, TSA2 (tsa1/2Δ cells), and we found that neither Ctt1 nor Ahp1 overexpression suppressed the impaired cell growth at the stationary phase, although the ROS levels were successfully suppressed. Furthermore, the cell cycle profile was not altered by Tsa1/2 loss, at least in the late exponential phase; however, the glucose consumption rate slowed in the late exponential phase. Our results suggest that ROS levels are not responsible for the growth phenotype. Tsa1 might have a specific function that could not be replaced by Ahp1. PMID:24418709

  14. The Drosophila cell adhesion molecule Klingon is required for long-term memory formation and is regulated by Notch

    OpenAIRE

    Matsuno, Motomi; Horiuchi, Junjiro; Tully, Tim; Saitoe, Minoru

    2008-01-01

    The ruslan (rus) mutant was previously identified in a behavioral screen for mutants defective in long-lasting memory, which consists of two consolidated memory types, anesthesia-resistant memory, and protein synthesis-dependent long-term memory (LTM). We demonstrate here that rus is a new allele of klingon (klg), which encodes a homophilic cell adhesion molecule. Klg is acutely required for LTM but not anesthesia-resistant memory formation, and Klg expression increases upon LTM induction. LT...

  15. Both asymmetric mitotic segregation and cell-to-cell invasion are required for stable germline transmission of Wolbachia in filarial nematodes

    Directory of Open Access Journals (Sweden)

    Frédéric Landmann

    2012-04-01

    Parasitic filarial nematodes that belong to the Onchocercidae family live in mutualism with Wolbachia endosymbionts. We developed whole-mount techniques to follow the segregation patterns of Wolbachia through the somatic and germline lineages of four filarial species. These studies reveal multiple evolutionarily conserved mechanisms that are required for Wolbachia localization to the germline. During the initial embryonic divisions, Wolbachia segregate asymmetrically such that they concentrate in the posteriorly localized P2 blastomere, a precursor to the adult germline and hypodermal lineages. Surprisingly, in the next division they are excluded from the germline precursor lineage. Rather, they preferentially segregate to the C blastomere, a source of posterior hypodermal cells. Localization to the germline is accomplished by a distinct mechanism in which Wolbachia invade first the somatic gonadal cells close to the ovarian distal tip cell, the nematode stem cell niche, from the hypodermis. This tropism is associated with a cortical F-actin disruption, suggesting an active engulfment. Significantly, germline invasion occurs only in females, explaining the lack of Wolbachia in the male germline. Once in the syncytial environment of the ovaries, Wolbachia rely on the rachis to multiply and disperse into the germ cells. The utilization of cell-to-cell invasion for germline colonization may indicate an ancestral mode of horizontal transfer that preceded the acquisition of the mutualism.

  16. Inflammation- and tumor-induced anorexia and weight loss require MyD88 in hematopoietic/myeloid cells but not in brain endothelial or neural cells.

    Science.gov (United States)

    Ruud, Johan; Wilhelms, Daniel Björk; Nilsson, Anna; Eskilsson, Anna; Tang, Yan-Juan; Ströhle, Peter; Caesar, Robert; Schwaninger, Markus; Wunderlich, Thomas; Bäckhed, Fredrik; Engblom, David; Blomqvist, Anders

    2013-05-01

    Loss of appetite is a hallmark of inflammatory diseases. The underlying mechanisms remain undefined, but it is known that myeloid differentiation primary response gene 88 (MyD88), an adaptor protein critical for Toll-like and IL-1 receptor family signaling, is involved. Here we addressed the question of determining in which cells the MyD88 signaling that results in anorexia development occurs by using chimeric mice and animals with cell-specific deletions. We found that MyD88-knockout mice, which are resistant to bacterial lipopolysaccharide (LPS)-induced anorexia, displayed anorexia when transplanted with wild-type bone marrow cells. Furthermore, mice with a targeted deletion of MyD88 in hematopoietic or myeloid cells were largely protected against LPS-induced anorexia and displayed attenuated weight loss, whereas mice with MyD88 deletion in hepatocytes or in neural cells or the cerebrovascular endothelium developed anorexia and weight loss of similar magnitude as wild-type mice. Furthermore, in a model for cancer-induced anorexia-cachexia, deletion of MyD88 in hematopoietic cells attenuated the anorexia and protected against body weight loss. These findings demonstrate that MyD88-dependent signaling within the brain is not required for eliciting inflammation-induced anorexia. Instead, we identify MyD88 signaling in hematopoietic/myeloid cells as a critical component for acute inflammatory-driven anorexia, as well as for chronic anorexia and weight loss associated with malignant disease.

  17. Protection against HPV-16-Associated Tumors Requires the Activation of CD8+ Effector Memory T Cells and the Control of Myeloid-Derived Suppressor Cells.

    Science.gov (United States)

    Diniz, Mariana O; Sales, Natiely S; Silva, Jamile R; Ferreira, Luís Carlos S

    2016-08-01

    Active anticancer immunotherapeutic approaches have been shown to induce cellular or humoral immune responses in patients, but, thus far, the observed outcomes did not ensure their recommendation for clinical use. The induction of tumor-specific CD8(+) T cells, although required for the clearance of most solid tumors, was shown to be insufficient for the development of a successful immunotherapeutic approach. The suppressive immune environment triggered by tumors, including the expansion of myeloid-derived suppressor cells (MDSC), is detrimental to the development of antitumor immune responses and precludes the generation of more promising clinical outcomes. In this work, we characterized the CD8(+) T-cell population specifically involved in the control of tumor growth and the role of MDSCs after administration of an antitumor therapeutic DNA vaccine targeting human papillomavirus type 16 (HPV-16)-associated tumors. Activation of cytotoxic high-avidity CD8(+) T cells with an effector memory phenotype was found in mice grafted with tumor cells expressing the HPV-16 oncoproteins. In addition, MDSC antibody depletion further enhanced the immunotherapeutic effects of the vaccine, resulting in the complete eradication of tumor cells. Collectively, the current results indicate that the simultaneous control of MDSCs and activation of high-avidity tumor-specific effector memory CD8(+) T cells are key features for tumor protection by immunotherapeutic approaches and deserve further testing under clinical conditions. Mol Cancer Ther; 15(8); 1920-30. ©2016 AACR. PMID:27222537

  18. Homologs of SCAR/WAVE complex components are required for epidermal cell morphogenesis in rice.

    Science.gov (United States)

    Zhou, Wenqi; Wang, Yuchuan; Wu, Zhongliang; Luo, Liang; Liu, Ping; Yan, Longfeng; Hou, Suiwen

    2016-07-01

    Filamentous actins (F-actins) play a vital role in epidermal cell morphogenesis. However, a limited number of studies have examined actin-dependent leaf epidermal cell morphogenesis events in rice. In this study, two recessive mutants were isolated: less pronounced lobe epidermal cell2-1 (lpl2-1) and lpl3-1, whose leaf and stem epidermis developed a smooth surface, with fewer serrated pavement cell (PC) lobes, and decreased papillae. The lpl2-1 also exhibited irregular stomata patterns, reduced plant height, and short panicles and roots. Molecular genetic studies demonstrated that LPL2 and LPL3 encode the PIROGI/Specifically Rac1-associated protein 1 (PIR/SRA1)-like and NCK-associated protein 1 (NAP1)-like proteins, respectively, two components of the suppressor of cAMP receptor/Wiskott-Aldrich syndrome protein-family verprolin-homologous protein (SCAR/WAVE) regulatory complex involved in actin nucleation and function. Epidermal cells exhibited abnormal arrangement of F-actins in both lpl2 and lpl3 expanding leaves. Moreover, the distorted trichomes of Arabidopsis pir could be partially restored by an overexpression of LPL2 A yeast two-hybrid assay revealed that LPL2 can directly interact with LPL3 in vitro Collectively, the results indicate that LPL2 and LPL3 are two functionally conserved homologs of the SCAR/WAVE complex components, and that they play an important role in controlling epidermal cell morphogenesis in rice by organising F-actin. PMID:27252469

  19. Signaling through urokinase and urokinase receptor in lung cancer cells requires interactions with beta1 integrins.

    Science.gov (United States)

    Tang, Chi-Hui; Hill, Marla L; Brumwell, Alexis N; Chapman, Harold A; Wei, Ying

    2008-11-15

    The urokinase receptor (uPAR) is upregulated upon tumor cell invasion and correlates with poor lung cancer survival. Although a cis-interaction with integrins has been ascribed to uPAR, whether this interaction alone is critical to urokinase (uPA)- and uPAR-dependent signaling and tumor promotion is unclear. Here we report the functional consequences of point mutations of uPAR (H249A-D262A) that eliminate beta1 integrin interactions but maintain uPA binding, vitronectin attachment and association with alphaV integrins, caveolin and epidermal growth factor receptor. Disruption of uPAR interactions with beta1 integrins recapitulated previously reported findings with beta1-integrin-derived peptides that attenuated matrix-dependent ERK activation, MMP expression and in vitro migration by human lung adenocarcinoma cell lines. The uPAR mutant cells acquired enhanced capacity to adhere to vitronectin via uPAR-alphaVbeta5-integrin, rather than through the uPAR-alpha3beta1-integrin complex and they were unable to initiate uPA signaling to activate ERK, Akt or Stat1. In an orthotopic lung cancer model, uPAR mutant cells exhibited reduced tumor size compared with cells expressing wild-type uPAR. Taken together, the results indicate that uPAR-beta1-integrin interactions are essential to signals induced by integrin matrix ligands or uPA that support lung cancer cell invasion in vitro and progression in vivo. PMID:18940913

  20. Sea urchin akt activity is Runx-dependent and required for post-cleavage stage cell division

    KAUST Repository

    Robertson, Anthony J.

    2013-03-25

    In animal development following the initial cleavage stage of embryogenesis, the cell cycle becomes dependent on intercellular signaling and controlled by the genomically encoded ontogenetic program. Runx transcription factors are critical regulators of metazoan developmental signaling, and we have shown that the sea urchin Runx gene runt-1, which is globally expressed during early embryogenesis, functions in support of blastula stage cell proliferation and expression of the mitogenic genes pkc1, cyclinD, and several wnts. To obtain a more comprehensive list of early runt-1 regulatory targets, we screened a Strongylocentrotus purpuratus microarray to identify genes mis-expressed in mid-blastula stage runt-1 morphants. This analysis showed that loss of Runx function perturbs the expression of multiple genes involved in cell division, including the pro-growth and survival kinase Akt (PKB), which is significantly underexpressed in runt-1 morphants. Further genomic analysis revealed that Akt is encoded by two genes in the S. purpuratus genome, akt-1 and akt-2, both of which contain numerous canonical Runx target sequences. The transcripts of both genes accumulate several fold during blastula stage, contingent on runt-1 expression. Inhibiting Akt expression or activity causes blastula stage cell cycle arrest, whereas overexpression of akt-1 mRNA rescues cell proliferation in runt-1 morphants. These results indicate that post-cleavage stage cell division requires Runx-dependent expression of akt.

  1. Omcg1 is critically required for mitosis in rapidly dividing mouse intestinal progenitors and embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Teddy Léguillier

    2012-05-01

    Recent studies have shown that factors involved in transcription-coupled mRNA processing are important for the maintenance of genome integrity. How these processes are linked and regulated in vivo remains largely unknown. In this study, we addressed in the mouse model the function of Omcg1, which has been shown to participate in co-transcriptional processes, including splicing and transcription-coupled repair. Using inducible mouse models, we found that Omcg1 is most critically required in intestinal progenitors. In absence of OMCG1, proliferating intestinal epithelial cells underwent abnormal mitosis followed by apoptotic cell death. As a consequence, the crypt proliferative compartment of the small intestine was quickly and totally abrogated leading to the rapid death of the mice. Lack of OMCG1 in embryonic stem cells led to a similar cellular phenotype, with multiple mitotic defects and rapid cell death. We showed that mutant intestinal progenitors and embryonic stem cells exhibited a reduced cell cycle arrest following irradiation, suggesting that mitotic defects may be consecutive to M phase entry with unrepaired DNA damages. These findings unravel a crucial role for pre-mRNA processing in the homeostasis of the small intestine and point to a major role of OMCG1 in the maintenance of genome integrity.

  2. Pancreatic cancer cells require an EGF receptor-mediated autocrine pathway for proliferation in serum-free conditions

    Science.gov (United States)

    Murphy, L O; Cluck, M W; Lovas, S; Ötvös, F; Murphy, R F; Schally, A V; Permert, J; Larsson, J; Knezetic, J A; Adrian, T E

    2001-01-01

    In-vitro and in-vivo studies have shown that autocrine growth factors and receptors are frequently expressed in human malignancies. Few of these studies, however, provide evidence that the identified autocrine pathway is functional. In this study, a functional autocrine growth pathway in pancreatic cancer has been identified using an in-vitro cell culture system. When pancreatic cancer cells were grown without change of medium, proliferation was greater than when either medium was replaced frequently (HPAF, CAPAN-2, PANC-1 or SW1990) or cells were grown in the presence of the EGF receptor tyrosine kinase inhibitor AG1478 or the MEK inhibitor PD098059 (HPAF or CAPAN-2). Activity of extracellular-regulated kinases (ERK) 1 and 2 and c- jun and c- fos mRNA levels were significantly elevated in CAPAN-2 cells cultured continuously in serum-free medium. Collectively, the observations indicate that the EGF receptor and the ERK MAP kinase pathway mediate autocrine signals. In contrast to previous reports, the GRP and IGF-I receptors were shown not to be required for autocrine effects on pancreatic cancer cell proliferation. Autocrine stimulation of the EGF receptor can contribute to sustained mitogenic activity and proliferation of pancreatic cancer cells. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11286473

  3. The V Protein of Canine Distemper Virus Is Required for Virus Replication in Human Epithelial Cells

    OpenAIRE

    Noriyuki Otsuki; Yuichiro Nakatsu; Toru Kubota; Tsuyoshi Sekizuka; Fumio Seki; Kouji Sakai; Makoto Kuroda; Ryoji Yamaguchi; Makoto Takeda

    2013-01-01

    Canine distemper virus (CDV) becomes able to use human receptors through a single amino acid substitution in the H protein. In addition, CDV strains possessing an intact C protein replicate well in human epithelial H358 cells. The present study showed that CDV strain 007Lm, which was isolated from lymph node tissue of a dog with distemper, failed to replicate in H358 cells, although it possessed an intact C protein. Sequence analyses suggested that a cysteine-to-tyrosine substitution at posit...

  4. Temporal artery biopsy is not required in all cases of suspected giant cell arteritis.

    LENUS (Irish Health Repository)

    Quinn, Edel Marie

    2012-07-01

    Temporal artery biopsy (TAB) is performed during the diagnostic workup for giant cell arteritis (GCA), a vasculitis with the potential to cause irreversible blindness or stroke. However, treatment is often started on clinical grounds, and TAB result frequently does not influence patient management. The aim of this study was to assess the need for TAB in cases of suspected GCA.

  5. Cell-penetrating TAT peptide in drug delivery systems: Proteolytic stability requirements

    Science.gov (United States)

    Koren, Erez; Apte, Anjali; Sawant, Rupa R.; Grunwald, Jacob; Torchilin, Vladimir P.

    2012-01-01

    The stability and activity of the HIV cell-penetrating TAT peptide (TATp) on the surface of TATp-modified micelles and liposomes in relation to its proteolytic cleavage was investigated. TATp moieties were attached to the surface of these nanocarriers using TATp modified with a conjugate of phosphatidyl ethanolamine with a ‘short’ PEG (PEG-PE). Following pre-incubation with trypsin, elastase, or collagenase, the proteolytic stability of TATp on the surface of these modified carriers was studied by HPLC with fluorescence detection using fluorenylmethyl chloroformate (FMOC) labeling. All tested enzymes produced a dose-dependent cleavage of TATp as shown by the presence of TATp Arg-Arg fragments. Inhibition of TATp cleavage occurred when these TATp-micelles were modified by the addition of longer PEG-PE blocks, indicating an effective shielding of TATp from proteolysis by these blocks. TATp-modified carriers were also tested for their ability to accumulate in EL-4, HeLa, and B16-F10 cells. Trypsin treatment of TATp-modified liposomes and micelles resulted in decreased uptake and cell interaction, as measured by fluorescence microscopy and fluorescence-activated cell sorting techniques. Furthermore, a decrease in the cytotoxicity of TATp-modified liposomes loaded with doxorubicin (Doxil) was observed following trypsin treatment. In conclusion, steric shielding of TATp is essential to ensure its in vivo therapeutic function. PMID:21438724

  6. CCDC-55 is required for larval development and distal tip cell migration in Caenorhabditis elegans.

    Science.gov (United States)

    Kovacevic, Ismar; Ho, Richard; Cram, Erin J

    2012-01-01

    The Caenorhabditis elegans distal tip cells (DTCs) are an in vivo model for the study of developmentally regulated cell migration. In this study, we characterize a novel role for CCDC-55, a conserved coiled-coil domain containing protein, in DTC migration and larval development in C. elegans. Although animals homozygous for a probable null allele, ccdc-55(ok2851), display an early larval arrest, RNAi depletion experiments allow the analysis of later phenotypes and suggest that CCDC-55 is needed within the DTC for migration to cease at the end of larval morphogenesis. The ccdc-55 gene is found in an operon with rnf-121 and rnf-5, E3 ubiquitin ligases that target cell migration genes such as the β-integrin PAT-3. Genetic interaction studies using RNAi depletion and the deletion alleles rnf-121(ok848) and rnf-5(tm794) indicate that CCDC-55 and the RNF genes act at least partially in parallel to promote termination of cell migration in the adult DTC.

  7. Lipid Raft is required for PSGL-1 ligation induced HL-60 cell adhesion on ICAM-1.

    Directory of Open Access Journals (Sweden)

    Tingshuang Xu

    Full Text Available P-selectin glycoprotein ligand-1 (PSGL-1 and integrins are adhesion molecules that play critical roles in host defense and innate immunity. PSGL-1 mediates leukocyte rolling and primes leukocytes for integrin-mediated adhesion. However, the mechanism that PSGL-1 as a rolling receptor in regulating integrin activation has not been well characterized. Here, we investigate the function of lipid raft in regulating PSGL-1 induced β2 integrin-mediated HL-60 cells adhesion. PSGL-1 ligation with antibody enhances the β2 integrin activation and β2 integrin-dependent adhesion to ICAM-1. Importantly, with the treatment of methyl-β-cyclodextrin (MβCD, we confirm the role of lipid raft in regulating the activation of β2 integrin. Furthermore, we find that the protein level of PSGL-1 decreased in raft fractions in MβCD treated cells. PSGL-1 ligation induces the recruitment of spleen tyrosine kinase (Syk, a tyrosine kinase and Vav1 (the pivotal downstream effector of Syk signaling pathway involved in cytoskeleton regulation to lipid raft. Inhibition of Syk activity with pharmacologic inhibitor strongly reduces HL-60 cells adhesion, implicating Syk is crucial for PSGL-1 mediated β2 integrin activation. Taken together, we report that ligation of PSGL-1 on HL-60 cells activates β2 integrin, for which lipid raft integrity and Syk activation are responsible. These findings have shed new light on the mechanisms that connect leukocyte initial rolling with subsequent adhesion.

  8. Polycystin Signaling Is Required for Directed Endothelial Cell Migration and Lymphatic Development

    Directory of Open Access Journals (Sweden)

    Patricia Outeda

    2014-05-01

    Full Text Available Autosomal dominant polycystic kidney disease is a common form of inherited kidney disease that is caused by mutations in two genes, PKD1 (polycystin-1 and PKD2 (polycystin-2. Mice with germline deletion of either gene die in midgestation with a vascular phenotype that includes profound edema. Although an endothelial cell defect has been suspected, the basis of this phenotype remains poorly understood. Here, we demonstrate that edema in Pkd1- and Pkd2-null mice is likely to be caused by defects in lymphatic development. Pkd1 and Pkd2 mutant embryos exhibit reduced lymphatic vessel density and vascular branching along with aberrant migration of early lymphatic endothelial cell precursors. We used cell-based assays to confirm that PKD1- and PKD2-depleted endothelial cells have an intrinsic defect in directional migration that is associated with a failure to establish front-rear polarity. Our studies reveal a role for polycystin signaling in lymphatic development.

  9. Cdc42-dependent structural development of auditory supporting cells is required for wound healing at adulthood

    DEFF Research Database (Denmark)

    Anttonen, Tommi; Kirjavainen, Anna; Belevich, Ilya;

    2012-01-01

    of a basolateral membrane protein in the apical domain were observed. These defects and changes in aPKCλ/ι expression suggested that apical polarization is impaired. Following a lesion at adulthood, supporting cells with Cdc42 loss-induced maturational defects collapsed and failed to remodel F-actin belts...

  10. Exercise-induced promotion of hippocampal cell proliferation requires beta-endorphin

    NARCIS (Netherlands)

    Koehl, M.; Meerlo, P.; Gonzales, D.; Rontal, A.; Turek, F. W.; Abrous, D. N.

    2008-01-01

    variety of stimuli, including exercise, but the mechanisms by which running affects neurogenesis are not yet fully understood. Because beta-endorphin, which is released in response to exercise, increases cell proliferation in vitro, we hypothesized that it could exert a similar effect in vivo and me

  11. Mitogen requirement for cell cycle progression in the absence of pocket protein activity

    NARCIS (Netherlands)

    Foijer, Floris; Wolthuis, Rob M F; Doodeman, Valerie; Medema, René H; te Riele, Hein

    2005-01-01

    Primary mouse embryonic fibroblasts lacking expression of all three retinoblastoma protein family members (TKO MEFs) have lost the G1 restriction point. However, in the absence of mitogens these cells become highly sensitive to apoptosis. Here, we show that TKO MEFs that survive serum depletion pass

  12. Changes in production control required for untended operation of a flexible manufacturing cell

    NARCIS (Netherlands)

    Slomp, Jannes; Stecke, Kathryn E.

    2012-01-01

    This article examines and improves the production control system of a firm that wants to operate its flexible manufacturing cell (FMC) in an untended third shift. The FMC consists of a machining centre, a pallet storage, a rail-guided transport vehicle for pallets and a clamping/unclamping station.

  13. 9 CFR 113.52 - Requirements for cell lines used for production of biologics.

    Science.gov (United States)

    2010-01-01

    ... of origin of the MCS may be used if approved by APHIS. (c) The MCS and either each subculture of... sources of cells in the batch. (d) The MCS and either each subculture used to prepare a biological product... not be used. If bacteria or fungi are found in a subculture, the subculture shall not be used. (e)...

  14. Meis1 regulates epidermal stem cells and is required for skin tumorigenesis.

    Directory of Open Access Journals (Sweden)

    Kazuhiro Okumura

    Full Text Available Previous studies have shown that Meis1 plays an important role in blood development and vascular homeostasis, and can induce blood cancers, such as leukemia. However, its role in epithelia remains largely unknown. Here, we uncover two roles for Meis1 in the epidermis: as a critical regulator of epidermal homeostasis in normal tissues and as a proto-oncogenic factor in neoplastic tissues. In normal epidermis, we show that Meis1 is predominantly expressed in the bulge region of the hair follicles where multipotent adult stem cells reside, and that the number of these stem cells is reduced when Meis1 is deleted in the epidermal tissue of mice. Mice with epidermal deletion of Meis1 developed significantly fewer DMBA/TPA-induced benign and malignant tumors compared with wild-type mice, suggesting that Meis1 plays a role in both tumor development and malignant progression. This is consistent with the observation that Meis1 expression increases as tumors progress from benign papillomas to malignant carcinomas. Interestingly, we found that Meis1 localization was altered to neoplasia development. Instead of being localized to the stem cell region, Meis1 is localized to more differentiated cells in tumor tissues. These findings suggest that, during the transformation from normal to neoplastic tissues, a functional switch occurs in Meis1.

  15. Postnatal epigenetic regulation of intestinal stem cells requires DNA methylation and is guided by the microbiome

    Science.gov (United States)

    DNA methylation is an epigenetic mechanism central to the development and maintenance of complex mammalian tissues, but our understanding of its role in intestinal development is limited. We used whole genome bisulfite sequencing, and found that differentiation of mouse colonic intestinal stem cell...

  16. TGF-β is required to maintain the pool of immature langerhans cells in the epidermis

    NARCIS (Netherlands)

    J.M. Kel (Junda); M.J.H. Girard-Madoux (Mathilde); B. Reizis (Boris); B.E. Clausen (Bjorn)

    2010-01-01

    textabstractThe pivotal role of TGF-β in Langerhans cell (LC) development has been previously established in TGF-β-deficient mice, which lack epidermal LCs. As to whether TGF-β also governs LC homeostasis and function remains elusive. To assess the role of TGF-β-mediated control of cutaneous dendrit

  17. Human neural stem cell-induced endothelial morphogenesis requires autocrine/paracrine and juxtacrine signaling

    Science.gov (United States)

    Chou, Chung-Hsing; Modo, Michel

    2016-01-01

    Transplanted neural stem cells (NSC) interact with the host brain microenvironment. A neovascularization is commonly observed in the vicinity of the cell deposit, which is correlated with behavioral improvements. To elucidate the signaling mechanisms between human NSCs and endothelial cells (ECs), these were cocultured in an in vitro model in which NSC-induced endothelial morphogenesis produced a neurovascular environment. Soluble (autocrine/paracrine) and contact–mediated (juxtacrine) signaling molecules were evaluated for two conditionally immortalized fetal NSC lines derived from the cortical anlage (CTXOE03) and ganglionic eminence (STROC05), as well as an adult EC line (D3) derived from the cerebral microvasculature of a hippocampal biopsy. STROC05 were 4 times as efficient to induce endothelial morphogenesis compared to CTXOE03. The cascade of reciprocal interactions between NSCs and ECs in this process was determined by quantifying soluble factors, receptor mapping, and immunocytochemistry for extracellular matrix molecules. The mechanistic significance of these was further evaluated by pharmacological blockade. The sequential cell-specific regulation of autocrine/paracrine and juxtacrine signaling accounted for the differential efficiency of NSCs to induce endothelial morphogenesis. These in vitro studies shed new light on the reciprocal interactions between NSCs and ECs, which are pivotal for our mechanistic understanding of the efficacy of NSC transplantation. PMID:27374240

  18. CCDC-55 is required for larval development and distal tip cell migration in Caenorhabditis elegans.

    Science.gov (United States)

    Kovacevic, Ismar; Ho, Richard; Cram, Erin J

    2012-01-01

    The Caenorhabditis elegans distal tip cells (DTCs) are an in vivo model for the study of developmentally regulated cell migration. In this study, we characterize a novel role for CCDC-55, a conserved coiled-coil domain containing protein, in DTC migration and larval development in C. elegans. Although animals homozygous for a probable null allele, ccdc-55(ok2851), display an early larval arrest, RNAi depletion experiments allow the analysis of later phenotypes and suggest that CCDC-55 is needed within the DTC for migration to cease at the end of larval morphogenesis. The ccdc-55 gene is found in an operon with rnf-121 and rnf-5, E3 ubiquitin ligases that target cell migration genes such as the β-integrin PAT-3. Genetic interaction studies using RNAi depletion and the deletion alleles rnf-121(ok848) and rnf-5(tm794) indicate that CCDC-55 and the RNF genes act at least partially in parallel to promote termination of cell migration in the adult DTC. PMID:22285439

  19. Histone acetyltransferase PCAF is required for Hedgehog-Gli-dependent transcription and cancer cell proliferation

    DEFF Research Database (Denmark)

    Malatesta, Martina; Steinhauer, Cornelia; Mohammad, Faizaan;

    2013-01-01

    that the histone acetyltransferase PCAF/KAT2B is an important factor of the Hh pathway. Specifically, we show that PCAF depletion impairs Hh activity and reduces expression of Hh target genes. Consequently, PCAF downregulation in medulloblastoma and glioblastoma cells leads to decreased proliferation and increased...

  20. The histone demethylase Jarid1b is required for hematopoietic stem cell self-renewal

    DEFF Research Database (Denmark)

    Stewart, Morag H; Albert, Mareike; Sroczynska, Patrycja;

    2015-01-01

    Jarid1b/KDM5b is a histone demethylase that regulates self-renewal and differentiation in stem cells and cancer, however its function in hematopoiesis is unclear. Here, we find that Jarid1b is highly expressed in primitive hematopoietic compartments and is overexpressed in acute myeloid leukemias...

  1. Pancreatic β-cell response to increased metabolic demand and to pharmacologic secretagogues requires EPAC2A.

    Science.gov (United States)

    Song, Woo-Jin; Mondal, Prosenjit; Li, Yuanyuan; Lee, Suh Eun; Hussain, Mehboob A

    2013-08-01

    Incretin hormone action on β-cells stimulates in parallel two different intracellular cyclic AMP-dependent signaling branches mediated by protein kinase A and exchange protein activated by cAMP islet/brain isoform 2A (EPAC2A). Both pathways contribute toward potentiation of glucose-stimulated insulin secretion (GSIS). However, the overall functional role of EPAC2A in β-cells as it relates to in vivo glucose homeostasis remains incompletely understood. Therefore, we have examined in vivo GSIS in global EPAC2A knockout mice. Additionally, we have conducted in vitro studies of GSIS and calcium dynamics in isolated EPAC2A-deficient islets. EPAC2A deficiency does not impact GSIS in mice under basal conditions. However, when mice are exposed to diet-induced insulin resistance, pharmacologic secretagogue stimulation of β-cells with an incretin hormone glucagon-like peptide-1 analog or with a fatty acid receptor 1/G protein-coupled receptor 40 selective activator, EPAC2A is required for the increased β-cell response to secretory demand. Under these circumstances, EPAC2A is required for potentiating the early dynamic increase in islet calcium levels after glucose stimulation, which is reflected in potentiated first-phase insulin secretion. These studies broaden our understanding of EPAC2A function and highlight its significance during increased secretory demand or drive on β-cells. Our findings advance the rationale for developing EPAC2A-selective pharmacologic activators for β-cell-targeted pharmacotherapy in type 2 diabetes.

  2. Histone demethylase JMJD2B is required for tumor cell proliferation and survival and is overexpressed in gastric cancer

    Energy Technology Data Exchange (ETDEWEB)

    Li, Wenjuan; Zhao, Li; Zang, Wen; Liu, Zhifang; Chen, Long; Liu, Tiantian [Department of Microbiology/Key Laboratory for Experimental Teratology of Chinese Ministry of Education, School of Medicine, Shandong University, 44 Wenhua Xi Road, Jinan 250012 (China); Xu, Dawei, E-mail: Dawei.Xu@ki.se [Department of Microbiology/Key Laboratory for Experimental Teratology of Chinese Ministry of Education, School of Medicine, Shandong University, 44 Wenhua Xi Road, Jinan 250012 (China); Department of Medicine, Division of Hematology, Karolinska University Hospital, Solna and Karolinska Institutet, Stockholm (Sweden); Jia, Jihui, E-mail: jiajihui@sdu.edu.cn [Department of Microbiology/Key Laboratory for Experimental Teratology of Chinese Ministry of Education, School of Medicine, Shandong University, 44 Wenhua Xi Road, Jinan 250012 (China)

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer JMJD2B is required for cell proliferation and in vivo tumorigenesis. Black-Right-Pointing-Pointer JMJD2B depletion induces apoptosis and/or cell cycle arrest. Black-Right-Pointing-Pointer JMJD2B depletion activates DNA damage response and enhances p53 stabilization. Black-Right-Pointing-Pointer JMJD2B is overexpressed in human primary gastric cancer. -- Abstract: Epigenetic alterations such as aberrant expression of histone-modifying enzymes have been implicated in tumorigenesis. Jumonji domain containing 2B (JMJD2B) is a newly identified histone demethylase that regulates chromatin structure or gene expression by removing methyl residues from trimethylated lysine 9 on histone H3. Recent observations have shown oncogenic activity of JMJD2B. We explored the functional role of JMJD2B in cancer cell proliferation, survival and tumorigenesis, and determined its expression profile in gastric cancer. Knocking down JMJD2B expression by small interfering RNA (siRNA) in gastric and other cancer cells inhibited cell proliferation and/or induced apoptosis and elevated the expression of p53 and p21{sup CIP1} proteins. The enhanced p53 expression resulted from activation of the DNA damage response pathway. JMJD2B knockdown markedly suppressed xenograft tumor growth in vivo in mice. Moreover, JMJD2B expression was increased in primary gastric-cancer tissues of humans. Thus, JMJD2B is required for sustained proliferation and survival of tumor cells in vitro and in vivo, and its aberrant expression may contribute to the pathogenesis of gastric cancer.

  3. The NG2 Protein Is Not Required for Glutamatergic Neuron-NG2 Cell Synaptic Signaling.

    Science.gov (United States)

    Passlick, Stefan; Trotter, Jacqueline; Seifert, Gerald; Steinhäuser, Christian; Jabs, Ronald

    2016-01-01

    NG2 glial cells (as from now NG2 cells) are unique in receiving synaptic input from neurons. However, the components regulating formation and maintenance of these neuron-glia synapses remain elusive. The transmembrane protein NG2 has been considered a potential mediator of synapse formation and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) clustering, because it contains 2 extracellular Laminin G/Neurexin/Sex Hormone-Binding Globulin domains, which in neurons are crucial for formation of transsynaptic neuroligin-neurexin complexes. NG2 is connected via Glutamate Receptor-Interacting Protein with GluA2/3-containing AMPARs, thereby possibly mediating receptor clustering in glial postsynaptic density. To elucidate the role of NG2 in neuron-glia communication, we investigated glutamatergic synaptic transmission in juvenile and aged hippocampal NG2 cells of heterozygous and homozygous NG2 knockout mice. Neuron-NG2 cell synapses readily formed in the absence of NG2. Short-term plasticity, synaptic connectivity, postsynaptic AMPAR current kinetics, and density were not affected by NG2 deletion. During development, an NG2-independent acceleration of AMPAR current kinetics and decreased synaptic connectivity were observed. Our results indicate that the lack of NG2 does not interfere with genesis and basic properties of neuron-glia synapses. In addition, we demonstrate frequent expression of neuroligins 1-3 in juvenile and aged NG2 cells, suggesting a role of these molecules in synapse formation between NG2 glia and neurons. PMID:25100858

  4. Fulvestrant-induced cell death and proteasomal degradation of estrogen receptor α protein in MCF-7 cells require the CSK c-Src tyrosine kinase.

    Directory of Open Access Journals (Sweden)

    Wei-Lan Yeh

    Full Text Available Fulvestrant is a representative pure antiestrogen and a Selective Estrogen Receptor Down-regulator (SERD. In contrast to the Selective Estrogen Receptor Modulators (SERMs such as 4-hydroxytamoxifen that bind to estrogen receptor α (ERα as antagonists or partial agonists, fulvestrant causes proteasomal degradation of ERα protein, shutting down the estrogen signaling to induce proliferation arrest and apoptosis of estrogen-dependent breast cancer cells. We performed genome-wide RNAi knockdown screenings for protein kinases required for fulvestrant-induced apoptosis of the MCF-7 estrogen-dependent human breast caner cells and identified the c-Src tyrosine kinase (CSK, a negative regulator of the oncoprotein c-Src and related protein tyrosine kinases, as one of the necessary molecules. Whereas RNAi knockdown of CSK in MCF-7 cells by shRNA-expressing lentiviruses strongly suppressed fulvestrant-induced cell death, CSK knockdown did not affect cytocidal actions of 4-hydroxytamoxifen or paclitaxel, a chemotherapeutic agent. In the absence of CSK, fulvestrant-induced proteasomal degradation of ERα protein was suppressed in both MCF-7 and T47D estrogen-dependent breast cancer cells whereas the TP53-mutated T47D cells were resistant to the cytocidal action of fulvestrant in the presence or absence of CSK. MCF-7 cell sensitivities to fulvestrant-induced cell death or ERα protein degradation was not affected by small-molecular-weight inhibitors of the tyrosine kinase activity of c-Src, suggesting possible involvement of other signaling molecules in CSK-dependent MCF-7 cell death induced by fulvestrant. Our observations suggest the importance of CSK in the determination of cellular sensitivity to the cytocidal action of fulvestrant.

  5. Nfil3 is required for the development of all innate lymphoid cell subsets.

    Science.gov (United States)

    Seillet, Cyril; Rankin, Lucille C; Groom, Joanna R; Mielke, Lisa A; Tellier, Julie; Chopin, Michael; Huntington, Nicholas D; Belz, Gabrielle T; Carotta, Sebastian

    2014-08-25

    Innate lymphoid cell (ILC) populations protect against infection and are essential for lymphoid tissue formation and tissue remodeling after damage. Nfil3 is implicated in the function of adaptive immune lineages and NK cell development, but it is not yet known if Nfil3 regulates other innate lymphoid lineages. Here, we identify that Nfil3 is essential for the development of Peyer's patches and ILC2 and ILC3 subsets. Loss of Nfil3 selectively reduced Peyer's patch formation and was accompanied by impaired recruitment and distribution of lymphocytes within the patches. ILC subsets exhibited high Nfil3 expression and genetic deletion of Nfil3 severely compromised the development of all subsets. Subsequently, Nfil3(-/-) mice were highly susceptible to disease when challenged with inflammatory or infectious agents. Thus, we demonstrate that Nfil3 is a key regulator of the development of ILC subsets essential for immune protection in the lung and gut.

  6. PML is required for telomere stability in non-neoplastic human cells

    OpenAIRE

    Marchesini, M.; Matocci, R; Tasselli, L; Cambiaghi, V; Orleth, A; Furia, L; Marinelli, C.; Lombardi, S.; Sammarelli, G; Aversa, F.; Minucci, S; Faretta, M; Pelicci, P.G.; Grignani, F

    2015-01-01

    Telomeres interact with numerous proteins, including components of the shelterin complex, whose alteration, similarly to proliferation-induced telomere shortening, initiates cellular senescence. In tumors, telomere length is maintained by Telomerase activity or by the Alternative Lengthening of Telomeres mechanism, whose hallmark is the telomeric localization of the promyelocytic leukemia (PML) protein. Whether PML contributes to telomeres maintenance in normal cells is unknown. We show that ...

  7. Activation of resting human T cells requires prolonged stimulation of protein kinase C.

    OpenAIRE

    Berry, N; Ase, K; Kishimoto, A.; Nishizuka, Y

    1990-01-01

    Purified resting human T cells can be induced to express the alpha subunit of the interleukin 2 receptor and to proliferate by treatment with 12-O-tetradecanoylphorbol-13-acetate plus ionomycin but not with 1,2-dioctanoylglycerol plus ionomycin. Determination of the translocation of protein kinase C showed that 12-O-tetradecanoylphorbol-13-acetate plus ionomycin caused a prolonged membrane association of the enzyme for more than 4 hr, whereas 1,2-dioctanoylglycerol plus ionomycin induced a tr...

  8. Sirt1 Is Required for Resveratrol-Mediated Chemopreventive Effects in Colorectal Cancer Cells

    Science.gov (United States)

    Buhrmann, Constanze; Shayan, Parviz; Popper, Bastian; Goel, Ajay; Shakibaei, Mehdi

    2016-01-01

    Sirt1 is a NAD+-dependent protein-modifying enzyme involved in regulating gene expression, DNA damage repair, metabolism and survival, as well as acts as an important subcellular target of resveratrol. The complex mechanisms underlying Sirt1 signaling during carcinogenesis remain controversial, as it can serve both as a tumor promoter and suppressor. Whether resveratrol-mediated chemopreventive effects are mediated via Sirt1 in CRC growth and metastasis remains unclear; which was the subject of this study. We found that resveratrol suppressed proliferation and invasion of two different human CRC cells in a dose-dependent manner, and interestingly, this was accompanied with a significant decrease in Ki-67 expression. By transient transfection of CRC cells with Sirt1-ASO, we demonstrated that the anti-tumor effects of resveratrol on cells was abolished, suggesting the essential role of this enzyme in the resveratrol signaling pathway. Moreover, resveratrol downregulated nuclear localization of NF-κB, NF-κB phosphorylation and its acetylation, causing attenuation of NF-κB-regulated gene products (MMP-9, CXCR4) involved in tumor-invasion and metastasis. Finally, Sirt1 was found to interact directly with NF-κB, and resveratrol did not suppress Sirt1-ASO-induced NF-κB phosphorylation, acetylation and NF-κB-regulated gene products. Overall, our results demonstrate that resveratrol can suppress tumorigenesis, at least in part by targeting Sirt1 and suppression of NF-κB activation. PMID:26959057

  9. Nubp1 is required for lung branching morphogenesis and distal progenitor cell survival in mice.

    Directory of Open Access Journals (Sweden)

    Carsten Schnatwinkel

    Full Text Available The lung is a complex system in biology and medicine alike. Whereas there is a good understanding of the anatomy and histology of the embryonic and adult lung, less is known about the molecular details and the cellular pathways that ultimately orchestrate lung formation and affect its health. From a forward genetic approach to identify novel genes involved in lung formation, we identified a mutated Nubp1 gene, which leads to syndactyly, eye cataract and lung hypoplasia. In the lung, Nubp1 is expressed in progenitor cells of the distal epithelium. Nubp1(m1Nisw mutants show increased apoptosis accompanied by a loss of the distal progenitor markers Sftpc, Sox9 and Foxp2. In addition, Nubp1 mutation disrupts localization of the polarity protein Par3 and the mitosis relevant protein Numb. Using knock-down studies in lung epithelial cells, we also demonstrate a function of Nubp1 in regulating centrosome dynamics and microtubule organization. Together, Nubp1 represents an essential protein for lung progenitor survival by coordinating vital cellular processes including cell polarity and centrosomal dynamics.

  10. Sirt1 Is Required for Resveratrol-Mediated Chemopreventive Effects in Colorectal Cancer Cells.

    Science.gov (United States)

    Buhrmann, Constanze; Shayan, Parviz; Popper, Bastian; Goel, Ajay; Shakibaei, Mehdi

    2016-03-05

    Sirt1 is a NAD⁺-dependent protein-modifying enzyme involved in regulating gene expression, DNA damage repair, metabolism and survival, as well as acts as an important subcellular target of resveratrol. The complex mechanisms underlying Sirt1 signaling during carcinogenesis remain controversial, as it can serve both as a tumor promoter and suppressor. Whether resveratrol-mediated chemopreventive effects are mediated via Sirt1 in CRC growth and metastasis remains unclear; which was the subject of this study. We found that resveratrol suppressed proliferation and invasion of two different human CRC cells in a dose-dependent manner, and interestingly, this was accompanied with a significant decrease in Ki-67 expression. By transient transfection of CRC cells with Sirt1-ASO, we demonstrated that the anti-tumor effects of resveratrol on cells was abolished, suggesting the essential role of this enzyme in the resveratrol signaling pathway. Moreover, resveratrol downregulated nuclear localization of NF-κB, NF-κB phosphorylation and its acetylation, causing attenuation of NF-κB-regulated gene products (MMP-9, CXCR4) involved in tumor-invasion and metastasis. Finally, Sirt1 was found to interact directly with NF-κB, and resveratrol did not suppress Sirt1-ASO-induced NF-κB phosphorylation, acetylation and NF-κB-regulated gene products. Overall, our results demonstrate that resveratrol can suppress tumorigenesis, at least in part by targeting Sirt1 and suppression of NF-κB activation.

  11. PML is required for telomere stability in non-neoplastic human cells.

    Science.gov (United States)

    Marchesini, M; Matocci, R; Tasselli, L; Cambiaghi, V; Orleth, A; Furia, L; Marinelli, C; Lombardi, S; Sammarelli, G; Aversa, F; Minucci, S; Faretta, M; Pelicci, P G; Grignani, F

    2016-04-01

    Telomeres interact with numerous proteins, including components of the shelterin complex, whose alteration, similarly to proliferation-induced telomere shortening, initiates cellular senescence. In tumors, telomere length is maintained by Telomerase activity or by the Alternative Lengthening of Telomeres mechanism, whose hallmark is the telomeric localization of the promyelocytic leukemia (PML) protein. Whether PML contributes to telomeres maintenance in normal cells is unknown. We show that in normal human fibroblasts the PML protein associates with few telomeres, preferentially when they are damaged. Proliferation-induced telomere attrition or their damage due to alteration of the shelterin complex enhances the telomeric localization of PML, which is increased in human T-lymphocytes derived from patients genetically deficient in telomerase. In normal fibroblasts, PML depletion induces telomere damage, nuclear and chromosomal abnormalities, and senescence. Expression of the leukemia protein PML/RARα in hematopoietic progenitors displaces PML from telomeres and induces telomere shortening in the bone marrow of pre-leukemic mice. Our work provides a novel view of the physiologic function of PML, which participates in telomeres surveillance in normal cells. Our data further imply that a diminished PML function may contribute to cell senescence, genomic instability, and tumorigenesis. PMID:26119943

  12. Sirt1 Is Required for Resveratrol-Mediated Chemopreventive Effects in Colorectal Cancer Cells

    Directory of Open Access Journals (Sweden)

    Constanze Buhrmann

    2016-03-01

    Full Text Available Sirt1 is a NAD+-dependent protein-modifying enzyme involved in regulating gene expression, DNA damage repair, metabolism and survival, as well as acts as an important subcellular target of resveratrol. The complex mechanisms underlying Sirt1 signaling during carcinogenesis remain controversial, as it can serve both as a tumor promoter and suppressor. Whether resveratrol-mediated chemopreventive effects are mediated via Sirt1 in CRC growth and metastasis remains unclear; which was the subject of this study. We found that resveratrol suppressed proliferation and invasion of two different human CRC cells in a dose-dependent manner, and interestingly, this was accompanied with a significant decrease in Ki-67 expression. By transient transfection of CRC cells with Sirt1-ASO, we demonstrated that the anti-tumor effects of resveratrol on cells was abolished, suggesting the essential role of this enzyme in the resveratrol signaling pathway. Moreover, resveratrol downregulated nuclear localization of NF-κB, NF-κB phosphorylation and its acetylation, causing attenuation of NF-κB-regulated gene products (MMP-9, CXCR4 involved in tumor-invasion and metastasis. Finally, Sirt1 was found to interact directly with NF-κB, and resveratrol did not suppress Sirt1-ASO-induced NF-κB phosphorylation, acetylation and NF-κB-regulated gene products. Overall, our results demonstrate that resveratrol can suppress tumorigenesis, at least in part by targeting Sirt1 and suppression of NF-κB activation.

  13. Capping protein beta is required for actin cytoskeleton organisation and cell migration during Drosophila oogenesis.

    Science.gov (United States)

    Ogienko, Anna A; Karagodin, Dmitry A; Lashina, Valentina V; Baiborodin, Sergey I; Omelina, Eugeniya S; Baricheva, Elina M

    2013-02-01

    Capping protein (CP) is a well-characterised actin-binding protein important for regulation of actin filament (AF) assembly. CP caps the barbed end of AFs, inhibiting the addition and loss of actin monomers. In Drosophila melanogaster, the gene encoding CP β-subunit is named capping protein beta (cpb; see Hopmann et al. [1996] J Cell Biol 133: 1293-305). The cpb level is reduced in the Drosophila bristle actin cytoskeleton and becomes disorganised with abnormal morphology. A reduced level of the CP protein in ovary results in disruption of oocyte determination, and disturbance of nurse cell (NC) cortical integrity and dumping. We describe novel defects appearing in cpb mutants during oogenesis, in which cpb plays an important role in border and centripetal follicle cell migration, ring canal development and cytoplasmic AF formation. The number of long cytoplasmic AFs was dramatically reduced in cpb hypomorphs and abnormal actin aggregates was seen on the inner side of NC membranes. A hypothesis to explain the formation of abnormal short-cut cytoplasmic AFs and actin aggregates in the cpb mutant NCs was proffered, along with a discussion of the reasons for 'dumpless' phenotype formation in the mutants.

  14. Cell wall degradation is required for normal starch mobilisation in barley endosperm.

    Science.gov (United States)

    Andriotis, Vasilios M E; Rejzek, Martin; Barclay, Elaine; Rugen, Michael D; Field, Robert A; Smith, Alison M

    2016-01-01

    Starch degradation in barley endosperm provides carbon for early seedling growth, but the control of this process is poorly understood. We investigated whether endosperm cell wall degradation is an important determinant of the rate of starch degradation. We identified iminosugar inhibitors of enzymes that degrade the cell wall component arabinoxylan. The iminosugar 1,4-dideoxy-1, 4-imino-l-arabinitol (LAB) inhibits arabinoxylan arabinofuranohydrolase (AXAH) but does not inhibit the main starch-degrading enzymes α- and β-amylase and limit dextrinase. AXAH activity in the endosperm appears soon after the onset of germination and resides in dimers putatively containing two isoforms, AXAH1 and AXAH2. Upon grain imbibition, mobilisation of arabinoxylan and starch spreads across the endosperm from the aleurone towards the crease. The front of arabinoxylan degradation precedes that of starch degradation. Incubation of grains with LAB decreases the rate of loss of both arabinoxylan and starch, and retards the spread of both degradation processes across the endosperm. We propose that starch degradation in the endosperm is dependent on cell wall degradation, which permeabilises the walls and thus permits rapid diffusion of amylolytic enzymes. AXAH may be of particular importance in this respect. These results provide new insights into the mobilization of endosperm reserves to support early seedling growth. PMID:27622597

  15. Cell wall degradation is required for normal starch mobilisation in barley endosperm

    Science.gov (United States)

    Andriotis, Vasilios M. E.; Rejzek, Martin; Barclay, Elaine; Rugen, Michael D.; Field, Robert A.; Smith, Alison M.

    2016-01-01

    Starch degradation in barley endosperm provides carbon for early seedling growth, but the control of this process is poorly understood. We investigated whether endosperm cell wall degradation is an important determinant of the rate of starch degradation. We identified iminosugar inhibitors of enzymes that degrade the cell wall component arabinoxylan. The iminosugar 1,4-dideoxy-1, 4-imino-l-arabinitol (LAB) inhibits arabinoxylan arabinofuranohydrolase (AXAH) but does not inhibit the main starch-degrading enzymes α- and β-amylase and limit dextrinase. AXAH activity in the endosperm appears soon after the onset of germination and resides in dimers putatively containing two isoforms, AXAH1 and AXAH2. Upon grain imbibition, mobilisation of arabinoxylan and starch spreads across the endosperm from the aleurone towards the crease. The front of arabinoxylan degradation precedes that of starch degradation. Incubation of grains with LAB decreases the rate of loss of both arabinoxylan and starch, and retards the spread of both degradation processes across the endosperm. We propose that starch degradation in the endosperm is dependent on cell wall degradation, which permeabilises the walls and thus permits rapid diffusion of amylolytic enzymes. AXAH may be of particular importance in this respect. These results provide new insights into the mobilization of endosperm reserves to support early seedling growth. PMID:27622597

  16. Induction of interleukin-8 by Naegleria fowleri lysates requires activation of extracellular signal-regulated kinase in human astroglial cells.

    Science.gov (United States)

    Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Sang-Hee; Kwon, Daeho; Shin, Ho-Joon

    2012-08-01

    Naegleria fowleri is a pathogenic free-living amoeba which causes primary amoebic meningoencephalitis in humans and experimental animals. To investigate the mechanisms of such inflammatory diseases, potential chemokine gene activation in human astroglial cells was investigated following treatment with N. fowleri lysates. We demonstrated that N. fowleri are potent inducers for the expression of interleukin-8 (IL-8) genes in human astroglial cells which was preceded by activation of extracellular signal-regulated kinase (ERK). In addition, N. fowleri lysates induces the DNA binding activity of activator protein-1 (AP-1), an important transcription factor for IL-8 induction. The specific mitogen-activated protein kinase kinase/ERK inhibitor, U0126, blocks N. fowleri-mediated AP-1 activation and subsequent IL-8 induction. N. fowleri-induced IL-8 expression requires activation of ERK in human astroglial cells. These findings indicate that treatment of N. fowleri on human astroglial cells leads to the activation of AP-1 and subsequent expression of IL-8 which are dependent on ERK activation. These results may help understand the N. fowleri-mediated upregulation of chemokine and cytokine expression in the astroglial cells.

  17. p53 is required for metformin-induced growth inhibition, senescence and apoptosis in breast cancer cells.

    Science.gov (United States)

    Li, Puyu; Zhao, Ming; Parris, Amanda B; Feng, Xiaoshan; Yang, Xiaohe

    2015-09-01

    The p53 tumor repressor gene is commonly mutated in human cancers. The tumor inhibitory effect of metformin on p53-mutated breast cancer cells remains unclear. Data from the present study demonstrated that p53 knockdown or mutation has a negative effect on metformin or phenformin-induced growth inhibition, senescence and apoptosis in breast cancer cells. We also found that p53 reactivating agent nutlin-3α and CP/31398 promoted metformin-induced growth inhibition, senescence and apoptosis in MCF-7 (wt p53) and MDA-MB-231 (mt p53) cells, respectively. Treatment of MCF-7 cells with metformin or phenformin induced increase in p53 protein levels and the transcription of its downstream target genes, Bax and p21, in a dose-dependent manner. Moreover, we demonstrated that AMPK-mTOR signaling played a role in metformin-induced p53 up-regulation. The present study showed that p53 is required for metformin or phenformin-induced growth inhibition, senescence and apoptosis in breast cancer cells. The combination of metformin with p53 reactivating agents, like nutlin-3α and CP/31398, is a promising strategy for improving metformin-mediated anti-cancer therapy, especially for tumors with p53 mutations. PMID:26225749

  18. The Drosophila melanogaster Apaf-1 homologue ARK is required for most, but not all, programmed cell death.

    Science.gov (United States)

    Mills, Kathryn; Daish, Tasman; Harvey, Kieran F; Pfleger, Cathie M; Hariharan, Iswar K; Kumar, Sharad

    2006-03-13

    The Apaf-1 protein is essential for cytochrome c-mediated caspase-9 activation in the intrinsic mammalian pathway of apoptosis. Although Apaf-1 is the only known mammalian homologue of the Caenorhabditis elegans CED-4 protein, the deficiency of apaf-1 in cells or in mice results in a limited cell survival phenotype, suggesting that alternative mechanisms of caspase activation and apoptosis exist in mammals. In Drosophila melanogaster, the only Apaf-1/CED-4 homologue, ARK, is required for the activation of the caspase-9/CED-3-like caspase DRONC. Using specific mutants that are deficient for ark function, we demonstrate that ARK is essential for most programmed cell death (PCD) during D. melanogaster development, as well as for radiation-induced apoptosis. ark mutant embryos have extra cells, and tissues such as brain lobes and wing discs are enlarged. These tissues from ark mutant larvae lack detectable PCD. During metamorphosis, larval salivary gland removal was severely delayed in ark mutants. However, PCD occurred normally in the larval midgut, suggesting that ARK-independent cell death pathways also exist in D. melanogaster.

  19. Post-embryonic nerve-associated precursors to adult pigment cells: genetic requirements and dynamics of morphogenesis and differentiation.

    Directory of Open Access Journals (Sweden)

    Erine H Budi

    2011-05-01

    Full Text Available The pigment cells of vertebrates serve a variety of functions and generate a stunning variety of patterns. These cells are also implicated in human pathologies including melanoma. Whereas the events of pigment cell development have been studied extensively in the embryo, much less is known about morphogenesis and differentiation of these cells during post-embryonic stages. Previous studies of zebrafish revealed genetically distinct populations of embryonic and adult melanophores, the ectotherm homologue of amniote melanocytes. Here, we use molecular markers, vital labeling, time-lapse imaging, mutational analyses, and transgenesis to identify peripheral nerves as a niche for precursors to adult melanophores that subsequently migrate to the skin to form the adult pigment pattern. We further identify genetic requirements for establishing, maintaining, and recruiting precursors to the adult melanophore lineage and demonstrate novel compensatory behaviors during pattern regulation in mutant backgrounds. Finally, we show that distinct populations of latent precursors having differential regenerative capabilities persist into the adult. These findings provide a foundation for future studies of post-embryonic pigment cell precursors in development, evolution, and neoplasia.

  20. An O-Methyltransferase Is Required for Infection of Tick Cells by Anaplasma phagocytophilum.

    Directory of Open Access Journals (Sweden)

    Adela S Oliva Chávez

    Full Text Available Anaplasma phagocytophilum, the causative agent of Human Granulocytic Anaplasmosis (HGA, is an obligately intracellular α-proteobacterium that is transmitted by Ixodes spp ticks. However, the pathogen is not transovarially transmitted between tick generations and therefore needs to survive in both a mammalian host and the arthropod vector to complete its life cycle. To adapt to different environments, pathogens rely on differential gene expression as well as the modification of proteins and other molecules. Random transposon mutagenesis of A. phagocytophilum resulted in an insertion within the coding region of an o-methyltransferase (omt family 3 gene. In wild-type bacteria, expression of omt was up-regulated during binding to tick cells (ISE6 at 2 hr post-inoculation, but nearly absent by 4 hr p.i. Gene disruption reduced bacterial binding to ISE6 cells, and the mutant bacteria that were able to enter the cells were arrested in their replication and development. Analyses of the proteomes of wild-type versus mutant bacteria during binding to ISE6 cells identified Major Surface Protein 4 (Msp4, but also hypothetical protein APH_0406, as the most differentially methylated. Importantly, two glutamic acid residues (the targets of the OMT were methyl-modified in wild-type Msp4, whereas a single asparagine (not a target of the OMT was methylated in APH_0406. In vitro methylation assays demonstrated that recombinant OMT specifically methylated Msp4. Towards a greater understanding of the overall structure and catalytic activity of the OMT, we solved the apo (PDB_ID:4OA8, the S-adenosine homocystein-bound (PDB_ID:4OA5, the SAH-Mn2+ bound (PDB_ID:4PCA, and SAM- Mn2+ bound (PDB_ID:4PCL X-ray crystal structures of the enzyme. Here, we characterized a mutation in A. phagocytophilum that affected the ability of the bacteria to productively infect cells from its natural vector. Nevertheless, due to the lack of complementation, we cannot rule out secondary

  1. An O-Methyltransferase Is Required for Infection of Tick Cells by Anaplasma phagocytophilum.

    Science.gov (United States)

    Oliva Chávez, Adela S; Fairman, James W; Felsheim, Roderick F; Nelson, Curtis M; Herron, Michael J; Higgins, LeeAnn; Burkhardt, Nicole Y; Oliver, Jonathan D; Markowski, Todd W; Kurtti, Timothy J; Edwards, Thomas E; Munderloh, Ulrike G

    2015-01-01

    Anaplasma phagocytophilum, the causative agent of Human Granulocytic Anaplasmosis (HGA), is an obligately intracellular α-proteobacterium that is transmitted by Ixodes spp ticks. However, the pathogen is not transovarially transmitted between tick generations and therefore needs to survive in both a mammalian host and the arthropod vector to complete its life cycle. To adapt to different environments, pathogens rely on differential gene expression as well as the modification of proteins and other molecules. Random transposon mutagenesis of A. phagocytophilum resulted in an insertion within the coding region of an o-methyltransferase (omt) family 3 gene. In wild-type bacteria, expression of omt was up-regulated during binding to tick cells (ISE6) at 2 hr post-inoculation, but nearly absent by 4 hr p.i. Gene disruption reduced bacterial binding to ISE6 cells, and the mutant bacteria that were able to enter the cells were arrested in their replication and development. Analyses of the proteomes of wild-type versus mutant bacteria during binding to ISE6 cells identified Major Surface Protein 4 (Msp4), but also hypothetical protein APH_0406, as the most differentially methylated. Importantly, two glutamic acid residues (the targets of the OMT) were methyl-modified in wild-type Msp4, whereas a single asparagine (not a target of the OMT) was methylated in APH_0406. In vitro methylation assays demonstrated that recombinant OMT specifically methylated Msp4. Towards a greater understanding of the overall structure and catalytic activity of the OMT, we solved the apo (PDB_ID:4OA8), the S-adenosine homocystein-bound (PDB_ID:4OA5), the SAH-Mn2+ bound (PDB_ID:4PCA), and SAM- Mn2+ bound (PDB_ID:4PCL) X-ray crystal structures of the enzyme. Here, we characterized a mutation in A. phagocytophilum that affected the ability of the bacteria to productively infect cells from its natural vector. Nevertheless, due to the lack of complementation, we cannot rule out secondary mutations

  2. Dicer is required for haploid male germ cell differentiation in mice.

    Directory of Open Access Journals (Sweden)

    Hanna M Korhonen

    Full Text Available BACKGROUND: The RNase III endonuclease Dicer is an important regulator of gene expression that processes microRNAs (miRNAs and small interfering RNAs (siRNAs. The best-characterized function of miRNAs is gene repression at the post-transcriptional level through the pairing with mRNAs of protein-encoding genes. Small RNAs can also act at the transcriptional level by controlling the epigenetic status of chromatin. Dicer and other mediators of small RNA pathways are present in mouse male germ cells, and several miRNAs and endogenous siRNAs are expressed in the testis, suggesting that Dicer-dependent small RNAs are involved in the control of the precisely timed and highly organised process of spermatogenesis. PRINCIPAL FINDINGS: Being interested in the Dicer-mediated functions during spermatogenesis, we have analysed here a male germ cell-specific Dicer1 knockout mouse model, in which the deletion of Dicer1 takes place during early postnatal development in spermatogonia. We found that Dicer1 knockout testes were reduced in size and spermatogenesis within the seminiferous tubules was disrupted. Dicer1 knockout epididymides contained very low number of mature sperm with pronounced morphological abnormalities. Spermatogonial differentiation appeared unaffected. However, the number of haploid cells was decreased in knockout testes, and an increased number of apoptotic spermatocytes was observed. The most prominent defects were found during late haploid differentiation, and Dicer was demonstrated to be critical for the normal organization of chromatin and nuclear shaping of elongating spermatids. CONCLUSIONS/SIGNIFICANCE: We demonstrate that Dicer and Dicer-dependent small RNAs are imperative regulators of haploid spermatid differentiation and essential for male fertility.

  3. Endogenous mesenchymal stromal cells in bone marrow are required to preserve muscle function in mdx mice.

    Science.gov (United States)

    Fujita, Ryo; Tamai, Katsuto; Aikawa, Eriko; Nimura, Keisuke; Ishino, Saki; Kikuchi, Yasushi; Kaneda, Yasufumi

    2015-03-01

    The physiological role of "endogenous" bone marrow (BM) mesenchymal stromal cells (MSCs) in tissue regeneration is poorly understood. Here, we show the significant contribution of unique endogenous BM-MSC populations to muscle regeneration in Duchenne muscular dystrophy (DMD) mice (mdx). Transplantation of BM cells (BMCs) from 10-week-old mdx into 3-4-week-old mdx mice increased inflammation and fibrosis and reduced muscle function compared with mdx mice that received BMCs from 10-week-old wild-type mice, suggesting that the alteration of BMC populations in mdx mice affects the progression of muscle pathology. Two distinct MSC populations in BM, that is, hematopoietic lineage (Lin)(-) /ckit(-) /CD106(+) /CD44(+) and Lin(-) /ckit(-) /CD106(+) /CD44(-) cells, were significantly reduced in 10-week-old mdx mice in disease progression. The results of a whole-transcriptome analysis indicated that these two MSC populations have distinct gene expression profiles, indicating that the Lin(-) /ckit(-) /CD106(+) /CD44(+) and Lin(-) /ckit(-) /CD106(+) /CD44(-) MSC populations are proliferative- and dormant-state populations in BM, respectively. BM-derived Lin(-) /CD106(+) /CD44(+) MSCs abundantly migrated to damaged muscles and highly expressed tumor necrosis factor-alpha-stimulated gene/protein-6 (TSG-6), an anti-inflammatory protein, in damaged muscles. We also demonstrated that TSG-6 stimulated myoblast proliferation. The injection of Lin(-) /ckit(-) /CD106(+) /CD44(+) MSCs into the muscle of mdx mice successfully ameliorated muscle dysfunction by decreasing inflammation and enhancing muscle regeneration through TSG-6-mediated activities. Thus, we propose a novel function of the unique endogenous BM-MSC population, which countered muscle pathology progression in a DMD model.

  4. Lipid rafts are required for GLUT4 internalization in adipose cells

    OpenAIRE

    Ros-Baró, Anna; López-Iglesias, Carmen; Peiró, Sandra; Bellido, David; Palacín, Manuel; Zorzano, Antonio; Camps, Marta

    2001-01-01

    It has been recently reported that insulin recruits a novel signaling machinery to lipid rafts required for insulin-stimulated GLUT4 translocation [Baumann, A., Ribon, V., Kanzaki, M., Thurmond, D. C., Mora, S., Shigematsu, S., Bickel, P. E., Pessin, J. E. & Saltiel, A. R. (2001) Nature 407, 202–207, 2000; Chiang, S. H., Baumann, C. A., Kanzaki, M., Thurmond, D. C., Watson, R. T., Neudauer, C. L., Macara, I. G., Pessin, J. E. & Saltiel, A. R. (2001) Nature 410, 944–948]. We have assessed the ...

  5. γ-Secretase inhibitor-resistant glioblastoma stem cells require RBPJ to propagate.

    Science.gov (United States)

    Fan, Xing

    2016-07-01

    Targeting glioblastoma stem cells with γ-secretase inhibitors (GSIs) disrupts the Notch pathway and has shown some benefit in both pre-clinical models and in patients during phase I/II clinical trials. However, it is largely unknown why some glioblastoma (GBM) does not respond to GSI treatment. In this issue of the JCI, Xie et al. determined that GSI-resistant brain tumor-initiating cells (BTICs) from GBM express a higher level of the gene RBPJ, which encodes a mediator of canonical Notch signaling, compared to non-BTICs. Knockdown of RBPJ in BTICs decreased propagation in vitro and in vivo by inducing apoptosis. Interestingly, RBPJ was shown to regulate a different transcription program than Notch in BTICs by binding CDK9, thereby affecting Pol II-regulated transcript elongation. Targeting CDK9 or c-MYC, an upstream regulator of RBPJ, with small molecules also decreased BTIC propagation, and prolonged survival in mice bearing orthotopic GBM xenografts. This study not only provides a mechanism for GSI treatment resistance, but also identifies two potential therapeutic strategies to target GSI-resistant BTICs. PMID:27322058

  6. Serine protease inhibitor 6 is required to protect dendritic cells from the kiss of death.

    Science.gov (United States)

    Lovo, Elena; Zhang, Manling; Wang, Lihui; Ashton-Rickardt, Philip G

    2012-02-01

    How dendritic cells (DC) present Ag to cytotoxic T cells (CTL) without themselves being killed through contact-mediated cytotoxicity (so-called kiss of death) has proved to be controversial. Using mice deficient in serine protease inhibitor 6 (Spi6), we show that Spi6 protects DC from the kiss of death by inhibiting granzyme B (GrB) delivered by CTL. Infection of Spi6 knockout mice with lymphocytic choriomeningitis virus revealed impaired survival of CD8α DC. The impaired survival of Spi6 knockout CD8α DC resulted in impaired priming and expansion of both primary and memory lymphocytic choriomeningitis virus-specific CTL, which could be corrected by GrB deficiency. The rescue in the clonal burst obtained by GrB elimination demonstrated that GrB was the physiological target through which Spi6 protected DC from CTL. We conclude that the negative regulation of DC priming of CD8 T lymphocyte immunity by CTL killing is mitigated by the physiological inhibition of GrB by Spi6.

  7. crumbs and stardust, two genes of Drosophila required for the development of epithelial cell polarity.

    Science.gov (United States)

    Knust, E; Tepass, U; Wodarz, A

    1993-01-01

    Loss-of-function mutations in the Drosophila genes crumbs and stardust are embryonic lethal and cause a breakdown of ectodermally derived epithelia during organogenesis, leading to formation of irregular cell clusters and extensive cell death in some epithelia. The mutant phenotype develops gradually and affects the various epithelia to different extents. crumbs encodes a large transmembrane protein with 30 EGF-like repeats and four laminin A G-domain-like repeats in its extracellular domain, suggesting its participation in protein-protein interactions. The CRUMBS protein is exclusively expressed on the apical membrane of all ectodermally derived epithelia, the tissues affected in crumbs and stardust mutant embryos. The gene function is completely abolished by a crumbs mutation that causes production of a protein with a truncated cytoplasmic domain. Instead of being apically localized as in wild-type, the mutant CRUMBS protein is diffusely distributed in the cytoplasm; this occurs before any morphologically detectable cellular phenotype is visible, suggesting that targeting of proteins is affected in crumbs mutant embryos. Later, the protein can be detected on the apical and basolateral membranes. Mutations in stardust produce a phenotype nearly identical to that associated with crumbs mutations, suggesting that both genes are functionally related. Double mutant combinations and gene dosage studies suggest that both genes are part of a common genetic pathway, in which stardust acts downstream of crumbs.

  8. Arabidopsis VARIEGATED 3 encodes a chloroplast-targeted, zinc-finger protein required for chloroplast and palisade cell development

    DEFF Research Database (Denmark)

    Næsted, Henrik; Holm, Agnethe; Jenkins, Tom;

    2004-01-01

    The stable, recessive Arabidopsis variegated 3 (var3) mutant exhibits a variegated phenotype due to somatic areas lacking or containing developmentally retarded chloroplasts and greatly reduced numbers of palisade cells. The VAR3 gene, isolated by transposon tagging, encodes the 85.9 kDa VAR3...... protein containing novel repeats and zinc fingers described as protein interaction domains. VAR3 interacts specifically in yeast and in vitro with NCED4, a putative polyene chain or carotenoid dioxygenase, and both VAR3 and NCED4 accumulate in the chloroplast stroma. Metabolic profiling demonstrates...... that pigment profiles are qualitatively similar in wild type and var3, although var3 accumulates lower levels of chlorophylls and carotenoids. These results indicate that VAR3 is a part of a protein complex required for normal chloroplast and palisade cell development....

  9. Arabidopsis VARIEGATED 3 encodes a chloroplasttargeted, zinc-finger protein required for chloroplast and palisade cell development

    DEFF Research Database (Denmark)

    Næsted, Henrik; Holm, A.; Jenkins, T.;

    2004-01-01

    The stable, recessive Arabidopsis variegated 3 (var3) mutant exhibits a variegated phenotype due to somatic areas lacking or containing developmentally retarded chloroplasts and greatly reduced numbers of palisade cells. The VAR3 gene, isolated by transposon tagging, encodes the 85.9 kDa VAR3...... protein containing novel repeats and zinc fingers described as protein interaction domains. VAR3 interacts specifically in yeast and in vitro with NCED4, a putative polyene chain or carotenoid dioxygenase, and both VAR3 and NCED4 accumulate in the chloroplast stroma. Metabolic profiling demonstrates...... that pigment profiles are qualitatively similar in wild type and var3, although var3 accumulates lower levels of chlorophylls and carotenoids. These results indicate that VAR3 is a part of a protein complex required for normal chloroplast and palisade cell development....

  10. Jmjd2/Kdm4 demethylases are required for expression of Il3ra and survival of acute myeloid leukemia cells

    DEFF Research Database (Denmark)

    Agger, Karl; Miyagi, Satoru; Pedersen, Marianne Terndrup;

    2016-01-01

    Acute myeloid leukemias (AMLs) with a rearrangement of the mixed-linage leukemia (MLL) gene are aggressive hematopoietic malignancies. Here, we explored the feasibility of using the H3K9- and H3K36-specific demethylases Jmjd2/Kdm4 as putative drug targets in MLL-AF9 translocated leukemia. Using...... a mechanism involving removal of H3K9me3 from the promoter of the Il3ra gene. Importantly, ectopic expression of Il3ra in Jmjd2/Kdm4 knockout cells alleviates the requirement of Jmjd2/Kdm4 for the survival of AML cells, showing that Il3ra is a critical downstream target of Jmjd2/Kdm4 in leukemia...

  11. Polarization of the epithelial layer and apical localization of integrins are required for engulfment of apoptotic cells in the Drosophila ovary

    Directory of Open Access Journals (Sweden)

    Tracy L. Meehan

    2015-12-01

    Full Text Available Inefficient clearance of dead cells or debris by epithelial cells can lead to or exacerbate debilitating conditions such as retinitis pigmentosa, macular degeneration, chronic obstructive pulmonary disease and asthma. Despite the importance of engulfment by epithelial cells, little is known about the molecular changes that are required within these cells. The misregulation of integrins has previously been associated with disease states, suggesting that a better understanding of the regulation of receptor trafficking could be key to treating diseases caused by defects in phagocytosis. Here, we demonstrate that the integrin heterodimer αPS3/βPS becomes apically enriched and is required for engulfment by the epithelial follicle cells of the Drosophila ovary. We found that integrin heterodimer localization and function is largely directed by the α-subunit. Moreover, proper cell polarity promotes asymmetric integrin enrichment, suggesting that αPS3/βPS trafficking occurs in a polarized fashion. We show that several genes previously known for their roles in trafficking and cell migration are also required for engulfment. Moreover, as in mammals, the same α-integrin subunit is required by professional and non-professional phagocytes and migrating cells in Drosophila. Our findings suggest that migrating and engulfing cells use common machinery, and demonstrate a crucial role for integrin function and polarized trafficking of integrin subunits during engulfment. This study also establishes the epithelial follicle cells of the Drosophila ovary as a powerful model for understanding the molecular changes required for engulfment by a polarized epithelium.

  12. Central Giant Cell Granuloma of the Mandible Requiring Multiple Treatment Modalities: A Case Report.

    Science.gov (United States)

    Jerkins, David; Malotky, Maximilian; Miremadi, Reza; Dole, Mukund

    2016-08-01

    Central giant cell granuloma (CGCG) is a relatively rare non-neoplastic, intraosseous lesion that exhibits a wide spectrum of clinical behavior, and its management can be particularly challenging even for experienced clinicians. The etiopathogenesis of this disease process remains unclear, although factors such as trauma, inflammatory foci, and a genetic predisposition have been implicated. Although multiple treatment modalities have been used with varying degrees of success, there is no accepted algorithm for therapeutic intervention and little is known about the reasons for success or failure of a given treatment. This article reviews the epidemiology, presentation, classification, and currently used therapies for CGCG while describing the clinical course and successful therapeutic outcome of a young female patient with an aggressive CGCG of the mandible. PMID:27000410

  13. Light-stimulated cell expansion in bean (Phaseolus vulgaris L.) leaves. II. Quantity and quality of light required

    Science.gov (United States)

    Van Volkenburgh, E.; Cleland, R. E.; Watanabe, M.

    1990-01-01

    The quantity and quality of light required for light-stimulated cell expansion in leaves of Phaseolus vulgaris L. have been determined. Seedlings were grown in dim red light (RL; 4 micromoles photons m-2 s-1) until cell division in the primary leaves was completed, then excised discs were incubated in 10 mM sucrose plus 10 mM KCl in a variety of light treatments. The growth response of discs exposed to continuous white light (WL) for 16 h was saturated at 100 micromoles m-2 s-1, and did not show reciprocity. Extensive, but not continuous, illumination was needed for maximal growth. The wavelength dependence of disc expansion was determined from fluence-response curves obtained from 380 to 730 nm provided by the Okazaki Large Spectrograph. Blue (BL; 460 nm) and red light (RL; 660 nm) were most effective in promoting leaf cell growth, both in photosynthetically active and inhibited leaf discs. Far-red light (FR; 730 nm) reduced the effectiveness of RL, but not BL, indicating that phytochrome and a separate blue-light receptor mediate expansion of leaf cells.

  14. Stem cell-dependent formation of a functional anterior regeneration pole in planarians requires Zic and Forkhead transcription factors.

    Science.gov (United States)

    Vogg, Matthias C; Owlarn, Suthira; Pérez Rico, Yuvia A; Xie, Jianlei; Suzuki, Yoko; Gentile, Luca; Wu, Wei; Bartscherer, Kerstin

    2014-06-15

    Planarians can regenerate their head within days. This process depends on the direction of adult stem cells to wound sites and the orchestration of their progenitors to commit to appropriate lineages and to arrange into patterned tissues. We identified a zinc finger transcription factor, Smed-ZicA, as a downstream target of Smed-FoxD, a Forkhead transcription factor required for head regeneration. Smed-zicA and Smed-FoxD are co-expressed with the Wnt inhibitor notum and the Activin inhibitor follistatin in a cluster of cells at the anterior-most tip of the regenerating head - the anterior regeneration pole - and in surrounding stem cell progeny. Depletion of Smed-zicA and Smed-FoxD by RNAi abolishes notum and follistatin expression at the pole and inhibits head formation downstream of initial polarity decisions. We suggest a model in which ZicA and FoxD transcription factors synergize to control the formation of Notum- and Follistatin-producing anterior pole cells. Pole formation might constitute an early step in regeneration, resulting in a signaling center that orchestrates cellular events in the growing tissue. PMID:24704339

  15. Sterilizing immunity to influenza virus infection requires local antigen-specific T cell response in the lungs

    Science.gov (United States)

    Dutta, Avijit; Huang, Ching-Tai; Lin, Chun-Yen; Chen, Tse-Ching; Lin, Yung-Chang; Chang, Chia-Shiang; He, Yueh-Chia

    2016-09-01

    Sterilizing immunity is a unique immune status, which prevents effective virus infection into the host. It is different from the immunity that allows infection but with subsequent successful eradication of the virus. Pre-infection induces sterilizing immunity to homologous influenza virus challenge in ferret. In our antigen-specific experimental system, mice pre-infected with PR8 influenza virus through nasal route are likewise resistant to reinfection of the same strain of virus. The virus is cleared before establishment of effective infection. Intramuscular influenza virus injection confers protection against re-infection with facilitated virus clearance but not sterilizing immunity. Pre-infection and intramuscular injection generates comparable innate immunity and antibody response, but only pre-infection induces virus receptor reduction and efficient antigen-specific T cell response in the lungs. Pre-infection with nH1N1 influenza virus induces virus receptor reduction but not PR8-specific T cell immune response in the lungs and cannot prevent infection of PR8 influenza virus. Pre-infection with PR8 virus induced PR8-specific T cell response in the lungs but cannot prevent infection of nH1N1 virus either. These results reveal that antigen-specific T cell immunity is required for sterilizing immunity.

  16. The ClpP protease homologue is required for the transmission traits and cell division of the pathogen Legionella pneumophila

    Directory of Open Access Journals (Sweden)

    Zhang Qin-fen

    2010-02-01

    Full Text Available Abstract Background Legionella pneumophila, the intracellular bacterial pathogen that causes Legionnaires' disease, exhibit characteristic transmission traits such as elevated stress tolerance, shortened length and virulence during the transition from the replication phase to the transmission phase. ClpP, the catalytic core of the Clp proteolytic complex, is widely involved in many cellular processes via the regulation of intracellular protein quality. Results In this study, we showed that ClpP was required for optimal growth of L. pneumophila at high temperatures and under several other stress conditions. We also observed that cells devoid of clpP exhibited cell elongation, incomplete cell division and compromised colony formation. Furthermore, we found that the clpP-deleted mutant was more resistant to sodium stress and failed to proliferate in the amoebae host Acanthamoeba castellanii. Conclusions The data present in this study illustrate that the ClpP protease homologue plays an important role in the expression of transmission traits and cell division of L. pneumophila, and further suggest a putative role of ClpP in virulence regulation.

  17. Stem cell-dependent formation of a functional anterior regeneration pole in planarians requires Zic and Forkhead transcription factors.

    Science.gov (United States)

    Vogg, Matthias C; Owlarn, Suthira; Pérez Rico, Yuvia A; Xie, Jianlei; Suzuki, Yoko; Gentile, Luca; Wu, Wei; Bartscherer, Kerstin

    2014-06-15

    Planarians can regenerate their head within days. This process depends on the direction of adult stem cells to wound sites and the orchestration of their progenitors to commit to appropriate lineages and to arrange into patterned tissues. We identified a zinc finger transcription factor, Smed-ZicA, as a downstream target of Smed-FoxD, a Forkhead transcription factor required for head regeneration. Smed-zicA and Smed-FoxD are co-expressed with the Wnt inhibitor notum and the Activin inhibitor follistatin in a cluster of cells at the anterior-most tip of the regenerating head - the anterior regeneration pole - and in surrounding stem cell progeny. Depletion of Smed-zicA and Smed-FoxD by RNAi abolishes notum and follistatin expression at the pole and inhibits head formation downstream of initial polarity decisions. We suggest a model in which ZicA and FoxD transcription factors synergize to control the formation of Notum- and Follistatin-producing anterior pole cells. Pole formation might constitute an early step in regeneration, resulting in a signaling center that orchestrates cellular events in the growing tissue.

  18. Sterilizing immunity to influenza virus infection requires local antigen-specific T cell response in the lungs

    Science.gov (United States)

    Dutta, Avijit; Huang, Ching-Tai; Lin, Chun-Yen; Chen, Tse-Ching; Lin, Yung-Chang; Chang, Chia-Shiang; He, Yueh-Chia

    2016-01-01

    Sterilizing immunity is a unique immune status, which prevents effective virus infection into the host. It is different from the immunity that allows infection but with subsequent successful eradication of the virus. Pre-infection induces sterilizing immunity to homologous influenza virus challenge in ferret. In our antigen-specific experimental system, mice pre-infected with PR8 influenza virus through nasal route are likewise resistant to reinfection of the same strain of virus. The virus is cleared before establishment of effective infection. Intramuscular influenza virus injection confers protection against re-infection with facilitated virus clearance but not sterilizing immunity. Pre-infection and intramuscular injection generates comparable innate immunity and antibody response, but only pre-infection induces virus receptor reduction and efficient antigen-specific T cell response in the lungs. Pre-infection with nH1N1 influenza virus induces virus receptor reduction but not PR8-specific T cell immune response in the lungs and cannot prevent infection of PR8 influenza virus. Pre-infection with PR8 virus induced PR8-specific T cell response in the lungs but cannot prevent infection of nH1N1 virus either. These results reveal that antigen-specific T cell immunity is required for sterilizing immunity. PMID:27596047

  19. Microdomains of High Calcium Are Not Required for Exocytosis in Rbl-2h3 Mucosal Mast Cells

    OpenAIRE

    Mahmoud, Sahar F.; Fewtrell, Clare

    2001-01-01

    We have previously shown that store-associated microdomains of high Ca2+ are not essential for exocytosis in RBL-2H3 mucosal mast cells. We have now examined whether Ca2+ microdomains near the plasma membrane are required, by comparing the secretory responses seen when Ca2+ influx was elicited by two very different mechanisms. In the first, antigen was used to activate the Ca2+ release–activated Ca2+ (CRAC) current (ICRAC) through CRAC channels. In the second, a Ca2+ ionophore was used to tra...

  20. AltMV TGB1 nucleolar localization requires homologous interaction and correlates with cell wall localization associated with cell-to-cell movement

    Science.gov (United States)

    The Potexvirus Alternanthera mosaic virus has multifunctional triple gene block (TGB) proteins, among which our studies have focused on the properties of the TGB1 protein. The TGB1 of AltMV has functions including RNA binding, RNA silencing suppression, and cell-to-cell movement, and is known to for...

  1. Epidermal growth factor receptor is required for estradiol-stimulated bovine satellite cell proliferation.

    Science.gov (United States)

    Reiter, B C; Kamanga-Sollo, E; Pampusch, M S; White, M E; Dayton, W R

    2014-07-01

    The objective of this study was to assess the role of the epidermal growth factor receptor (EGFR) in estradiol-17β (E2)-stimulated proliferation of cultured bovine satellite cells (BSCs). Treatment of BSC cultures with AG1478 (a specific inhibitor of EGFR tyrosine kinase activity) suppresses E2-stimulated BSC proliferation (P LR3-IGF-1 (an IGF1 analogue that binds normally to the insulin-like growth factor receptor (IGFR)-1 but has little or no affinity for IGF binding proteins) in cultured BSCs (P < 0.05). Even though EGFR siRNA treatment has no effect on IGFR-1β mRNA expression in cultured BSCs, IGFR-1β protein level is substantially reduced in BSCs treated with EGFR siRNA. These data suggest that EGFR silencing results in post-transcriptional modifications that result in decreased IGFR-1β protein levels. Although it is clear that functional EGFR is necessary for E2-stimulated proliferation of BSCs, the role of EGFR is not clear. Transactivation of EGFR may directly stimulate proliferation, or EGFR may function to maintain the level of IGFR-1β which is necessary for E2-stimulated proliferation. It also is possible that the role of EGFR in E2-stimulated BSC proliferation may involve both of these mechanisms. PMID:24906928

  2. Precise temporal regulation of roughest is required for correct salivary gland autophagic cell death in Drosophila.

    Science.gov (United States)

    Simon, Claudio R; Moda, Livia M R; Octacilio-Silva, Shirlei; Anhezini, Lucas; Machado-Gitai, Luciana C H; Ramos, Ricardo Guelerman P

    2009-07-01

    The Drosophila roughest (rst) locus encodes an immunoglobulin superfamily transmembrane glycoprotein implicated in a variety of embryonic and postembryonic developmental processes. Here we demonstrate a previously unnoticed role for this gene in the autophagic elimination of larval salivary glands during early pupal stages by showing that overexpression of the Rst protein ectodomain in early pupa leads to persistence of salivary glands up to at least 12 hours after head eversion, although with variable penetrance. The same phenotype is observed in individuals carrying the dominant regulatory allele rst(D), but not in loss of function alleles. Analysis of persistent glands at the ultrastructural level showed that programmed cell death starts at the right time but is arrested at an early stage of the process. Finally we describe the expression pattern and intracellular distribution of Rst in wild type and rst(D) mutants, showing that its downregulation in salivary glands at the beginning of pupal stage is an important factor in the correct implementation of the autophagic program of this tissue in space and time.

  3. Smad3 signaling is required for satellite cell function and myogenic differentiation of myoblasts

    Institute of Scientific and Technical Information of China (English)

    Xiaojia Ge; Ravi Kambadur; Craig McFarlane; Anuradha Vajjala; Sudarsanareddy Lokireddy; Zhi Hui Ng; Chek Kun Tan; Nguan Soon Tan; Walter Wahli; Mridula Sharma

    2011-01-01

    TGF-β and myostatin are the two most important regulators of muscle growth.Both growth factors have been shown to signal through a Smad3-dependent pathway.However to date,the role of Smad3 in muscle growth and differentiation is not investigated.Here,we demonstrate that Smad3-null mice have decreased muscle mass and pronounced skeletal muscle atrophy.Consistent with this,we also find increased protein ubiquitination and elevated levels of the ubiquitin E3 ligase MuRF1 in muscle tissue isolated from Smad3-null mice.Loss of Smad3 also led to defective satellite cell (SC) functionality.Smad3-null SCs showed reduced propensity for self-renewal,which may lead to a progressive loss of SC number.Indeed,decreased SC number was observed in skeletal muscle from Smad3- null mice showing signs of severe muscle wasting.Further in vitro analysis of primary myoblast cultures identified that Smad3-nuil myoblasts exhibit impaired proliferation,differentiation and fusion,resulting in the formation of atrophied myotubes.A search for the molecular mechanism revealed that loss of Smad3 results in increased myostatin expression in Smad3-null muscle and myoblasts.Given that myostatin is a negative regulator,we hypothesize that increased myostatin levels are responsible for the atrophic phenotype in Smad3-null mice.Consistent with this theory,inactivation of myostatin in Smad3-null mice rescues the muscle atrophy phenotype.

  4. Lymphocytes and macrophages are infected by Theileria equi, but T cells and B cells are not required to establish infection in vivo.

    Directory of Open Access Journals (Sweden)

    Joshua D Ramsay

    Full Text Available Theileria equi has a biphasic life cycle in horses, with a period of intraleukocyte development followed by patent erythrocytic parasitemia that causes acute and sometimes fatal hemolytic disease. Unlike Theileria spp. that infect cattle (Theileria parva and Theileria annulata, the intraleukocyte stage (schizont of Theileria equi does not cause uncontrolled host cell proliferation or other significant pathology. Nevertheless, schizont-infected leukocytes are of interest because of their potential to alter host cell function and because immune responses directed against this stage could halt infection and prevent disease. Based on cellular morphology, Theileria equi has been reported to infect lymphocytes in vivo and in vitro, but the specific phenotype of schizont-infected cells has yet to be defined. To resolve this knowledge gap in Theileria equi pathogenesis, peripheral blood mononuclear cells were infected in vitro and the phenotype of infected cells determined using flow cytometry and immunofluorescence microscopy. These experiments demonstrated that the host cell range of Theileria equi was broader than initially reported and included B lymphocytes, T lymphocytes and monocyte/macrophages. To determine if B and T lymphocytes were required to establish infection in vivo, horses affected with severe combined immunodeficiency (SCID, which lack functional B and T lymphocytes, were inoculated with Theileria equi sporozoites. SCID horses developed patent erythrocytic parasitemia, indicating that B and T lymphocytes are not necessary to complete the Theileria equi life cycle in vivo. These findings suggest that the factors mediating Theileria equi leukocyte invasion and intracytoplasmic differentiation are common to several leukocyte subsets and are less restricted than for Theileria annulata and Theileria parva. These data will greatly facilitate future investigation into the relationships between Theileria equi leukocyte tropism and pathogenesis

  5. Lymphocytes and macrophages are infected by Theileria equi, but T cells and B cells are not required to establish infection in vivo.

    Science.gov (United States)

    Ramsay, Joshua D; Ueti, Massaro W; Johnson, Wendell C; Scoles, Glen A; Knowles, Donald P; Mealey, Robert H

    2013-01-01

    Theileria equi has a biphasic life cycle in horses, with a period of intraleukocyte development followed by patent erythrocytic parasitemia that causes acute and sometimes fatal hemolytic disease. Unlike Theileria spp. that infect cattle (Theileria parva and Theileria annulata), the intraleukocyte stage (schizont) of Theileria equi does not cause uncontrolled host cell proliferation or other significant pathology. Nevertheless, schizont-infected leukocytes are of interest because of their potential to alter host cell function and because immune responses directed against this stage could halt infection and prevent disease. Based on cellular morphology, Theileria equi has been reported to infect lymphocytes in vivo and in vitro, but the specific phenotype of schizont-infected cells has yet to be defined. To resolve this knowledge gap in Theileria equi pathogenesis, peripheral blood mononuclear cells were infected in vitro and the phenotype of infected cells determined using flow cytometry and immunofluorescence microscopy. These experiments demonstrated that the host cell range of Theileria equi was broader than initially reported and included B lymphocytes, T lymphocytes and monocyte/macrophages. To determine if B and T lymphocytes were required to establish infection in vivo, horses affected with severe combined immunodeficiency (SCID), which lack functional B and T lymphocytes, were inoculated with Theileria equi sporozoites. SCID horses developed patent erythrocytic parasitemia, indicating that B and T lymphocytes are not necessary to complete the Theileria equi life cycle in vivo. These findings suggest that the factors mediating Theileria equi leukocyte invasion and intracytoplasmic differentiation are common to several leukocyte subsets and are less restricted than for Theileria annulata and Theileria parva. These data will greatly facilitate future investigation into the relationships between Theileria equi leukocyte tropism and pathogenesis, breed

  6. FGFR2IIIb-MAPK Activity Is Required for Epithelial Cell Fate Decision in the Lower Müllerian Duct.

    Science.gov (United States)

    Terakawa, Jumpei; Rocchi, Altea; Serna, Vanida A; Bottinger, Erwin P; Graff, Jonathan M; Kurita, Takeshi

    2016-07-01

    Cell fate of lower Müllerian duct epithelium (MDE), to become uterine or vaginal epithelium, is determined by the absence or presence of ΔNp63 expression, respectively. Previously, we showed that SMAD4 and runt-related transcription factor 1 (RUNX1) were independently required for MDE to express ΔNp63. Here, we report that vaginal mesenchyme directs vaginal epithelial cell fate in MDE through paracrine activation of fibroblast growth factor (FGF) receptor-MAPK pathway. In the developing reproductive tract, FGF7 and FGF10 were enriched in vaginal mesenchyme, whereas FGF receptor 2IIIb was expressed in epithelia of both the uterus and vagina. When Fgfr2 was inactivated, vaginal MDE underwent uterine cell fate, and this differentiation defect was corrected by activation of MEK-ERK pathway. In vitro, FGF10 in combination with bone morphogenetic protein 4 and activin A (ActA) was sufficient to induce ΔNp63 in MDE, and ActA was essential for induction of RUNX1 through SMAD-independent pathways. Accordingly, inhibition of type 1 receptors for activin in neonatal mice induced uterine differentiation in vaginal epithelium by down-regulating RUNX1, whereas conditional deletion of Smad2 and Smad3 had no effect on vaginal epithelial differentiation. In conclusion, vaginal epithelial cell fate in MDE is induced by FGF7/10-MAPK, bone morphogenetic protein 4-SMAD, and ActA-RUNX1 pathway activities, and the disruption in any one of these pathways results in conversion from vaginal to uterine epithelial cell fate. PMID:27164167

  7. Protease activated receptor signaling is required for African trypanosome traversal of human brain microvascular endothelial cells.

    Directory of Open Access Journals (Sweden)

    Dennis J Grab

    Full Text Available BACKGROUND: Using human brain microvascular endothelial cells (HBMECs as an in vitro model for how African trypanosomes cross the human blood-brain barrier (BBB we recently reported that the parasites cross the BBB by generating calcium activation signals in HBMECs through the activity of parasite cysteine proteases, particularly cathepsin L (brucipain. In the current study, we examined the possible role of a class of protease stimulated HBMEC G protein coupled receptors (GPCRs known as protease activated receptors (PARs that might be implicated in calcium signaling by African trypanosomes. METHODOLOGY/PRINCIPAL FINDINGS: Using RNA interference (RNAi we found that in vitro PAR-2 gene (F2RL1 expression in HBMEC monolayers could be reduced by over 95%. We also found that the ability of Trypanosoma brucei rhodesiense to cross F2RL1-silenced HBMEC monolayers was reduced (39%-49% and that HBMECs silenced for F2RL1 maintained control levels of barrier function in the presence of the parasite. Consistent with the role of PAR-2, we found that HBMEC barrier function was also maintained after blockade of Galpha(q with Pasteurella multocida toxin (PMT. PAR-2 signaling has been shown in other systems to have neuroinflammatory and neuroprotective roles and our data implicate a role for proteases (i.e. brucipain and PAR-2 in African trypanosome/HBMEC interactions. Using gene-profiling methods to interrogate candidate HBMEC pathways specifically triggered by brucipain, several pathways that potentially link some pathophysiologic processes associated with CNS HAT were identified. CONCLUSIONS/SIGNIFICANCE: Together, the data support a role, in part, for GPCRs as molecular targets for parasite proteases that lead to the activation of Galpha(q-mediated calcium signaling. The consequence of these events is predicted to be increased permeability of the BBB to parasite transmigration and the initiation of neuroinflammation, events precursory to CNS disease.

  8. Prediction of Neck Dissection Requirement After Definitive Radiotherapy for Head-and-Neck Squamous Cell Carcinoma

    International Nuclear Information System (INIS)

    Background: This analysis was undertaken to assess the need for planned neck dissection in patients with a complete response (CR) of involved nodes after irradiation and to determine the benefit of a neck dissection in those with less than CR by tumor site. Methods: Our cohort included 880 patients with T1-4, N1-3M0 squamous cell carcinoma of the oropharynx, larynx, or hypopharynx who received treatment between 1994 and 2004. Survival curves were calculated by the Kaplan-Meier Method, comparisons of rates with the log–rank test and prognostic factors by Cox's proportional hazard model. Results: Nodal CR occurred in 377 (43%) patients, of whom 365 patients did not undergo nodal dissection. The 5-year actuarial regional control rate of patients with CR was 92%. Two hundred sixty-eight of the remaining patients (53%) underwent neck dissections. The 5-year actuarial regional control rate for patients without a CR was 84%. Those who had a neck dissection fared better with 5-year actuarial regional control rates of 90% and 76% for those operated and those not operated (p < 0.001). Variables associated with poorer regional control rates included higher T and N stage, non-oropharynx cancers, non-CR, both clinical and pathological. Conclusions: With 92% 5-year neck control rate without neck dissection after CR, there is little justification for systematic neck dissection. The addition of a neck dissection resulted in higher neck control after partial response though patients with viable tumor on pathology specimens had poorer outcomes. The identification of that subgroup that benefits from additional treatment remains a challenge.

  9. Meis1 Is Required for Adult Mouse Erythropoiesis, Megakaryopoiesis and Hematopoietic Stem Cell Expansion.

    Science.gov (United States)

    Miller, Michelle Erin; Rosten, Patty; Lemieux, Madeleine E; Lai, Courteney; Humphries, R Keith

    2016-01-01

    Meis1 is recognized as an important transcriptional regulator in hematopoietic development and is strongly implicated in the pathogenesis of leukemia, both as a Hox transcription factor co-factor and independently. Despite the emerging recognition of Meis1's importance in the context of both normal and leukemic hematopoiesis, there is not yet a full understanding of Meis1's functions and the relevant pathways and genes mediating its functions. Recently, several conditional mouse models for Meis1 have been established. These models highlight a critical role for Meis1 in adult mouse hematopoietic stem cells (HSCs) and implicate reactive oxygen species (ROS) as a mediator of Meis1 function in this compartment. There are, however, several reported differences between these studies in terms of downstream progenitor populations impacted and effectors of function. In this study, we describe further characterization of a conditional knockout model based on mice carrying a loxP-flanked exon 8 of Meis1 which we crossed onto the inducible Cre localization/expression strains, B6;129-Gt(ROSA)26Sor(tm1(Cre/ERT)Nat)/J or B6.Cg-Tg(Mx1-Cre)1Cgn/J. Findings obtained from these two inducible Meis1 knockout models confirm and extend previous reports of the essential role of Meis1 in adult HSC maintenance and expansion and provide new evidence that highlights key roles of Meis1 in both megakaryopoiesis and erythropoiesis. Gene expression analyses point to a number of candidate genes involved in Meis1's role in hematopoiesis. Our data additionally support recent evidence of a role of Meis1 in ROS regulation. PMID:26986211

  10. Differential requirement of CD28 costimulation for activation of murine CD8+ intestinal intraepithelial lymphocyte subsets and lymph node cells.

    Science.gov (United States)

    Gelfanov, V; Lai, Y G; Gelfanova, V; Dong, J Y; Su, J P; Liao, N S

    1995-07-01

    The CD8+CD4- (CD8+) murine small intestinal intraepithelial lymphocytes (IELs) contain two subpopulations, one expressing alpha alpha-CD8 homodimers and another alpha beta-CD8 heterodimers. In this study, plate-bound anti-TCR beta-chain (TCR-beta) mAb alone or combined with anti-CD28 mAb is used as a model system to study activation requirement of these two CD8+ IEL subsets. In contrast to CD8+ lymph node (LN) cells that require both TCR and CD28 triggering for activation, alpha beta-CD8+ IELs proliferate and produce IL-2 and IFN-gamma when stimulated with anti-TCR-beta mAb alone, and soluble CTLA-4 Ig has no effect on their responses. On the other hand, alpha alpha-CD8+ IELs neither make IL-2 or IFN-gamma nor proliferate even when both stimuli are provided. However, alpha alpha-CD8+ IELs are capable of proliferation in both CD8+ IEL subsets is lower than in CD8+ LN cells, which contributes to the weaker and delayed response of CD8+ IELs. PMID:7602124

  11. The N-terminal domain is a transcriptional activation domain required for Nanog to maintain ES cell self-renewal

    Institute of Scientific and Technical Information of China (English)

    GUO YunQian; ZHANG Juan; YE Li; CHEN Mo; YAO Dong; PAN GuangJin; ZHANG JieQiong; PEI DuanQing

    2009-01-01

    Nanog is a transcription factor identified by its ability to maintain the self-renewal of ES cells in the absence of leukemia inhibitory factor (LIF). Nanog protein contains an N-terminal domain (ND),a DNA-binding homeobox domain (HD) and a C-terminal domain (CD). We previously reported that the CD in Nanog is a transcriptional activation domain essential for the in vivo function of Nanog. Here we demonstrated that the ND in Nanog is also functionally important. Deletion of the ND reduces the transcriptional activity of Nanog on either artificial reporters or native Nanog promoters. This truncated Nanog is also less effective in regulating the endogenous Nanog target genes. Furthermore,the ND truncation disrupted the ability of Nanog to maintain ES cell self-renewal as well. We found that the ND Is not required for the nuclear localization of Nanog. These results suggest that the regulation of endogenous pluripotent genes such as oct3/4 and rex-1 is required for the in vivo function of Nanog.

  12. Efficient induction of CD25- iTreg by co-immunization requires strongly antigenic epitopes for T cells

    Directory of Open Access Journals (Sweden)

    Li Jinyao

    2011-05-01

    Full Text Available Abstract Background We previously showed that co-immunization with a protein antigen and a DNA vaccine coding for the same antigen induces CD40low IL-10high tolerogenic DCs, which in turn stimulates the expansion of antigen-specific CD4+CD25-Foxp3+ regulatory T cells (CD25- iTreg. However, it was unclear how to choose the antigen sequence to maximize tolerogenic antigen presentation and, consequently, CD25- iTreg induction. Results In the present study, we demonstrated the requirement of highly antigenic epitopes for CD25- iTreg induction. Firstly, we showed that the induction of CD25- iTreg by tolerogenic DC can be blocked by anti-MHC-II antibody. Next, both the number and the suppressive activity of CD25- iTreg correlated positively with the overt antigenicity of an epitope to activate T cells. Finally, in a mouse model of dermatitis, highly antigenic epitopes derived from a flea allergen not only induced more CD25- iTreg, but also more effectively prevented allergenic reaction to the allergen than did weakly antigenic epitopes. Conclusions Our data thus indicate that efficient induction of CD25- iTreg requires highly antigenic peptide epitopes. This finding suggests that highly antigenic epitopes should be used for efficient induction of CD25- iTreg for clinical applications such as flea allergic dermatitis.

  13. Epinephrine Activation of the β2-Adrenoceptor Is Required for IL-13-Induced Mucin Production in Human Bronchial Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Nour Al-Sawalha

    Full Text Available Mucus hypersecretion by airway epithelium is a hallmark of inflammation in allergic asthma and results in airway narrowing and obstruction. Others have shown that administration a TH2 cytokine, IL-13 is sufficient to cause mucus hypersecretion in vivo and in vitro. Asthma therapy often utilizes β2-adrenoceptor (β2AR agonists, which are effective acutely as bronchodilators, however chronic use may lead to a worsening of asthma symptoms. In this study, we asked whether β2AR signaling in normal human airway epithelial (NHBE cells affected mucin production in response to IL-13. This cytokine markedly increased mucin production, but only in the presence of epinephrine. Mucin production was blocked by ICI-118,551, a preferential β2AR antagonist, but not by CGP-20712A, a preferential β1AR antagonist. Constitutive β2AR activity was not sufficient for IL-13 induced mucin production and β-agonist-induced signaling is required. A clinically important long-acting β-agonist, formoterol, was as effective as epinephrine in potentiating IL-13 induced MUC5AC transcription. IL-13 induced mucin production in the presence of epinephrine was significantly reduced by treatment with selective inhibitors of ERK1/2 (FR180204, p38 (SB203580 and JNK (SP600125. Replacement of epinephrine with forskolin + IBMX resulted in a marked increase in mucin production in NHBE cells in response to IL-13, and treatment with the inhibitory cAMP analogue Rp-cAMPS decreased mucin levels induced by epinephrine + IL-13. Our findings suggest that β2AR signaling is required for mucin production in response to IL-13, and that mitogen activated protein kinases and cAMP are necessary for this effect. These data lend support to the notion that β2AR-agonists may contribute to asthma exacerbations by increasing mucin production via activation of β2ARs on epithelial cells.

  14. Rabies Internalizes into Primary Peripheral Neurons via Clathrin Coated Pits and Requires Fusion at the Cell Body

    Science.gov (United States)

    Piccinotti, Silvia; Whelan, Sean P. J.

    2016-01-01

    The single glycoprotein (G) of rabies virus (RABV) dictates all viral entry steps from receptor engagement to membrane fusion. To study the uptake of RABV into primary neuronal cells in culture, we generated a recombinant vesicular stomatitis virus in which the G protein was replaced with that of the neurotropic RABV CVS-11 strain (rVSV CVS G). Using microfluidic compartmentalized culture, we examined the uptake of single virions into the termini of primary neurons of the dorsal root ganglion and ventral spinal cord. By pharmacologically disrupting endocytosis at the distal neurites, we demonstrate that rVSV CVS G uptake and infection are dependent on dynamin. Imaging of single virion uptake with fluorescent endocytic markers further identifies endocytosis via clathrin-coated pits as the predominant internalization mechanism. Transmission electron micrographs also reveal the presence of viral particles in vesicular structures consistent with incompletely coated clathrin pits. This work extends our previous findings of clathrin-mediated uptake of RABV into epithelial cells to two neuronal subtypes involved in rabies infection in vivo. Chemical perturbation of endosomal acidification in the neurite or somal compartment further shows that establishment of infection requires pH-dependent fusion of virions at the cell body. These findings correlate infectivity to existing single particle evidence of long-range endosomal transport of RABV and clathrin dependent uptake at the plasma membrane. PMID:27463226

  15. TopBP1 is required at mitosis to reduce transmission of DNA damage to G1 daughter cells.

    Science.gov (United States)

    Pedersen, Rune Troelsgaard; Kruse, Thomas; Nilsson, Jakob; Oestergaard, Vibe H; Lisby, Michael

    2015-08-17

    Genome integrity is critically dependent on timely DNA replication and accurate chromosome segregation. Replication stress delays replication into G2/M, which in turn impairs proper chromosome segregation and inflicts DNA damage on the daughter cells. Here we show that TopBP1 forms foci upon mitotic entry. In early mitosis, TopBP1 marks sites of and promotes unscheduled DNA synthesis. Moreover, TopBP1 is required for focus formation of the structure-selective nuclease and scaffold protein SLX4 in mitosis. Persistent TopBP1 foci transition into 53BP1 nuclear bodies (NBs) in G1 and precise temporal depletion of TopBP1 just before mitotic entry induced formation of 53BP1 NBs in the next cell cycle, showing that TopBP1 acts to reduce transmission of DNA damage to G1 daughter cells. Based on these results, we propose that TopBP1 maintains genome integrity in mitosis by controlling chromatin recruitment of SLX4 and by facilitating unscheduled DNA synthesis.

  16. Large isoforms of UNC-89 (obscurin are required for muscle cell architecture and optimal calcium release in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Patrick M Spooner

    Full Text Available Calcium, a ubiquitous intracellular signaling molecule, controls a diverse array of cellular processes. Consequently, cells have developed strategies to modulate the shape of calcium signals in space and time. The force generating machinery in muscle is regulated by the influx and efflux of calcium ions into the muscle cytoplasm. In order for efficient and effective muscle contraction to occur, calcium needs to be rapidly, accurately and reliably regulated. The mechanisms underlying this highly regulated process are not fully understood. Here, we show that the Caenorhabditis elegans homolog of the giant muscle protein obscurin, UNC-89, is required for normal muscle cell architecture. The large immunoglobulin domain-rich isoforms of UNC-89 are critical for sarcomere and sarcoplasmic reticulum organization. Furthermore, we have found evidence that this structural organization is crucial for excitation-contraction coupling in the body wall muscle, through the coordination of calcium signaling. Thus, our data implicates UNC-89 in maintaining muscle cell architecture and that this precise organization is essential for optimal calcium mobilization and efficient and effective muscle contraction.

  17. Indian hedgehog B function is required for the specification of oligodendrocyte progenitor cells in the zebrafish CNS.

    Science.gov (United States)

    Chung, Ah-Young; Kim, Suhyun; Kim, Eunmi; Kim, Dohyun; Jeong, Inyoung; Cha, Young Ryun; Bae, Young-ki; Park, Seung Woo; Lee, Jehee; Park, Hae-Chul

    2013-01-23

    A subset of ventral spinal cord precursors, known as pMN precursor cells, initially generate motor neurons and then oligodendrocyte progenitor cells (OPCs), which migrate and differentiate as myelinating oligodendrocytes in the developing neural tube. The switch between motor neuron and oligodendrocyte production by the pMN neural precursors is an important step in building a functional nervous system. However, the precise mechanism that orchestrates the sequential generation of motor neurons and oligodendrocytes within the common population of pMN precursors is still unclear. The current study demonstrates that Indian Hedgehog b (Ihhb), previously known as Echidna Hedgehog, begins to be expressed in the floor plate cells of the ventral spinal cord at the time of OPC specification in zebrafish embryos. Ihhb loss-of-function analysis revealed that Ihhb function is required for OPC specification from pMN precursors by negatively regulating the proliferation of neural precursors. Finally, results showed that Sonic Hedgehog (Shh) could not replace Ihhb function in OPC specification, suggesting that Ihhb and Shh play separate roles in OPC specification. Altogether, data from the present study suggested a novel mechanism, mediated by Ihhb, for the sequential generation of motor neurons and oligodendrocytes from pMN precursors in the ventral spinal cord of zebrafish embryos.

  18. TopBP1 is required at mitosis to reduce transmission of DNA damage to G1 daughter cells

    DEFF Research Database (Denmark)

    Pedersen, Rune Troelsgaard; Kruse, Thomas; Nilsson, Jakob;

    2015-01-01

    Genome integrity is critically dependent on timely DNA replication and accurate chromosome segregation. Replication stress delays replication into G2/M, which in turn impairs proper chromosome segregation and inflicts DNA damage on the daughter cells. Here we show that TopBP1 forms foci upon...... mitotic entry. In early mitosis, TopBP1 marks sites of and promotes unscheduled DNA synthesis. Moreover, TopBP1 is required for focus formation of the structure-selective nuclease and scaffold protein SLX4 in mitosis. Persistent TopBP1 foci transition into 53BP1 nuclear bodies (NBs) in G1 and precise...... temporal depletion of TopBP1 just before mitotic entry induced formation of 53BP1 NBs in the next cell cycle, showing that TopBP1 acts to reduce transmission of DNA damage to G1 daughter cells. Based on these results, we propose that TopBP1 maintains genome integrity in mitosis by controlling chromatin...

  19. CD4+ T Cells Are Not Required for Suppression of Hepatitis B Virus Replication in the Liver of Vaccinated Chimpanzees.

    Science.gov (United States)

    Rybczynska, Jolanta; Campbell, Katherine; Kamili, Saleem; Locarnini, Stephen; Krawczynski, Krzysztof; Walker, Christopher M

    2016-01-01

    Humans vaccinated with hepatitis B virus (HBV) surface antigen (HBsAg) sometimes develop humoral and cellular immunity to HBV proteins such as core and polymerase that are not vaccine components, providing indirect evidence that vaccine-induced immunity is not sterilizing. We previously described CD4(+) T-cell immunity against HBsAg and polymerase in chimpanzees after vaccination and HBV challenge. Here, vaccinated chimpanzees with protective levels of anti-HBsAg antibodies were rechallenged with HBV after antibody-mediated CD4(+) T-cell depletion. HBV DNA was detected in liver for at least 3 months after rechallenge, but virus replication was suppressed, as revealed by the absence of HBV DNA and HBsAg in serum. These observations provide direct virological evidence for nonsterilizing immunity in individuals with anti-HBsAg antibodies and are consistent with translation of HBV proteins to prime immune responses. They also indicate that CD4(+) T cells were not required for suppression of HBV replication in previously vaccinated individuals. PMID:26324781

  20. Non-cysteine linked MUC1 cytoplasmic dimers are required for Src recruitment and ICAM-1 binding induced cell invasion

    Directory of Open Access Journals (Sweden)

    Gunasekara Nirosha

    2011-07-01

    dimerization or ICAM-1 induced signalling. Conclusions Our data implicates non-cysteine linked MUC1 dimerization in cell signalling pathways required for cancer cell migration.

  1. Rotavirus structural proteins and dsRNA are required for the human primary plasmacytoid dendritic cell IFNalpha response.

    Directory of Open Access Journals (Sweden)

    Emily M Deal

    Full Text Available Rotaviruses are the leading cause of severe dehydrating diarrhea in children worldwide. Rotavirus-induced immune responses, especially the T and B cell responses, have been extensively characterized; however, little is known about innate immune mechanisms involved in the control of rotavirus infection. Although increased levels of systemic type I interferon (IFNalpha and beta correlate with accelerated resolution of rotavirus disease, multiple rotavirus strains, including rhesus rotavirus (RRV, have been demonstrated to antagonize type I IFN production in a variety of epithelial and fibroblast cell types through several mechanisms, including degradation of multiple interferon regulatory factors by a viral nonstructural protein. This report demonstrates that stimulation of highly purified primary human peripheral plasmacytoid dendritic cells (pDCs with either live or inactivated RRV induces substantial IFNalpha production by a subset of pDCs in which RRV does not replicate. Characterization of pDC responses to viral stimulus by flow cytometry and Luminex revealed that RRV replicates in a small subset of human primary pDCs and, in this RRV-permissive small subset, IFNalpha production is diminished. pDC activation and maturation were observed independently of viral replication and were enhanced in cells in which virus replicates. Production of IFNalpha by pDCs following RRV exposure required viral dsRNA and surface proteins, but neither viral replication nor activation by trypsin cleavage of VP4. These results demonstrate that a minor subset of purified primary human peripheral pDCs are permissive to RRV infection, and that pDCs retain functionality following RRV stimulus. Additionally, this study demonstrates trypsin-independent infection of primary peripheral cells by rotavirus, which may allow for the establishment of extraintestinal viremia and antigenemia. Importantly, these data provide the first evidence of IFNalpha induction in primary

  2. Epidermal Fatty Acid Binding Protein (E-FABP) Is Not Required for the Generation or Maintenance of Effector and Memory T Cells following Infection with Listeria monocytogenes.

    Science.gov (United States)

    Li, Bing; Schmidt, Nathan W

    2016-01-01

    Following activation of naïve T cells there are dynamic changes in the metabolic pathways used by T cells to support both the energetic needs of the cell and the macromolecules required for growth and proliferation. Among other changes, lipid metabolism undergoes dynamic transitions between fatty acid oxidation and fatty acid synthesis as cells progress from naïve to effector and effector to memory T cells. The hydrophobic nature of lipids requires that they be bound to protein chaperones within a cell. Fatty acid binding proteins (FABPs) represent a large class of lipid chaperones, with epidermal FABP (E-FABP) expressed in T cells. The objective of this study was to determine the contribution of E-FABP in antigen-specific T cell responses. Following infection with Listeria monocytogenes, we observed similar clonal expansion, contraction and formation of memory CD8 T cells in WT and E-FABP-/- mice, which also exhibited similar phenotypic and functional characteristics. Analysis of Listeria-specific CD4 T cells also revealed no defect in the expansion, contraction, and formation of memory CD4 T cells in E-FABP-/- mice. These data demonstrate that E-FABP is dispensable for antigen-specific T cell responses following a bacterial infection. PMID:27588422

  3. Cell-autonomous requirement of the USP/EcR-B ecdysone receptor for mushroom body neuronal remodeling in Drosophila.

    Science.gov (United States)

    Lee, T; Marticke, S; Sung, C; Robinow, S; Luo, L

    2000-12-01

    Neuronal process remodeling occurs widely in the construction of both invertebrate and vertebrate nervous systems. During Drosophila metamorphosis, gamma neurons of the mushroom bodies (MBs), the center for olfactory learning in insects, undergo pruning of larval-specific dendrites and axons followed by outgrowth of adult-specific processes. To elucidate the underlying molecular mechanisms, we conducted a genetic mosaic screen and identified one ultraspiracle (usp) allele defective in larval process pruning. Consistent with the notion that USP forms a heterodimer with the ecdysone receptor (EcR), we found that the EcR-B1 isoform is specifically expressed in the MB gamma neurons, and is required for the pruning of larval processes. Surprisingly, most identified primary EcR/USP targets are dispensable for MB neuronal remodeling. Our study demonstrates cell-autonomous roles for EcR/USP in controlling neuronal remodeling, potentially through novel downstream targets. PMID:11163268

  4. Cell cycle-dependent deposition of CENP-A requires the Dos1/2-Cdc20 complex.

    Science.gov (United States)

    Gonzalez, Marlyn; He, Haijin; Sun, Siyu; Li, Chen; Li, Fei

    2013-01-01

    Centromeric histone CENP-A, a variant of canonical histone H3, plays a central role in proper chromosome segregation. Loading of CENP-A at centromeres is cell cycle-regulated: parental CENP-A is deposited at centromeres during S phase, whereas newly synthesized CENP-A is deposited during later stages of the cell cycle. The mechanisms involved in deposition of CENP-A at centromeres during S phase remain poorly understood. In fission yeast, loading of CENP-A during S phase is regulated by the GATA-type factor, Ams2. Here we show that the Dos1/2-Cdc20 complex, previously characterized as a silencing complex essential for inheritance of H3K9 methylation during S phase, is also required for localization of CENP-A(cnp1) at centromeres at this stage. Disruption of Dos1 (also known as Raf1/Clr8/Cmc1), Dos2 (also known as Raf2/Clr7/Cmc2), or Cdc20, a DNA polymerase epsilon subunit, results in dissociation of CENP-A from centromeres and mislocalization of the protein to noncentromeric sites. All three mutants display spindle disorganization and mitotic defects. Inactivation of Dos1 or Cdc20 also results in accumulation of noncoding RNA transcripts from centromeric cores, a feature common to mutants affecting kinetochore integrity. We further find that Dos1 physically associates with Ams2 and is required for the association of Ams2 with centromeric cores during S phase. Finally, we show that Dos2 associates with centromeric cores during S phase and that its recruitment to centromeric cores depends on Cdc20. This study identifies a physical link between DNA replication and CENP-A assembly machinery and provides mechanistic insight into how CENP-A is faithfully inherited during S phase. PMID:23267073

  5. 40 CFR Table 5 to Subpart IIIii of... - Required Elements of Floor-Level Mercury Vapor Measurement and Cell Room Monitoring Plans

    Science.gov (United States)

    2010-07-01

    ... Mercury Vapor Measurement and Cell Room Monitoring Plans 5 Table 5 to Subpart IIIII of Part 63 Protection... Hazardous Air Pollutants: Mercury Emissions From Mercury Cell Chlor-Alkali Plants Pt. 63, Subpt. IIIII, Table 5 Table 5 to Subpart IIIII of Part 63—Required Elements of Floor-Level Mercury Vapor...

  6. Pro-recombination role of Srs2 protein requires SUMO (small ubiquitin-like modifier) but is independent of PCNA (proliferating cell nuclear antigen) interaction

    DEFF Research Database (Denmark)

    Kolesar, Peter; Altmannova, Veronika; Pinela da Silva, Sonia Cristina;

    2016-01-01

    -interacting motif (SIM) of Srs2 is important for the interaction with several recombination factors. Lack of SIM, but not proliferating cell nuclear antigen (PCNA)-interacting motif (PIM), leads to increased cell death under circumstances requiring homologous recombination for DNA repair. Simultaneous mutation...

  7. Grp/DChk1 is required for G(2)-M checkpoint activation in Drosophila S2 cells, whereas Dmnk/DChk2 is dispensable

    NARCIS (Netherlands)

    de Vries, HI; Uyetake, L; Lemstra, W; Brunsting, JF; Su, TT; Kampinga, HH; Sibon, OCM

    2005-01-01

    Cell-cycle checkpoints are signal-transduction pathways required to maintain genomic stability in dividing cells. Previously, it was reported that two kinases essential for checkpoint signalling, Chk1 and Chk2 are structurally conserved. In contrast to yeast, Xenopus and mammals, the Chk1- and Chk2-

  8. Phosphorylation at tyrosine 114 of Proliferating Cell Nuclear Antigen (PCNA) is required for adipogenesis in response to high fat diet

    Energy Technology Data Exchange (ETDEWEB)

    Lo, Yuan-Hung; Ho, Po-Chun; Chen, Min-Shan; Hugo, Eric; Ben-Jonathan, Nira [Department of Cancer Biology, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267-0521 (United States); Department of Environmental Health, University of Cincinnati College of Medicine, 3223 Eden Avenue, Kettering Laboratory, Cincinnati, OH 45267-0056 (United States); Wang, Shao-Chun, E-mail: shao-chun.wang@uc.edu [Department of Cancer Biology, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267-0521 (United States); Department of Environmental Health, University of Cincinnati College of Medicine, 3223 Eden Avenue, Kettering Laboratory, Cincinnati, OH 45267-0056 (United States)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Proliferating Cell Nuclear Antigen (PCNA) is phosphorylated at Y114. Black-Right-Pointing-Pointer Phospho-Y114 of PCNA is not required for cell proliferation for normal growth. Black-Right-Pointing-Pointer MCE during adipogenesis is abolished in the lack of the phosphorylation. Black-Right-Pointing-Pointer Homozygous Y114F mice are resistant to high fat diet induced obesity. Black-Right-Pointing-Pointer Our results shed light on the interface between proliferation and differentiation. -- Abstract: Clonal proliferation is an obligatory component of adipogenesis. Although several cell cycle regulators are known to participate in the transition between pre-adipocyte proliferation and terminal adipocyte differentiation, how the core DNA synthesis machinery is coordinately regulated in adipogenesis remains elusive. PCNA (Proliferating Cell Nuclear Antigen) is an indispensable component for DNA synthesis during proliferation. Here we show that PCNA is subject to phosphorylation at the highly conserved tyrosine residue 114 (Y114). Replacing the Y114 residue with phenylalanine (Y114F), which is structurally similar to tyrosine but cannot be phosphorylated, does not affect normal animal development. However, when challenged with high fat diet, mice carrying homozygous Y114F alleles (PCNA{sup F/F}) are resistant to adipose tissue enlargement in comparison to wild-type (WT) mice. Mouse embryonic fibroblasts (MEFs) harboring WT or Y114F mutant PCNA proliferate at similar rates. However, when subjected to adipogenesis induction in culture, PCNA{sup F/F} MEFs are not able to re-enter the cell cycle and fail to form mature adipocytes, while WT MEFs undergo mitotic clonal expansion in response to the adipogenic stimulation, accompanied by enhanced Y114 phosphorylation of PCNA, and differentiate to mature adipocytes. Consistent with the function of Y114 phosphorylation in clonal proliferation in adipogenesis, fat tissues isolated from WT

  9. The phosphoenolpyruvate/phosphate translocator is required for phenolic metabolism, palisade cell development, and plastid-dependent nuclear gene expression.

    Science.gov (United States)

    Streatfield, S J; Weber, A; Kinsman, E A; Häusler, R E; Li, J; Post-Beittenmiller, D; Kaiser, W M; Pyke, K A; Flügge, U I; Chory, J

    1999-09-01

    The Arabidopsis chlorophyll a/b binding protein (CAB) gene underexpressed 1 (cue1) mutant underexpresses light-regulated nuclear genes encoding chloroplast-localized proteins. cue1 also exhibits mesophyll-specific chloroplast and cellular defects, resulting in reticulate leaves. Both the gene underexpression and the leaf cell morphology phenotypes are dependent on light intensity. In this study, we determine that CUE1 encodes the plastid inner envelope phosphoenolpyruvate/phosphate translocator (PPT) and define amino acid residues that are critical for translocator function. The biosynthesis of aromatics is compromised in cue1, and the reticulate phenotype can be rescued by feeding aromatic amino acids. Determining that CUE1 encodes PPT indicates the in vivo role of the translocator in metabolic partitioning and reveals a mesophyll cell-specific requirement for the translocator in Arabidopsis leaves. The nuclear gene expression defects in cue1 suggest that a light intensity-dependent interorganellar signal is modulated through metabolites dependent on a plastid supply of phosphoenolpyruvate.

  10. Rapid structural changes and acidification of guard cell vacuoles during stomatal closure require phosphatidylinositol 3,5-bisphosphate.

    Science.gov (United States)

    Bak, Gwangbae; Lee, Eun-Jung; Lee, Yuree; Kato, Mariko; Segami, Shoji; Sze, Heven; Maeshima, Masayoshi; Hwang, Jae-Ung; Lee, Youngsook

    2013-06-01

    Rapid stomatal closure is essential for water conservation in plants and is thus critical for survival under water deficiency. To close stomata rapidly, guard cells reduce their volume by converting a large central vacuole into a highly convoluted structure. However, the molecular mechanisms underlying this change are poorly understood. In this study, we used pH-indicator dyes to demonstrate that vacuolar convolution is accompanied by acidification of the vacuole in fava bean (Vicia faba) guard cells during abscisic acid (ABA)-induced stomatal closure. Vacuolar acidification is necessary for the rapid stomatal closure induced by ABA, since a double mutant of the vacuolar H(+)-ATPase vha-a2 vha-a3 and vacuolar H(+)-PPase mutant vhp1 showed delayed stomatal closure. Furthermore, we provide evidence for the critical role of phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] in changes in pH and morphology of the vacuole. Single and double Arabidopsis thaliana null mutants of phosphatidylinositol 3-phosphate 5-kinases (PI3P5Ks) exhibited slow stomatal closure upon ABA treatment compared with the wild type. Moreover, an inhibitor of PI3P5K reduced vacuolar acidification and convolution and delayed stomatal closure in response to ABA. Taken together, these results suggest that rapid ABA-induced stomatal closure requires PtdIns(3,5)P2, which is essential for vacuolar acidification and convolution. PMID:23757398

  11. S1P-Yap1 signaling regulates endoderm formation required for cardiac precursor cell migration in zebrafish.

    Science.gov (United States)

    Fukui, Hajime; Terai, Kenta; Nakajima, Hiroyuki; Chiba, Ayano; Fukuhara, Shigetomo; Mochizuki, Naoki

    2014-10-13

    To form the primary heart tube in zebrafish, bilateral cardiac precursor cells (CPCs) migrate toward the midline beneath the endoderm. Mutants lacking endoderm and fish with defective sphingosine 1-phosphate (S1P) signaling exhibit cardia bifida. Endoderm defects lead to the lack of foothold for the CPCs, whereas the cause of cardia bifida in S1P signaling mutants remains unclear. Here we show that S1P signaling regulates CPC migration through Yes-associated protein 1 (Yap1)-dependent endoderm survival. Cardia bifida seen in spns2 (S1P transporter) morphants and s1pr2 (S1P receptor-2) morphants could be rescued by endodermal expression of nuclear localized form of yap1. yap1 morphants had decreased expression of the Yap1/Tead target connective tissue growth factor a (Ctgfa) and consequently increased endodermal cell apoptosis. Consistently, ctgfa morphants showed defects of the endodermal sheet and cardia bifida. Collectively, we show that S1pr2/Yap1-regulated ctgfa expression is essential for the proper endoderm formation required for CPC migration.

  12. Isoleucine requirement of 80- to 120-kilogram barrows fed corn-soybean meal or corn-blood cell diets.

    Science.gov (United States)

    Dean, D W; Southern, L L; Kerr, B J; Bidner, T D

    2005-11-01

    Six experiments were conducted to validate an Ile-deficient diet and determine the Ile requirement of 80- to 120-kg barrows. Experiment 1 had five replications, and Exp. 2 through 6 had four replications per treatment; all pen replicates had four crossbred barrows each (initial BW were 93, 83, 85, 81, 81, and 88 kg, respectively). All dietary additions were on an as-fed basis. In Exp. 1, pigs were fed a corn-soybean meal diet (C-SBM) or a corn-5% blood cell (BC) diet with or without 0.26% supplemental Ile (C-BC or C-BC+Ile) in a 28-d growth assay. On d 14, pigs receiving the C-BC diet were taken off experiment as a result of a severe decrease in ADFI. Growth performance did not differ for pigs fed C-SBM or C-BC + Ile (P = 0.36) over the 28-d experiment. In Exp. 2, pigs were fed the C-BC diet containing 0.24, 0.26, 0.28, 0.30, or 0.32% true ileal digestible (TD) Ile for 7 d in an attempt to estimate the Ile requirement using plasma urea N (PUN) as the response variable. Because of incremental increases in ADFI as TD Ile increased, PUN could not be used to estimate the Ile requirement. In Exp. 3, pigs were fed the C-BC diet containing 0.28, 0.30, 0.32, 0.34, or 0.36% TD Ile. Daily gain, ADFI, and G:F increased linearly (P kilograms of lean increased linearly (P kilograms of lean is not < 0.34% in a C-BC diet, but may be as low as 0.24% in a C-SBM diet.

  13. Requirement of B-Raf, C-Raf, and A-Raf for the growth and survival of mouse embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Wenjing; Hao, Baixia; Wang, Qian; Lu, Yingying; Yue, Jianbo, E-mail: jbyue@me.com

    2013-11-01

    Extracellular signal-regulated kinases (ERKs) have been implicated to be dispensable for self-renewal of mouse embryonic stem (ES) cells, and simultaneous inhibition of both ERK signaling and glycogen synthase kinase 3 (GSK3) not only allows mouse ES cells to self-renew independent of extracellular stimuli but also enables more efficient derivation of naïve ES cells from mouse and rat strains. Interestingly, some ERKs stay active in mouse ES cells which are maintained in regular medium containing leukemia inhibitory factor (LIF) and bone morphogenetic protein (BMP). Yet, the upstream signaling for ERK activation and their roles in mouse ES cells, other than promoting or priming differentiation, have not been determined. Here we found that mouse ES cells express three forms of Raf kinases, A-Raf, B-Raf, and C-Raf. Knocking-down each single Raf member failed to affect the sustained ERK activity, neither did A-Raf and B-Raf double knockdown or B-Raf and C-Raf double knockdown change it in ES cells. Interestingly, B-Raf and C-Raf double knockdown, not A-Raf and B-Raf knockdown, inhibited the maximal ERK activation induced by LIF, concomitant with the slower growth of ES cells. On the other hand, A-Raf, B-Raf, and C-Raf triple knockdown markedly inhibited both the maximal and sustained ERK activity in ES cells. Moreover, Raf triple knockdown, similar to the treatment of U-0126, an MEK inhibitor, significantly inhibited the survival and proliferation of ES cells, thereby compromising the colony propagation of mouse ES cells. In summary, our data demonstrate that all three Raf members are required for ERK activation in mouse ES cells and are involved in growth and survival of mouse ES cells. - Highlights: ●Mouse ES (mES) cells express all three Raf members, A-Raf, B-Raf, and C-Raf. ●Leukemia inhibitory factor (LIF) temporally activates ERKs in mES cells. ●B-Raf and C-Raf are required for LIF-induced maximal ERKs activity in mES cells. ●All Raf members are

  14. ICOS is required for the generation of both central and effector CD4+ memory T‐cell populations following acute bacterial infection

    Science.gov (United States)

    Marriott, Clare L.; Carlesso, Gianluca; Herbst, Ronald

    2015-01-01

    Interactions between ICOS and ICOS ligand (ICOSL) are essential for the development of T follicular helper (Tfh) cells and thus the formation and maintenance of GC reactions. Given the conflicting reports on the requirement of other CD4+ T‐cell populations for ICOS signals, we have employed a range of in vivo approaches to dissect requirements for ICOS signals in mice during an endogenous CD4+ T‐cell response and contrasted this with CD28 signals. Genetic absence of ICOSL only modestly reduced the total number of antigen‐specific CD4+ T cells at the peak of the primary response, but resulted in a severely diminished number of both T central memory and T effector memory cells. Treatment with blocking anti‐ICOS mAb during the primary response recapitulated these effects and caused a more substantial reduction than blocking CD28 signals with CTLA4Ig. During the memory phase of the response further signals through ICOS or CD28 were not required for survival. However, upon secondary challenge only Tfh cell expansion remained heavily ICOS‐dependent, while CD28 signals were required for optimal expansion of all subsets. These data demonstrate the importance of ICOS signals specifically for memory CD4+ T‐cell formation, while highlighting the potential of therapeutically targeting this pathway. PMID:25754933

  15. Glycoprotein H of herpes simplex virus type 1 requires glycoprotein L for transport to the surfaces of insect cells

    NARCIS (Netherlands)

    Westra, DF; Glazenburg, KL; Harmsen, MC; Tiran, A; Scheffer, AJ; Welling, GW; The, TH; WellingWester, S

    1997-01-01

    In mammalian cells, formation of heterooligomers consisting of the glycoproteins H and L (gH and gL) of herpes simplex virus type 1 is essential for the cell-to-cell spread of virions and for the penetration of virions into cells. We examined whether formation of gH1/gL1 heterooligomers and cell sur

  16. Mannose Receptor Is Required for Optimal Induction of Vaccine-Induced T-Helper Type 17 Cells and Resistance to Blastomyces dermatitidis Infection.

    Science.gov (United States)

    Wang, Huafeng; LeBert, Vanessa; Li, Mengyi; Lerksuthirat, Tassanee; Galles, Kevin; Klein, Bruce; Wüthrich, Marcel

    2016-06-01

    We investigated how innate sensing by the mannose receptor (MR) influences the development of antifungal immunity. We demonstrate that MR senses mannan on the surface of attenuated Blastomyces dermatitidis vaccine yeast and that MR(-/-) mice demonstrate impaired vaccine immunity against lethal experimental blastomycosis, compared with wild-type control mice. Using naive Blastomyces-specific transgenic CD4(+) T cells, we found that MR regulates differentiation of naive T cells into T-helper type 17 (Th17) effector cells, which are essential in vaccine immunity against systemic dimorphic fungi. Thus, MR regulates differentiation of Th17 cells and is required to induce vaccine immunity against lethal pulmonary blastomycosis. PMID:26931447

  17. Micro-RNA-155-mediated control of heme oxygenase 1 (HO-1) is required for restoring adaptively tolerant CD4+ T-cell function in rodents.

    OpenAIRE

    Zhang, Jinyu; Vandevenne, Patricia; Hamdi, Haifa; Van Puyvelde, Merry; Zucchi, Alessandro; Bettonville, Marie; Weatherly, Kathleen; Braun, Michel Y

    2015-01-01

    T cells chronically stimulated by a persistent antigen often become dysfunctional and lose effector functions and proliferative capacity. To identify the importance of micro-RNA-155 (miR-155) in this phenomenon, we analyzed mouse miR-155-deficient CD4(+) T cells in a model where the chronic exposure to a systemic antigen led to T-cell functional unresponsiveness. We found that miR-155 was required for restoring function of T cells after programmed death receptor 1 blockade. Heme oxygenase 1 (...

  18. Rac1 is required for Prkar1a-mediated Nf2 suppression in Schwann cell tumors.

    Science.gov (United States)

    Manchanda, P K; Jones, G N; Lee, A A; Pringle, D R; Zhang, M; Yu, L; La Perle, K M D; Kirschner, L S

    2013-07-25

    Schwannomas are peripheral nerve sheath tumors that often occur in the setting of an inherited tumor predisposition syndrome, including neurofibromatosis types 1 (NF1) and 2 (NF2), familial schwannomatosis and Carney complex. Loss of the NF2 tumor suppressor (encoding NF2, or Merlin) is associated with upregulation of the Rac1 small GTPase, which is thought to have a key role in mediating tumor formation. In prior studies, we generated a mouse model of schwannomas by performing tissue-specific knockout (KO) of the Carney complex gene Prkar1a, which encodes the type 1A regulatory subunit of protein kinase A. These tumors exhibited down-regulation of Nf2 protein and an increase in activated Rac1. To assess the requirement for Rac1 in schwannoma formation, we generated a double KO (DKO) of Prkar1a and Rac1 in Schwann cells and monitored tumor formation. Loss of Rac1 reduced tumor formation by reducing proliferation and enhancing apoptosis. Surprisingly, the reduction of tumor formation was accompanied by re-expression of the Nf2 protein. Furthermore, activated Rac1 was able to downregulate Nf2 in vitro in a Pak-dependent manner. These in vivo data indicate that activation of Rac1 is responsible for suppression of Nf2 protein production; deficiency of Nf2 in Schwann cells leads to loss of cellular growth control and tumor formation. Further, PKA activation through mutation in Prkar1a is sufficient to initiate Rac1 signaling, with subsequent reduction of Nf2 and schwannomagenesis. Although in vitro evidence has shown that loss of Nf2 activates Rac1, our data indicate that signaling between Nf2 and Rac1 occurs in a bidirectional fashion, and these interactions are modulated by PKA. PMID:23045281

  19. Actin-associated protein palladin is required for migration behavior and differentiation potential of C2C12 myoblast cells

    International Nuclear Information System (INIS)

    Highlights: • Palladin is involved in myogenesis in vitro. • Palladin knockdown by siRNA increases myoblast proliferation, viability and differentiation. • Palladin knockdown decreases C2C12 myoblast migration ability. - Abstract: The actin-associated protein palladin has been shown to be involved in differentiation processes in non-muscle tissues. However, but its function in skeletal muscle has rarely been studied. Palladin plays important roles in the regulation of diverse actin-related signaling in a number of cell types. Since intact actin-cytoskeletal remodeling is necessary for myogenesis, in the present study, we pursue to investigate the role of actin-associated palladin in skeletal muscle differentiation. Palladin in C2C12 myoblasts is knocked-down using specific small interfering RNA (siRNA). The results show that down-regulation of palladin decreased migratory activity of mouse skeletal muscle C2C12 myoblasts. Furthermore, the depletion of palladin enhances C2C12 vitality and proliferation. Of note, the loss of palladin promotes C2C12 to express the myosin heavy chain, suggesting that palladin has a role in the modulation of C2C12 differentiation. It is thus proposed that palladin is required for normal C2C12 myogenesis in vitro

  20. CRISPR reveals a distal super-enhancer required for Sox2 expression in mouse embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Yan Li

    Full Text Available The pluripotency of embryonic stem cells (ESCs is maintained by a small group of master transcription factors including Oct4, Sox2 and Nanog. These core factors form a regulatory circuit controlling the transcription of a number of pluripotency factors including themselves. Although previous studies have identified transcriptional regulators of this core network, the cis-regulatory DNA sequences required for the transcription of these key pluripotency factors remain to be defined. We analyzed epigenomic data within the 1.5 Mb gene-desert regions around the Sox2 gene and identified a 13kb-long super-enhancer (SE located 100kb downstream of Sox2 in mouse ESCs. This SE is occupied by Oct4, Sox2, Nanog, and the mediator complex, and physically interacts with the Sox2 locus via DNA looping. Using a simple and highly efficient double-CRISPR genome editing strategy we deleted the entire 13-kb SE and characterized transcriptional defects in the resulting monoallelic and biallelic deletion clones with RNA-seq. We showed that the SE is responsible for over 90% of Sox2 expression, and Sox2 is the only target gene along the chromosome. Our results support the functional significance of a SE in maintaining the pluripotency transcription program in mouse ESCs.

  1. Actin-associated protein palladin is required for migration behavior and differentiation potential of C2C12 myoblast cells

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Ngoc Uyen Nhi; Liang, Vincent Roderick; Wang, Hao-Ven, E-mail: hvwang@mail.ncku.edu.tw

    2014-09-26

    Highlights: • Palladin is involved in myogenesis in vitro. • Palladin knockdown by siRNA increases myoblast proliferation, viability and differentiation. • Palladin knockdown decreases C2C12 myoblast migration ability. - Abstract: The actin-associated protein palladin has been shown to be involved in differentiation processes in non-muscle tissues. However, but its function in skeletal muscle has rarely been studied. Palladin plays important roles in the regulation of diverse actin-related signaling in a number of cell types. Since intact actin-cytoskeletal remodeling is necessary for myogenesis, in the present study, we pursue to investigate the role of actin-associated palladin in skeletal muscle differentiation. Palladin in C2C12 myoblasts is knocked-down using specific small interfering RNA (siRNA). The results show that down-regulation of palladin decreased migratory activity of mouse skeletal muscle C2C12 myoblasts. Furthermore, the depletion of palladin enhances C2C12 vitality and proliferation. Of note, the loss of palladin promotes C2C12 to express the myosin heavy chain, suggesting that palladin has a role in the modulation of C2C12 differentiation. It is thus proposed that palladin is required for normal C2C12 myogenesis in vitro.

  2. Outcome of Recipients of Hematopoietic Stem Cell Transplants Who Require Intensive Care Unit Support: A Single Institution Experience.

    Science.gov (United States)

    Galindo-Becerra, Samantha; Labastida-Mercado, Nancy; Rosales-Padrón, Jaime; García-Chavez, Jessica; Soto-Vega, Elena; Rivadeneyra-Espinoza, Liliana; León-Peña, Andres A; Fernández-Lara, Danitza; Dominguez-Cid, Monica; Anthon-Méndez, Javier; Arizpe-Bravo, Daniel; Ruiz-Delgado, Guillermo J; Ruiz-Argüelles, Guillermo J

    2015-01-01

    Admission to the intensive care unit (ICU) of a patient who has been grafted with hematopoietic stem cells is a serious event, but the role of the ICU in this setting remains controversial. Data were analyzed from patients who underwent autologous or allogeneic bone marrow transplantation at the Centro de Hematología y Medicina Interna de Puebla, México, between May 1993 and October 2014. In total, 339 patients were grafted: 150 autografts and 189 allografts; 68 of the grafted patients (20%) were admitted to the ICU after transplantation: 27% of the allografted and 11% of the autografted patients (p = 0.2). Two of 17 autografted patients (12%) and 5 of 51 allografted patients (10%) survived. All patients who required insertion of an endotracheal tube died, whereas 7 of 11 patients without invasive mechanical ventilation survived (p = 0.001). Only 10% of the grafted patients survived their stay in the ICU; this figure is lower than those reported from other centers and may reflect several facts, varying from the quality of the ICU support to ICU admission criteria to the initial management of all the grafts in an outpatient setting, which could somehow delay the arrival of patients to the hospital.

  3. Thyroid Hormone-Otx2 Signaling Is Required for Embryonic Ventral Midbrain Neural Stem Cells Differentiated into Dopamine Neurons.

    Science.gov (United States)

    Chen, Chunhai; Ma, Qinglong; Chen, Xiaowei; Zhong, Min; Deng, Ping; Zhu, Gang; Zhang, Yanwen; Zhang, Lei; Yang, Zhiqi; Zhang, Kuan; Guo, Lu; Wang, Liting; Yu, Zhengping; Zhou, Zhou

    2015-08-01

    Midbrain dopamine (DA) neurons are essential for maintaining multiple brain functions. These neurons have also been implicated in relation with diverse neurological disorders. However, how these neurons are developed from neuronal stem cells (NSCs) remains largely unknown. In this study, we provide both in vivo and in vitro evidence that the thyroid hormone, an important physiological factor for brain development, promotes DA neuron differentiation from embryonic ventral midbrain (VM) NSCs. We find that thyroid hormone deficiency during development reduces the midbrain DA neuron number, downregulates the expression of tyrosine hydroxylase (TH) and the dopamine transporter (DAT), and impairs the DA neuron-dependent motor behavior. In addition, thyroid hormone treatment during VM NSC differentiation in vitro increases the production of DA neurons and upregulates the expression of TH and DAT. We also found that the thyroid hormone enhances the expression of Otx2, an important determinant of DA neurogenesis, during DA neuron differentiation. Our in vitro gene silencing experiments indicate that Otx2 is required for thyroid hormone-dependent DA neuron differentiation from embryonic VM NSCs. Finally, we revealed both in vivo and in vitro that the thyroid hormone receptor alpha 1 is expressed in embryonic VM NSCs. Furthermore, it participates in the effects of thyroid hormone-induced Otx2 upregulation and DA neuron differentiation. These data demonstrate the role and molecular mechanisms of how the thyroid hormone regulates DA neuron differentiation from embryonic VM NSCs, particularly providing new mechanisms and a potential strategy for generating dopamine neurons from NSCs.

  4. Retinoic acid signalling is required for the efficient differentiation of CD4+ T cells into pathogenic effector cells during the development of intestinal inflammation

    DEFF Research Database (Denmark)

    Rivollier, Aymeric Marie Christian; Pool, Lieneke; Frising, Ulrika;

    compromised. In vitro studies confirm the inefficacy of RA signalling-deficient T cells to generate bona fide Th1 cells and demonstrate their aberrant increased RORγt expression while their differentiation into Th17 remains unaffected. Surprisingly, RA signalling-deficient CD45RBlo regulatory T cells (Tregs...... cells, while it is dispensable for the protective function of Treg cells. We are currently deciphering the mechanisms of these effects of RA on CD4+ T cells....

  5. A late requirement for Wnt and FGF signalling during activin-induced formation of foregut endoderm from mouse embryonic stem cells

    DEFF Research Database (Denmark)

    Hansson, Mattias; Petersen, Dorthe Rønn; Peterslund, Janny M.L.;

    2009-01-01

    requires FGF signaling in adherent but not aggregate culture. Lastly, we demonstrate that activin-induced definitive endoderm derived from mouse ES cells can incorporate into the developing foregut endoderm in vivo and adopt a mostly anterior foregut character after further culture in vitro.......Here we examine how BMP, Wnt, and FGF signaling modulate activin-induced mesendodermal differentiation of mouse ES cells grown under defined conditions in adherent monoculture. We monitor ES cells containing reporter genes for markers of primitive streak (PS) and its progeny and extend previous...... findings on the ability of increasing concentrations of activin to progressively induce more ES cell progeny to anterior PS and endodermal fates. We find that the number of Sox17- and Gsc-expressing cells increases with increasing activin concentration while the highest number of T-expressing cells...

  6. Evidence that the Dictyostelium Dd-STATa protein is a repressor that regulates commitment to stalk cell differentiation and is also required for efficient chemotaxis.

    Science.gov (United States)

    Mohanty, S; Jermyn, K A; Early, A; Kawata, T; Aubry, L; Ceccarelli, A; Schaap, P; Williams, J G; Firtel, R A

    1999-08-01

    developmentally arrested for several days before forming very small spore masses supported by a column of apparently undifferentiated cells. Thus, complete stalk cell differentiation appears to require at least two events: a commitment step, whereby the repression exerted by Dd-STATa is lifted, and a second step that is blocked in a Dd-STATa null organism. This latter step may involve extracellular cAMP, a known repressor of stalk cell differentiation, because Dd-STATa null cells are abnormally sensitive to the inhibitory effects of extracellular cyclic AMP.

  7. Nkx6.1 controls a gene regulatory network required for establishing and maintaining pancreatic Beta cell identity.

    Directory of Open Access Journals (Sweden)

    Ashleigh E Schaffer

    Full Text Available All pancreatic endocrine cell types arise from a common endocrine precursor cell population, yet the molecular mechanisms that establish and maintain the unique gene expression programs of each endocrine cell lineage have remained largely elusive. Such knowledge would improve our ability to correctly program or reprogram cells to adopt specific endocrine fates. Here, we show that the transcription factor Nkx6.1 is both necessary and sufficient to specify insulin-producing beta cells. Heritable expression of Nkx6.1 in endocrine precursors of mice is sufficient to respecify non-beta endocrine precursors towards the beta cell lineage, while endocrine precursor- or beta cell-specific inactivation of Nkx6.1 converts beta cells to alternative endocrine lineages. Remaining insulin(+ cells in conditional Nkx6.1 mutants fail to express the beta cell transcription factors Pdx1 and MafA and ectopically express genes found in non-beta endocrine cells. By showing that Nkx6.1 binds to and represses the alpha cell determinant Arx, we identify Arx as a direct target of Nkx6.1. Moreover, we demonstrate that Nkx6.1 and the Arx activator Isl1 regulate Arx transcription antagonistically, thus establishing competition between Isl1 and Nkx6.1 as a critical mechanism for determining alpha versus beta cell identity. Our findings establish Nkx6.1 as a beta cell programming factor and demonstrate that repression of alternative lineage programs is a fundamental principle by which beta cells are specified and maintained. Given the lack of Nkx6.1 expression and aberrant activation of non-beta endocrine hormones in human embryonic stem cell (hESC-derived insulin(+ cells, our study has significant implications for developing cell replacement therapies.

  8. IL-15 cis Presentation Is Required for Optimal NK Cell Activation in Lipopolysaccharide-Mediated Inflammatory Conditions

    Directory of Open Access Journals (Sweden)

    Ivan Zanoni

    2013-09-01

    Full Text Available Natural killer (NK cells have antitumor, antiviral, and antibacterial functions, and efforts are being made to manipulate them in immunotherapeutic approaches. However, their activation mechanisms remain poorly defined, particularly during bacterial infections. Here, we show that upon lipopolysaccharide or E. coli exposure, dendritic cells (DCs produce three cytokines—interleukin 2 (IL-2, IL-18, and interferon β (IFN-β—necessary and sufficient for NK cell activation. IFN-β enhances NK cell activation by inducing IL-15 and IL-15 receptor α not only in DCs but, surprisingly, also in NK cells. This process allows the transfer of IL-15 on NK cell surface and its cis presentation. cis-presented NK cell-derived and trans-presented DC-derived IL-15 contribute equally to optimal NK cell activation.

  9. Requirement of 8-mercaptoguanosine as a costimulus for IL-4-dependent μ to γ1 class switch recombination in CD38-activated B cells

    International Nuclear Information System (INIS)

    Mature B-2 cells expressing surface IgM and IgD proliferate upon stimulation by CD38, CD40 or lipopolysaccharide (LPS) and differentiate into IgG1-producing plasma cells in the presence of cytokines. The process of class switch recombination (CSR) from IgM to other isotypes is highly regulated by cytokines and activation-induced cytidine deaminase (AID). Blimp-1 and XBP-1 play an essential role in the terminal differentiation of switched B-2 cells to Ig-producing plasma cells. IL-5 induces AID and Blimp-1 expression in CD38- and CD40-activated B-2 cells, leading to μ to γ1 CSR at DNA level and IgG1 production. IL-4, a well-known IgG1-inducing factor, does not induce μ to γ1 CSR in CD38-activated B-2 cells or Blimp-1, while IL-4 induces μ to γ1 CSR, XBP-1 expression, and IgG1 production expression in CD40-activated B-2 cells. Interestingly, the addition of 8-mercaptoguanosine (8-SGuo) with IL-4 to the culture of CD38-activated B cells can induce μ to γ1 CSR, Blimp-1 expression, and IgG1 production. Intriguingly, 8-SGuo by itself induces AID expression in CD38-activated B cells. However, it does not induce μ to γ1 CSR. These results imply that the mode of B-cell activation for extracellular stimulation affects the outcome of cytokine stimulation with respect to the efficiency and direction of CSR, and the requirements of the transcriptional regulator and the generation of antibody-secreting cells. Furthermore, our data suggest the requirement of additional molecules in addition to AID for CSR

  10. Differential requirements of MyD88 and TRIF pathways in TLR4-mediated immune responses in murine B cells.

    Science.gov (United States)

    Yanagibashi, Tsutomu; Nagai, Yoshinori; Watanabe, Yasuharu; Ikutani, Masashi; Hirai, Yoshikatsu; Takatsu, Kiyoshi

    2015-01-01

    LPS stimulates the TLR4/Myeloid differentiation protein-2 (MD-2) complex and promotes a variety of immune responses in B cells. TLR4 has two main signaling pathways, MyD88 and Toll/IL-1R (TIR)-domain-containing adaptor-inducing interferon-β (TRIF) pathways, but relatively few studies have examined these pathways in B cells. In this study, we investigated MyD88- or TRIF-dependent LPS responses in B cells by utilizing their knockout mice. Compared with wild-type (WT) B cells, MyD88(-/-) B cells were markedly impaired in up-regulation of CD86 and proliferation induced by lipid A moiety of LPS. TRIF(-/-) B cells were also impaired in these responses compared with WT B cells, but showed better responses than MyD88(-/-) B cells. Regarding class switch recombination (CSR) elicited by lipid A plus IL-4, MyD88(-/-) B cells showed similar patterns of CSR to WT B cells. However, TRIF(-/-) B cells showed the impaired in the CSR. Compared with WT and MyD88(-/-) B cells, TRIF(-/-) B cells exhibited reduced cell division, fewer IgG1(+) cells per division, and decreased activation-induced cytidine deaminase (Aicda) mRNA expression in response to lipid A plus IL-4. Finally, IgG1 production to trinitrophenyl (TNP)-LPS immunization was impaired in TRIF(-/-) mice, while MyD88(-/-) mice exhibited increased IgG1 production. Thus, MyD88 and TRIF pathways differently regulate TLR4-induced immune responses in B cells. PMID:25448706

  11. Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

    DEFF Research Database (Denmark)

    Jørgensen, Niklas Rye; Teilmann, Stefan Cuoni; Henriksen, Zanne;

    2003-01-01

    in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal...

  12. Atypical Cadherin Fat1 Is Required for Lens Epithelial Cell Polarity and Proliferation but Not for Fiber Differentiation

    OpenAIRE

    Sugiyama, Yuki; Shelley, Elizabeth J.; Badouel, Caroline; McNeill, Helen; McAvoy, John W.

    2015-01-01

    Using knockout mice, we show that atypical cadherin Fat1 is essential for lens epithelial cells to maintain cell junctions, polarity, and proliferation but not for terminal fiber cell differentiation. We also report that Fat4 does not exhibit functional redundancy with Fat1 during lens development.

  13. Mitogen-activated protein kinase kinase activity is required for the G2/M transition of the cell cycle in mammalian fibroblasts

    OpenAIRE

    Wright, Jocelyn H.; Munar, Erlynda; Jameson, Damon R; Andreassen, Paul R.; Margolis, Robert L.; Seger, Rony; Krebs, Edwin G.

    1999-01-01

    The mitogen-activated protein kinase (MAPK) cascade is required for mitogenesis in somatic mammalian cells and is activated by a wide variety of oncogenic stimuli. Specific roles for this signaling module in growth were dissected by inhibiting MAPK kinase 1 (MAPKK1) activity in highly synchronized NIH 3T3 cells. In addition to the known role of this kinase in cell-cycle entry from G0, the level of MAPKK activity was observed to affect the kinetics of progression through both the G1 and G2 pha...

  14. The Polycomb group protein RING1B is overexpressed in ductal breast carcinoma and is required to sustain FAK steady state levels in breast cancer epithelial cells

    Science.gov (United States)

    Bosch, Almudena; Panoutsopoulou, Konstantina; Corominas, Josep Maria; Gimeno, Ramón; Moreno-Bueno, Gema; Martín-Caballero, Juan; Morales, Saleta; Lobato, Tania; Martínez-Romero, Carles; Farias, Eduardo F.; Mayol, Xavier; Cano, Amparo; Hernández-Muáoz, Inmaculada

    2014-01-01

    In early stages of metastasis malignant cells must acquire phenotypic changes to enhance their migratory behavior and their ability to breach the matrix surrounding tumors and blood vessel walls. Epigenetic regulation of gene expression allows the acquisition of these features that, once tumoral cells have escape from the primary tumor, can be reverted. Here we report that the expression of the Polycomb epigenetic repressor Ring1B is enhanced in tumoral cells that invade the stroma in human ductal breast carcinoma and its expression is coincident with that of Fak in these tumors. Ring1B knockdown in breast cancer cell lines revealed that Ring1B is required to sustain Fak expression in basal conditions as well as in Tgfβ-treated cells. Functionally, endogenous Ring1B is required for cell migration and invasion in vitro and for in vivo invasion of the mammary fat pad by tumoral cells. Finally we identify p63 as a target of Ring1B to regulate Fak expression: Ring1B depletion results in enhanced p63 expression, which in turns represses Fak expression. Importantly, Fak downregulation upon Ring1B depletion is dependent on p63 expression. Our findings provide new insights in the biology of the breast carcinoma and open new avenues for breast cancer prognosis and therapy. PMID:24742605

  15. The calcium-sensing receptor-dependent regulation of cell-cell adhesion and keratinocyte differentiation requires Rho and Filamin A

    OpenAIRE

    Tu, Chia-Ling; Chang, Wenhan; Bikle, Daniel D.

    2011-01-01

    Extracellular Ca2+ (Ca2+o) acting through the calcium-sensing receptor (CaR) induces E-cadherin mediated cell-cell adhesion and cellular signals mediating cell differentiation in epidermal keratinocytes. Previous studies indicate that the CaR regulates cell-cell adhesion through the Fyn/Src tyrosine kinases. Here we investigate whether Rho GTPase is a part of the CaR-mediated signaling cascade regulating cell adhesion and differentiation. Suppressing endogenous Rho A expression by small inter...

  16. T cell receptor stimulation-induced epigenetic changes and Foxp3 expression are independent and complementary events required for Treg cell development.

    OpenAIRE

    Ohkura, Naganari; Hamaguchi, Masahide; Morikawa, Hiromasa; Sugimura, Kyoko; Tanaka, Atsushi; Ito, Yoshinaga; Osaki, Motonao; Tanaka, Yoshiaki; Yamashita, Riu; Nakano, Naoko; Huehn, Jochen; Fehling, Hans Joerg; Sparwasser, Tim; Nakai, Kenta; Sakaguchi, Shimon

    2012-01-01

    The transcription factor Foxp3 is essential for the development of regulatory T (Treg) cells, yet its expression is insufficient for establishing the Treg cell lineage. Here we showed that Treg cell development was achieved by the combination of two independent processes, i.e., the expression of Foxp3 and the establishment of Treg cell-specific CpG hypomethylation pattern. Both events were induced by T cell receptor stimulation. The Treg cell-type CpG hypomethylation began in the thymus and c...

  17. IL-34 is a tissue-restricted ligand of CSF1R required for the development of Langerhans cells and microglia.

    Science.gov (United States)

    Wang, Yaming; Szretter, Kristy J; Vermi, William; Gilfillan, Susan; Rossini, Cristina; Cella, Marina; Barrow, Alexander D; Diamond, Michael S; Colonna, Marco

    2012-08-01

    The differentiation of bone marrow-derived progenitor cells into monocytes, tissue macrophages and some dendritic cell (DC) subtypes requires the growth factor CSF1 and its receptor, CSF1R. Langerhans cells (LCs) and microglia develop from embryonic myeloid precursor cells that populate the epidermis and central nervous system (CNS) before birth. Notably, LCs and microglia are present in CSF1-deficient mice but absent from CSF1R-deficient mice. Here we investigated whether an alternative CSF1R ligand, interleukin 34 (IL-34), is responsible for this discrepancy. Through the use of IL-34-deficient (Il34(LacZ/LacZ)) reporter mice, we found that keratinocytes and neurons were the main sources of IL-34. Il34(LacZ/LacZ) mice selectively lacked LCs and microglia and responded poorly to skin antigens and viral infection of the CNS. Thus, IL-34 specifically directs the differentiation of myeloid cells in the skin epidermis and CNS.

  18. Agonist-Driven Development of CD4+CD25+Foxp3+Regulatory T Cells Requires a Second Signal Mediated by Stat6

    DEFF Research Database (Denmark)

    Sanchez-Guajardo, Vanesa Maria; Tanshot, C.; O'Malley, J.T.;

    2007-01-01

    The factors that induce Foxp3 expression and regulatory T (Treg) cell development remain unknown. In this study, we investigated the role of STAT4 and STAT6 in agonist-driven generation of Ag-specific Foxp3-expressing Treg cells. Our findings indicate that fully efficient induction of Foxp3...... expression and development of Ag-specific Treg cells requires the synergistic action of two signals: a TCR-mediated signal and a second signal mediated by STAT6. Indeed, by comparing the development of wild-type and STAT4- and STAT6-deficient hemagglutinin-specific T cells in the presence of hemagglutinin Ag...... a role for the STAT6 pathway in Treg cell development and maintenance....

  19. Kinesin 5B (KIF5B is required for progression through female meiosis and proper chromosomal segregation in mitotic cells.

    Directory of Open Access Journals (Sweden)

    Dawit Kidane

    Full Text Available The fidelity of chromosomal segregation during cell division is important to maintain chromosomal stability in order to prevent cancer and birth defects. Although several spindle-associated molecular motors have been shown to be essential for cell division, only a few chromosome arm-associated motors have been described. Here, we investigated the role of Kinesin 5b (Kif5b during female mouse meiotic cell development and mitotic cell division. RNA interference (RNAi-mediated silencing of Kif5b in mouse oocytes induced significant delay in germinal vesicle breakdown (GVBD and failure in extrusion of the first polar body (PBE. In mitotic cells, knockdown of Kif5b leads to centrosome amplification and a chromosomal segregation defect. These data suggest that KIF5B is critical in suppressing chromosomal instability at the early stages of female meiotic cell development and mitotic cell division.

  20. Kinesin 5B (KIF5B) is required for progression through female meiosis and proper chromosomal segregation in mitotic cells.

    Science.gov (United States)

    Kidane, Dawit; Sakkas, Denny; Nottoli, Timothy; McGrath, James; Sweasy, Joann B

    2013-01-01

    The fidelity of chromosomal segregation during cell division is important to maintain chromosomal stability in order to prevent cancer and birth defects. Although several spindle-associated molecular motors have been shown to be essential for cell division, only a few chromosome arm-associated motors have been described. Here, we investigated the role of Kinesin 5b (Kif5b) during female mouse meiotic cell development and mitotic cell division. RNA interference (RNAi)-mediated silencing of Kif5b in mouse oocytes induced significant delay in germinal vesicle breakdown (GVBD) and failure in extrusion of the first polar body (PBE). In mitotic cells, knockdown of Kif5b leads to centrosome amplification and a chromosomal segregation defect. These data suggest that KIF5B is critical in suppressing chromosomal instability at the early stages of female meiotic cell development and mitotic cell division.

  1. Mouse hepatic oval cells require Met-dependent PI3K to impair TGF-β-induced oxidative stress and apoptosis.

    Directory of Open Access Journals (Sweden)

    Adoración Martínez-Palacián

    Full Text Available We have previously shown that oval cells harboring a genetically inactivated Met tyrosine kinase (Met(-/- oval cells are more sensitive to TGF-β-induced apoptosis than cells expressing a functional Met (Met(flx/flx, demonstrating that the HGF/Met axis plays a pivotal role in oval cell survival. Here, we have examined the mechanism behind this effect and have found that TGF-β induced a mitochondria-dependent apoptotic cell death in Met(flx/flx and Met(-/- oval cells, associated with a marked increase in levels of the BH3-only proteins Bim and Bmf. Bmf plays a key role during TGF-β-mediated apoptosis since knocking down of BMF significantly diminished the apoptotic response in Met(-/- oval cells. TGF-β also induced oxidative stress accompanied by NADPH oxidase 4 (Nox4 mRNA up-regulation and decreased protein levels of antioxidant enzymes. Antioxidants inhibit both TGF-β-induced caspase 3 activity and Bmf up-regulation, revealing an oxidative stress-dependent Bmf regulation by TGF-β. Notably, oxidative stress-related events were strongly amplified in Met(-/- oval cells, emphasizing the critical role of Met in promoting survival. Pharmacological inhibition of PI3K did impair HGF-driven protection from TGF-β-induced apoptosis and increased sensitivity of Met(flx/flx oval cells to TGF-ß by enhancing oxidative stress, reaching apoptotic indices similar to those obtained in Met(-/- oval cells. Interestingly, both PI3K inhibition and/or knockdown itself resulted in caspase-3 activation and loss of viability in Met(flx/flx oval cells, whereas no effect was observed in Met(-/- oval cells. Altogether, results presented here provide solid evidences that both paracrine and autocrine HGF/Met signaling requires PI3K to promote mouse hepatic oval cell survival against TGF-β-induced oxidative stress and apoptosis.

  2. Persistence of autoreactive T cell drive is required to elicit anti-chromatin antibodies in a murine model of drug-induced lupus.

    Science.gov (United States)

    Kretz-Rommel, A; Rubin, R L

    1999-01-15

    Long-term treatment with procainamide and numerous other medications is occasionally associated with the development of drug-induced lupus. We recently established a murine model for this syndrome by disrupting central T cell tolerance. Two intrathymic injections of procainamide-hydroxylamine (PAHA), a reactive metabolite of procainamide, into (C57BL/6 x DBA/2)F1 mice resulted in the appearance of chromatin-reactive T cells and anti-chromatin autoantibodies. The current study explores in this model the role of autoreactive T cells in autoantibody production and examines why autoantibodies after a single intrathymic drug injection were much more limited in isotype and specificity. Injection of as few as 5000 chromatin-reactive T cells into naive, syngeneic mice induced a rapid IgM anti-denatured DNA response, while injection of at least 100-fold greater number of activated T cells was required for induction of IgG anti-chromatin Abs, suggesting that small numbers of autoreactive T cells can be homeostatically controlled. Mice subjected to a single intrathymic PAHA injection after receiving splenic B cells from an intrathymic PAHA-injected syngeneic donor also developed anti-chromatin Abs, but adoptive transfer of similarly primed T cells or of B cells without intrathymic PAHA injection of the recipient failed to produce an anti-chromatin response. However, anti-chromatin Abs developed after a single intrathymic PAHA injection in Fas-deficient C57BL/6-lpr/lpr mice, suggesting that activation-induced cell death limited autoimmunity in normal mice. Taken together, these results imply that chromatin-reactive T cells produced by intrathymic PAHA created a B cell population primed to somatically mutate and Ig class switch when subjected to a heavy load or second wave of autoreactive T cells.

  3. Integrin α PAT-2/CDC-42 signaling is required for muscle-mediated clearance of apoptotic cells in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Hsiao-Han Hsieh

    Full Text Available Clearance of apoptotic cells by engulfment plays an important role in the homeostasis and development of multicellular organisms. Despite the fact that the recognition of apoptotic cells by engulfment receptors is critical in inducing the engulfment process, the molecular mechanisms are still poorly understood. Here, we characterize a novel cell corpse engulfment pathway mediated by the integrin α subunit PAT-2 in Caenorhabditis elegans and show that it specifically functions in muscle-mediated engulfment during embryogenesis. Inactivation of pat-2 results in a defect in apoptotic cell internalization. The PAT-2 extracellular region binds to the surface of apoptotic cells in vivo, and the intracellular region may mediate signaling for engulfment. We identify essential roles of small GTPase CDC-42 and its activator UIG-1, a guanine-nucleotide exchange factor, in PAT-2-mediated cell corpse removal. PAT-2 and CDC-42 both function in muscle cells for apoptotic cell removal and are co-localized in growing muscle pseudopods around apoptotic cells. Our data suggest that PAT-2 functions through UIG-1 for CDC-42 activation, which in turn leads to cytoskeletal rearrangement and apoptotic cell internalization by muscle cells. Moreover, in contrast to PAT-2, the other integrin α subunit INA-1 and the engulfment receptor CED-1, which signal through the conserved signaling molecules CED-5 (DOCK180/CED-12 (ELMO or CED-6 (GULP respectively, preferentially act in epithelial cells to mediate cell corpse removal during mid-embryogenesis. Our results show that different engulfing cells utilize distinct repertoires of receptors for engulfment at the whole organism level.

  4. Lethal(2)giant larvae is required in the follicle cells for formation of the initial AP asymmetry and the oocyte polarity during Drosophila oogenesis

    Institute of Scientific and Technical Information of China (English)

    Qi Li; Tianchi Xin; Wenlian Chen; Mingwei Zhu; Mingfa Li

    2008-01-01

    The intricately regulated differentiation of the somatic follicle cell lineages into distinct subpopulations with specific functions plays an essential role in Drosophila egg development. At early oogenesis, induction of the stalk cells generates the first anteroposterior (AP) asymmetry in the egg chamber by inducing the posterior localization of the oocyte. Later, the properly specified posterior follicle cells signal to polarize the oocyte along the AP and dorsoventral (DV) axes at mid-oogenesis. Here, we show that lethal(2)giant larvae (Igt), a Drosophila tumor suppressor gene, is required in the follicle cells for the differentiation of both stalk cells and posterior follicle cells. Loss-of-function mutations in Igl cause oocyte mispositioning in the younger one of the fused chambers, due to lack of the stalk. Removal of Igl function from the posterior follicle cells using the FLP/FRT system results in loss of the oocyte polarity that is elicited by the failure of those posterior cells to differentiate normally. Thus, we provide the first demonstration that Igl is implicated in the formation of the initial AP asymmetry and the patterning of the AP and DV axes in the oocyte by acting in the specification of a subset of somatic follicle cells.

  5. Butyrate-induced proapoptotic and antiangiogenic pathways in EAT cells require activation of CAD and downregulation of VEGF

    International Nuclear Information System (INIS)

    Butyrate, a short-chain fatty acid produced in the colon, induces cell cycle arrest, differentiation, and apoptosis in transformed cell lines. In this report, we study the effects of butyrate (BuA) on the growth of Ehrlich ascites tumor (EAT) cells in vivo. BuA, when injected intraperitoneally (i.p) into mice, inhibited proliferation of EAT cells. Further, induction of apoptosis in EAT cells was monitored by nuclear condensation, annexin-V staining, DNA fragmentation, and translocation of caspase-activated DNase into nucleus upon BuA-treatment. Ac-DEVD-CHO, a caspase-3 inhibitor, completely inhibited BuA-induced apoptosis, indicating that activation of caspase-3 mediates the apoptotic pathway in EAT cells. The proapoptotic effect of BuA also reflects on the antiangiogenic pathway in EAT cells. The antiangiogenic effect of BuA in vivo was demonstrated by the downregulation of the secretion of VEGF in EAT cells. CD31 immunohistochemical staining of peritoneum sections clearly indicated a potential angioinhibitory effect of BuA in EAT cells. These results suggest that BuA, besides regulating other fundamental cellular processes, is able to modulate the expression/secretion of the key angiogenic growth factor VEGF in EAT cells

  6. Amoebic PI3K and PKC is required for Jurkat T cell death induced by Entamoeba histolytica.

    Science.gov (United States)

    Lee, Young Ah; Kim, Kyeong Ah; Min, Arim; Shin, Myeong Heon

    2014-08-01

    The enteric protozoan parasite Entamoeba histolytica is the causative agent of human amebiasis. During infection, adherence of E. histolytica through Gal/GalNAc lectin on the surface of the amoeba can induce caspase-3-dependent or -independent host cell death. Phosphorylinositol 3-kinase (PI3K) and protein kinase C (PKC) in E. histolytica play an important function in the adhesion, killing, or phagocytosis of target cells. In this study, we examined the role of amoebic PI3K and PKC in amoeba-induced apoptotic cell death in Jurkat T cells. When Jurkat T cells were incubated with E. histolytica trophozoites, phosphatidylserine (PS) externalization and DNA fragmentation in Jurkat cells were markedly increased compared to those of cells incubated with medium alone. However, when amoebae were pretreated with a PI3K inhibitor, wortmannin before being incubated with E. histolytica, E. histolytica-induced PS externalization and DNA fragmentation in Jurkat cells were significantly reduced compared to results for amoebae pretreated with DMSO. In addition, pretreatment of amoebae with a PKC inhibitor, staurosporine strongly inhibited Jurkat T cell death. However, E. histolytica-induced cleavage of caspase-3, -6, and -7 were not inhibited by pretreatment of amoebae with wortmannin or staurosporin. In addition, we found that amoebic PI3K and PKC have an important role on amoeba adhesion to host compartment. These results suggest that amebic PI3K and PKC activation may play an important role in caspase-independent cell death in Entamoeba-induced apoptosis.

  7. α-1,6-Mannosylation of N-Linked Oligosaccharide Present on Cell Wall Proteins Is Required for Their Incorporation into the Cell Wall in the Filamentous Fungus Neurospora crassa▿†

    OpenAIRE

    Maddi, Abhiram; Free, Stephen J.

    2010-01-01

    The enzyme α-1,6-mannosyltransferase (OCH-1) is required for the synthesis of galactomannans attached to the N-linked oligosaccharides of Neurospora crassa cell wall proteins. The Neurospora crassa och-1 mutant has a tight colonial phenotype and a defective cell wall. A carbohydrate analysis of the och-1 mutant cell wall revealed a 10-fold reduction in the levels of mannose and galactose and a total lack of 1,6-linked mannose residues. Analysis of the integral cell wall protein from wild-type...

  8. The rotamase Pin1 is up-regulated, hypophosphorylated and required for cell cycle progression in head and neck squamous cell carcinomas.

    Science.gov (United States)

    Wiegand, Susanne; Dakic, Branka; Rath, Ariane F E; Makarova, Galina; Sterz, Carolina; Meissner, Wolfgang; Bette, Michael; Adamkiewicz, Jürgen; Müller-Brüsselbach, Sabine; Müller, Rolf; Werner, Jochen A; Mandic, Robert

    2009-10-01

    The peptidyl-prolyl cis/trans isomerase Pin1 has been implicated in malignant transformation in multiple studies, however, little is known about its potential impact in head and neck cancer. This study evaluates the role of Pin1 in head and neck squamous cell carcinomas (HNSCCs). Pin1 expression and level of phosphorylation was evaluated by Western blot analysis and 2D-gel-electrophoresis. Pin1 was inhibited with juglone (5-hydroxy-1,4-naphthalenedione) or Pin1 specific siRNA and its influence on cell cycle checkpoint distribution was assessed by FACS analysis. Pin1 overexpression was found in HNSCC tissues and cell lines. 2D-gel-electrophoresis data pointed to Pin1 being hypophosphorylated in HNSCC cells which is consistent with overactivation of this rotamase. Inhibition of HNSCC cells with juglone or Pin1 siRNA induced the cell cycle inhibitor p21(WAF1/Cip1) with a concomitant reduction of cells in G2/M and an increased fraction of cells with fragmented DNA. Cell death did not correlate with significant levels of apoptosis in Pin1 depleted HNSCC cells. In summary, the data shows that Pin1 is overexpressed and hypophosphorylated in HNSCC, and that inhibition of Pin1 blocks cell cycle progression and triggers tumor cell death. Pin1 therefore could represent a new target for the development of improved HNSCC targeting drugs.

  9. Regulations of Gene Expression in Medullary Thymic Epithelial Cells Required for Preventing the Onset of Autoimmune Diseases

    OpenAIRE

    Akiyama, Taishin; Shinzawa, Miho; Qin, Junwen; Akiyama, Nobuko

    2013-01-01

    Elimination of potential self-reactive T cells in the thymus is crucial for preventing the onset of autoimmune diseases. Epithelial cell subsets localized in thymic medulla [medullary thymic epithelial cells (mTECs)] contribute to this process by supplying a wide range of self-antigens that are otherwise expressed in a tissue-specific manner (TSAs). Expression of some TSAs in mTECs is controlled by the autoimmune regulator (AIRE) protein, of which dysfunctional mutations are the causative fac...

  10. Characterization of promoter elements required for cell-specific expression of the neurotensin/neuromedin N gene in a human endocrine cell line.

    OpenAIRE

    Evers, B M; X Wang; Zhou, Z; Townsend, C M; McNeil, G P; Dobner, P R

    1995-01-01

    Expression of the gene encoding neurotensin/neuromedin N (NT/N) is mostly limited to the brain and specialized enteroendocrine cells (N cells) of the distal small intestine. We have analyzed the NT/N DNA sequences upstream of the RNA start site that direct cell-specific expression using a novel human endocrine cell line, BON, that resembles intestinal N cells in several important aspects, including NT/N precursor protein processing, ratios of different NT/N mRNA forms, and high levels of cons...

  11. The cytokines interleukin 27 and interferon-γ promote distinct Treg cell populations required to limit infection-induced pathology.

    Science.gov (United States)

    Hall, Aisling O'Hara; Beiting, Daniel P; Tato, Cristina; John, Beena; Oldenhove, Guillaume; Lombana, Claudia Gonzalez; Pritchard, Gretchen Harms; Silver, Jonathan S; Bouladoux, Nicolas; Stumhofer, Jason S; Harris, Tajie H; Grainger, John; Wojno, Elia D Tait; Wagage, Sagie; Roos, David S; Scott, Philip; Turka, Laurence A; Cherry, Sara; Reiner, Steven L; Cua, Daniel; Belkaid, Yasmine; Elloso, M Merle; Hunter, Christopher A

    2012-09-21

    Interferon-γ (IFN-γ) promotes a population of T-bet(+) CXCR3(+) regulatory T (Treg) cells that limit T helper 1 (Th1) cell-mediated pathology. Our studies demonstrate that interleukin-27 (IL-27) also promoted expression of T-bet and CXCR3 in Treg cells. During infection with Toxoplasma gondii, a similar population emerged that limited T cell responses and was dependent on IFN-γ in the periphery but on IL-27 at mucosal sites. Transfer of Treg cells ameliorated the infection-induced pathology observed in Il27(-/-) mice, and this was dependent on their ability to produce IL-10. Microarray analysis revealed that Treg cells exposed to either IFN-γ or IL-27 have distinct transcriptional profiles. Thus, IFN-γ and IL-27 have different roles in Treg cell biology and IL-27 is a key cytokine that promotes the development of Treg cells specialized to control Th1 cell-mediated immunity at local sites of inflammation.

  12. A combination of upstream and proximal elements is required for efficient expression of the mouse renin promoter in cultured cells.

    OpenAIRE

    Tamura, K.; Tanimoto, K; Murakami, K.; Fukamizu, A

    1992-01-01

    Renin, a key enzyme controlling blood pressure, is produced mainly in the kidney. To identify the transcriptional regulatory elements of the mouse Ren-1c gene, the promoter regions were fused to the CAT reporter gene and transfected into embryonic kidney-derived 293 cells and four extrarenal cell lines, HeLa, HepG2, HT1080 and NIH3T3 cells. Transient transfection assay showed that sequences from -365 to +16 of the renin gene could direct transcription of the CAT hybrid gene only in 293 cells....

  13. Repression of the Integrated Papillomavirus E6/E7 Promoter Is Required for Growth Suppression of Cervical Cancer Cells

    OpenAIRE

    Francis, Delicia A.; Schmid, Susanne I.; Howley, Peter M.

    2000-01-01

    The human papillomavirus (HPV) E2 protein is an important regulator of viral E6 and E7 gene expression. E2 can repress the viral promoter for E6 and E7 expression as well as block progression of the cell cycle in cancer cells harboring the DNA of “high-risk” HPV types. Although the phenomenon of E2-mediated growth arrest of HeLa cells and other HPV-positive cancer cells has been well documented, the specific mechanism by which E2 affects cellular proliferation has not yet been elucidated. Her...

  14. In vivo IFN-γ secretion by NK cells in response to Salmonella typhimurium requires NLRC4 inflammasomes.

    Directory of Open Access Journals (Sweden)

    Andreas Kupz

    Full Text Available Natural killer (NK cells are a critical part of the innate immune defense against viral infections and for the control of tumors. Much less is known about how NK cells contribute to anti-bacterial immunity. NK cell-produced interferon gamma (IFN-γ contributes to the control of early exponential replication of bacterial pathogens, however the regulation of these events remains poorly resolved. Using a mouse model of invasive Salmonellosis, here we report that the activation of the intracellular danger sensor NLRC4 by Salmonella-derived flagellin within CD11c+ cells regulates early IFN-γ secretion by NK cells through the provision of interleukin 18 (IL-18, independently of Toll-like receptor (TLR-signaling. Although IL18-signalling deficient NK cells improved host protection during S. Typhimurium infection, this increased resistance was inferior to that provided by wild-type NK cells. These findings suggest that although NLRC4 inflammasome-driven secretion of IL18 serves as a potent activator of NK cell mediated IFN-γ secretion, IL18-independent NK cell-mediated mechanisms of IFN-γ secretion contribute to in vivo control of Salmonella replication.

  15. β3-integrin is required for differentiation in OC-2 cells derived from mammalian embryonic inner ear

    Directory of Open Access Journals (Sweden)

    Brunetta Ivan

    2012-03-01

    Full Text Available Abstract Background The mammalian inner ear contains the organ of Corti which is responsible for the conversion of sound into neuronal signals. This specialised epithelial tissue is the product of a complex developmental process where a common precursor cell type differentiates into the sound transducing hair cells and the non-innervated supporting cells. We hypothesised that integrin proteins, which are involved in cell attachment to extracellular matrix proteins and cellular signalling, play a role in the differentiation process of the precursor inner ear epithelial cells. To test our hypothesis we have utilised a cell line (OC-2 derived from E13 embryonic immortomouse inner ears. In vitro, by switching the incubation temperature from 33°C to 39°C, the OC-2 cells can be induced to differentiate and express hair cells markers, such as Myosin VIIa. The OC-2 cells are thus a useful model system for testing mechanism of hair cells differentiation. Results We have identified 4 integrin subunits which are expressed in OC-2 cells: α6, αv, β1 and β3. Among these, the relative level of expression of the αv, β1 and β3 subunits increased in a time dependent manner when the cells were exposed to the differentiating temperature of 39°C, most notably so for β3 which was not detectable at 33°C. Treatment of fully differentiated OC-2 cells with siRNA against the four integrin subunits reduced the expression of not only the respective integrin proteins but also of the hair cell marker Myosin VIIa. Conversely over-expression of β3 was sufficient to induce the expression of Myosin VIIa at 33°C. Conclusions Our data demonstrate that modulation of integrin expression is associated with the differentiation process of the OC-2 cells. This suggests that the maturation of the organ of Corti, from where OC-2 cells are derived, may also depend on changes of gene expression associated with integrin expression.

  16. Programming tumor-reactive effector memory CD8+ T cells in vitro obviates the requirement for in vivo vaccination

    OpenAIRE

    Klebanoff, Christopher A.; Yu, Zhiya; Hwang, Leroy N.; Douglas C Palmer; Gattinoni, Luca; Restifo, Nicholas P

    2009-01-01

    Naive and memory CD8+ T cells can undergo programmed activation and expansion in response to a short T-cell receptor stimulus, but the extent to which in vitro programming can qualitatively substitute for an in vivo antigen stimulation remains unknown. We show that self-/tumor-reactive effector memory CD8+ T cells (TEM) programmed in vitro either with peptide-pulsed antigen-presenting cells or plate-bound anti-CD3/anti-CD28 embark on a highly stereotyped response of in vivo clonal expansion a...

  17. Sample size requirements for studies of treatment effects on beta-cell function in newly diagnosed type 1 diabetes.

    Directory of Open Access Journals (Sweden)

    John M Lachin

    Full Text Available Preservation of β-cell function as measured by stimulated C-peptide has recently been accepted as a therapeutic target for subjects with newly diagnosed type 1 diabetes. In recently completed studies conducted by the Type 1 Diabetes Trial Network (TrialNet, repeated 2-hour Mixed Meal Tolerance Tests (MMTT were obtained for up to 24 months from 156 subjects with up to 3 months duration of type 1 diabetes at the time of study enrollment. These data provide the information needed to more accurately determine the sample size needed for future studies of the effects of new agents on the 2-hour area under the curve (AUC of the C-peptide values. The natural log(x, log(x+1 and square-root (√x transformations of the AUC were assessed. In general, a transformation of the data is needed to better satisfy the normality assumptions for commonly used statistical tests. Statistical analysis of the raw and transformed data are provided to estimate the mean levels over time and the residual variation in untreated subjects that allow sample size calculations for future studies at either 12 or 24 months of follow-up and among children 8-12 years of age, adolescents (13-17 years and adults (18+ years. The sample size needed to detect a given relative (percentage difference with treatment versus control is greater at 24 months than at 12 months of follow-up, and differs among age categories. Owing to greater residual variation among those 13-17 years of age, a larger sample size is required for this age group. Methods are also described for assessment of sample size for mixtures of subjects among the age categories. Statistical expressions are presented for the presentation of analyses of log(x+1 and √x transformed values in terms of the original units of measurement (pmol/ml. Analyses using different transformations are described for the TrialNet study of masked anti-CD20 (rituximab versus masked placebo. These results provide the information needed to

  18. Inducing P-selectin ligand formation in CD8 T cells: IL-2 and IL-12 are active in vitro but not required in vivo.

    Science.gov (United States)

    Carlow, Douglas A; Williams, Michael J; Ziltener, Hermann J

    2005-04-01

    In vitro studies have demonstrated that IL-2 and IL-12 can support formation of P-selectin ligands (P-SelL) in activated T cells, ligands that are variably required for efficient lymphocyte recruitment to sites of inflammation. To ascertain whether these cytokines were required for P-SelL formation in vivo, TCR transgenic CD8 T cells specific for male Ag (HY) were transferred into male mice under conditions in which either IL-2 and/or IL-15 or IL-12Rp40 were absent. P-SelL formation at day 2 was unperturbed in HY-TCR IL-2(null) CD8 T cells responding in doubly deficient IL-2(null)IL-12(null) or IL-2(null)IL-15(null) male recipients. HY-specific CD8 T cell proliferative responses detected in both spleen and peritoneum occurred vigorously, but only splenic CD8 T cells up-regulated P-SelL, demonstrating that in vivo induction of P-SelL is an active, nonprogrammed event following T cell activation and that despite the efficacy of IL-2 and IL-12 in supporting P-SelL formation in vitro, these cytokines appear to be dispensable for this purpose in vivo.

  19. Up-regulation of Bcl-2 is required for the progression of prostate cancer cells from an androgen-dependent to an androgen-independent growth stage

    Institute of Scientific and Technical Information of China (English)

    Yuting Lin; Junichi Fukuchi; Richard A Hiipakka; John M Kokontis; Jialing Xiang

    2007-01-01

    Bcl-2 is an anti-apoptotic oncoprotein and its protein levels are inversely correlated with prognosis in many cancers.However, the role of Bcl-2 in the progression of prostate cancer is not clear. Here we report that Bcl-2 is required for the progression of LNCaP prostate cancer cells from an androgen-dependent to an androgen-independent growth stage. The mRNA and protein levels of Bcl-2 are significantly increased in androgen-independent prostate cancer cells, shRNA-mediated gene silencing of Bcl-2 in androgen-independent prostate cancer cells promotes UV-induced apoptosis and suppresses the growth of prostate tumors in vivo. Growing androgen-dependent cells under androgen-deprivation conditions results in formation of androgen-independent colonies; and the transition from androgen-dependent to androgen-independent growth is blocked by ectopic expression of the Bcl-2 antagonist Bax or Bcl-2 shRNA. Thus, our results demonstrate that Bcl-2 is not only critical for the survival of androgen-independent prostate cancer cells, but is also required for the progression of prostate cancer cells from an androgen-dependent to an androgen-independent growth stage.

  20. One decade after the discovery of single-cell C4 species in terrestrial plants: what did we learn about the minimal requirements of C4 photosynthesis?

    Science.gov (United States)

    Sharpe, Richard M; Offermann, Sascha

    2014-02-01

    Until about 10 years ago the general accepted textbook knowledge was that terrestrial C4 photosynthesis requires separation of photosynthetic functions into two specialized cell types, the mesophyll and bundle sheath cells forming the distinctive Kranz anatomy typical for C4 plants. This paradigm has been broken with the discovery of Suaeda aralocaspica, a chenopod from central Asia, performing C4 photosynthesis within individual chlorenchyma cells. Since then, three more single-cell C4 (SCC4) species have been discovered in the genus Bienertia. They are interesting not only because of their unusual mode of photosynthesis but also present a puzzle for cell biologists. In these species, two morphological and biochemical specialized types of chloroplasts develop within individual chlorenchyma cells, a situation that has never been observed in plants before. Here we review recent literature concerning the biochemistry, physiology, and molecular biology of SCC4 photosynthesis. Particularly, we focus on what has been learned in relation to the following questions: How does the specialized morphology required for the operation of SCC4 develop and is there a C3 intermediate type of photosynthesis during development? What is the degree of specialization between the two chloroplast types and how does this compare to the chloroplasts of Kranz C4 species? How do nucleus-encoded proteins that are targeted to chloroplasts accumulate differentially in the two chloroplast types and how efficient is the CO2 concentrating mechanism in SCC4 species compared to the Kranz C4 forms?

  1. Concurrent MEK and autophagy inhibition is required to restore cell death associated danger-signalling in Vemurafenib-resistant melanoma cells.

    Science.gov (United States)

    Martin, S; Dudek-Perić, A M; Maes, H; Garg, A D; Gabrysiak, M; Demirsoy, S; Swinnen, J V; Agostinis, P

    2015-02-01

    Vemurafenib (PLX4032), an inhibitor of BRAF(V600E), has demonstrated significant clinical anti-melanoma effects. However, the majority of treated patients develop resistance, due to a variety of molecular mechanisms including MAPK reactivation through MEK. The induction of a cancer cell death modality associated with danger-signalling resulting in surface mobilization of crucial damage-associated-molecular-patterns (DAMPs), e.g. calreticulin (CRT) and heat shock protein-90 (HSP90), from dying cells, is emerging to be crucial for therapeutic success. Both cell death and danger-signalling are modulated by autophagy, a key adaptation mechanism stimulated during melanoma progression. However, whether melanoma cell death induced by MAPK inhibition is associated with danger-signalling, and the reliance of these mechanisms on autophagy, has not yet been scrutinized. Using a panel of isogenic PLX4032-sensitive and resistant melanoma cell lines we show that PLX4032-induced caspase-dependent cell death and DAMPs exposure in the drug-sensitive cells, but failed to do so in the drug-resistant cells, displaying heightened MEK activation. MEK inhibitor, U0126, treatment sensitized PLX4032-resistant cells to death and re-established their danger-signalling capacity. Only melanoma cells exposing death-induced danger-signals were phagocytosed and induced DC maturation. Although the PLX4032-resistant melanoma cells displayed higher basal and drug-induced autophagy, compromising autophagy, pharmacologically or by ATG5 knockdown, was insufficient to re-establish their PLX4032 sensitivity. Interestingly, autophagy abrogation was particularly efficacious in boosting cell death and ecto-CRT/ecto-HSP90 in PLX4032-resistant cells upon blockage of MEK hyper-activation by U0126. Thus combination of MEK inhibitors with autophagy blockers may represent a novel treatment regime to increase both cell death and danger-signalling in Vemurafenib-resistant metastatic melanoma.

  2. Caspase-1-independent interleukin-1β is required for clearance of Bordetella pertussis infections and whole-cell vaccine-mediated immunity.

    Science.gov (United States)

    Place, David E; Muse, Sarah J; Kirimanjeswara, Girish S; Harvill, Eric T

    2014-01-01

    Whooping cough remains a significant disease worldwide and its re-emergence in highly vaccinated populations has been attributed to a combination of imperfect vaccines and evolution of the pathogen. The focus of this study was to examine the role of IL-1α/β and the inflammasome in generation of the interleukin-1 (IL-1) response, which is required for the clearance of Bordetella pertussis. We show that IL-1β but not IL-1α is required for mediating the clearance of B. pertussis from the lungs of mice. We further found that IL-1β and IL-1R deficient mice, compared to wild-type, have similar but more persistent levels of inflammation, characterized by immune cell infiltration, with significantly increased IFNγ and a normal IL-17A response during B. pertussis infection. Contrary to expectations, the cleavage of precursor IL-1β to its mature form did not require caspase-1 during primary infections within the lung despite being required by bone marrow-derived macrophages exposed to live bacteria. We also found that the caspase-1 inflammasome was not required for protective immunity against a B. pertussis challenge following vaccination with heat-killed whole cell B. pertussis, despite IL-1R signaling being required. These findings demonstrate that caspase-1-independent host factors are involved in the processing of protective IL-1β responses that are critical for bacterial clearance and vaccine-mediated immunity.

  3. Caspase-1-independent interleukin-1β is required for clearance of Bordetella pertussis infections and whole-cell vaccine-mediated immunity.

    Directory of Open Access Journals (Sweden)

    David E Place

    Full Text Available Whooping cough remains a significant disease worldwide and its re-emergence in highly vaccinated populations has been attributed to a combination of imperfect vaccines and evolution of the pathogen. The focus of this study was to examine the role of IL-1α/β and the inflammasome in generation of the interleukin-1 (IL-1 response, which is required for the clearance of Bordetella pertussis. We show that IL-1β but not IL-1α is required for mediating the clearance of B. pertussis from the lungs of mice. We further found that IL-1β and IL-1R deficient mice, compared to wild-type, have similar but more persistent levels of inflammation, characterized by immune cell infiltration, with significantly increased IFNγ and a normal IL-17A response during B. pertussis infection. Contrary to expectations, the cleavage of precursor IL-1β to its mature form did not require caspase-1 during primary infections within the lung despite being required by bone marrow-derived macrophages exposed to live bacteria. We also found that the caspase-1 inflammasome was not required for protective immunity against a B. pertussis challenge following vaccination with heat-killed whole cell B. pertussis, despite IL-1R signaling being required. These findings demonstrate that caspase-1-independent host factors are involved in the processing of protective IL-1β responses that are critical for bacterial clearance and vaccine-mediated immunity.

  4. Lipid rafts are required for signal transduction by angiotensin II receptor type 1 in neonatal glomerular mesangial cells

    Energy Technology Data Exchange (ETDEWEB)

    Adebiyi, Adebowale, E-mail: aadebiyi@uthsc.edu; Soni, Hitesh; John, Theresa A.; Yang, Fen

    2014-05-15

    Angiotensin II (ANG-II) receptors (AGTRs) contribute to renal physiology and pathophysiology, but the underlying mechanisms that regulate AGTR function in glomerular mesangium are poorly understood. Here, we show that AGTR1 is the functional AGTR subtype expressed in neonatal pig glomerular mesangial cells (GMCs). Cyclodextrin (CDX)-mediated cholesterol depletion attenuated cell surface AGTR1 protein expression and ANG-II-induced intracellular Ca{sup 2+} ([Ca{sup 2+}]{sub i}) elevation in the cells. The COOH-terminus of porcine AGTR1 contains a caveolin (CAV)-binding motif. However, neonatal GMCs express CAV-1, but not CAV-2 and CAV-3. Colocalization and in situ proximity ligation assay detected an association between endogenous AGTR1 and CAV-1 in the cells. A synthetic peptide corresponding to the CAV-1 scaffolding domain (CSD) sequence also reduced ANG-II-induced [Ca{sup 2+}]{sub i} elevation in the cells. Real-time imaging of cell growth revealed that ANG-II stimulates neonatal GMC proliferation. ANG-II-induced GMC growth was attenuated by EMD 66684, an AGTR1 antagonist; BAPTA, a [Ca{sup 2+}]{sub i} chelator; KN-93, a Ca{sup 2+}/calmodulin-dependent protein kinase II inhibitor; CDX; and a CSD peptide, but not PD 123319, a selective AGTR2 antagonist. Collectively, our data demonstrate [Ca{sup 2+}]{sub i}-dependent proliferative effect of ANG-II and highlight a critical role for lipid raft microdomains in AGTR1-mediated signal transduction in neonatal GMCs. - Highlights: • AGTR1 is the functional AGTR subtype expressed in neonatal mesangial cells. • Endogenous AGTR1 associates with CAV-1 in neonatal mesangial cells. • Lipid raft disruption attenuates cell surface AGTR1 protein expression. • Lipid raft disruption reduces ANG-II-induced [Ca{sup 2+}]{sub i} elevation in neonatal mesangial cells. • Lipid raft disruption inhibits ANG-II-induced neonatal mesangial cell growth.

  5. FGF signaling via MAPK is required early and improves Activin A-induced definitive endoderm formation from human embryonic stem cells

    International Nuclear Information System (INIS)

    Highlights: ► Deep study the FGF signaling role during DE specification in the context of hESCs. ► DE differentiation from hESCs has an early dependence on FGF signaling. ► A serum-free DE protocol is developed based on the findings. ► The DE cells showed potential to differentiate into pancreatic progenitor cells. -- Abstract: Considering their unlimited proliferation and pluripotency properties, human embryonic stem cells (hESCs) constitute a promising resource applicable for cell replacement therapy. To facilitate this clinical translation, it is critical to study and understand the early stage of hESCs differentiation wherein germ layers are defined. In this study, we examined the role of FGF signaling in Activin A-induced definitive endoderm (DE) differentiation in the absence of supplemented animal serum. We found that activated FGF/MAPK signaling is required at the early time point of Activin A-induced DE formation. In addition, FGF activation increased the number of DE cells compared to Activin A alone. These DE cells could further differentiate into PDX1 and NKX6.1 positive pancreatic progenitors in vitro. We conclude that Activin A combined with FGF/MAPK signaling efficiently induce DE cells in the absence of serum. These findings improve our understanding of human endoderm formation, and constitute a step forward in the generation of clinical grade hESCs progenies for cell therapy.

  6. In a SLE mouse model the production of IgG autoantibody requires expression of activation-induced deaminase in early developing B cells

    Science.gov (United States)

    Umiker, Benjamin R.; McDonald, Gabrielle; Larbi, Amma; Medina, Carlos O.; Reth, Michael; Imanishi-Kari, Thereza

    2014-01-01

    Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of pathogenic IgG anti-nuclear antibodies. Pathogenic IgG autoantibody production requires B-cell activation, leading to the production of activation-induced deaminase (AID) and class switching of IgM genes to IgG. To understand how and when B cells are activated to produce these IgG autoantibodies, we studied cells from 564Igi, a mouse model of SLE. 564Igi mice develop a disease profile closely resembling that found in human SLE patients, including the presence of IgG anti-nucleic acid antibodies. We have generated 564Igi mice that conditionally express an activation-induced cytidine deaminase transgene (Aicdatg), either in all B cells or only in mature B cells. Here we show that class-switched pathogenic IgG autoantibodies were produced only in 564Igi mice in which AID was functional in early developing B cells, resulting in loss of tolerance. Furthermore, we show that the absence of AID in early developing B cells also results in increased production of self-reactive IgM, indicating that AID, through somatic hypermutation (SHM), contributes to tolerance. Our results suggest that the pathophysiology of clinical SLE might also be dependent on AID expression in early developing B cells. PMID:25044405

  7. FGF signaling via MAPK is required early and improves Activin A-induced definitive endoderm formation from human embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Sui, Lina, E-mail: linasui@vub.ac.be [Cell Differentiation Unit, Diabetes Research Center, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels (Belgium); Mfopou, Josue K. [Cell Differentiation Unit, Diabetes Research Center, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels (Belgium); Geens, Mieke; Sermon, Karen [Department of Embryology and Genetics, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels (Belgium); Bouwens, Luc [Cell Differentiation Unit, Diabetes Research Center, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels (Belgium)

    2012-09-28

    Highlights: Black-Right-Pointing-Pointer Deep study the FGF signaling role during DE specification in the context of hESCs. Black-Right-Pointing-Pointer DE differentiation from hESCs has an early dependence on FGF signaling. Black-Right-Pointing-Pointer A serum-free DE protocol is developed based on the findings. Black-Right-Pointing-Pointer The DE cells showed potential to differentiate into pancreatic progenitor cells. -- Abstract: Considering their unlimited proliferation and pluripotency properties, human embryonic stem cells (hESCs) constitute a promising resource applicable for cell replacement therapy. To facilitate this clinical translation, it is critical to study and understand the early stage of hESCs differentiation wherein germ layers are defined. In this study, we examined the role of FGF signaling in Activin A-induced definitive endoderm (DE) differentiation in the absence of supplemented animal serum. We found that activated FGF/MAPK signaling is required at the early time point of Activin A-induced DE formation. In addition, FGF activation increased the number of DE cells compared to Activin A alone. These DE cells could further differentiate into PDX1 and NKX6.1 positive pancreatic progenitors in vitro. We conclude that Activin A combined with FGF/MAPK signaling efficiently induce DE cells in the absence of serum. These findings improve our understanding of human endoderm formation, and constitute a step forward in the generation of clinical grade hESCs progenies for cell therapy.

  8. WNT/β-Catenin Signaling Is Required for Integration of CD24+ Renal Progenitor Cells into Glycerol-Damaged Adult Renal Tubules

    Directory of Open Access Journals (Sweden)

    Zhao Zhang

    2015-01-01

    Full Text Available During development, nephron progenitor cells (NPC are induced to differentiate by WNT9b signals from the ureteric bud. Although nephrogenesis ends in the perinatal period, acute kidney injury (AKI elicits repopulation of damaged nephrons. Interestingly, embryonic NPC infused into adult mice with AKI are incorporated into regenerating tubules. Since WNT/β-catenin signaling is crucial for primary nephrogenesis, we reasoned that it might also be needed for the endogenous repair mechanism and for integration of exogenous NPC. When we examined glycerol-induced AKI in adult mice bearing a β-catenin/TCF reporter transgene, endogenous tubular cells reexpressed the NPC marker, CD24, and showed widespread β-catenin/TCF signaling. We isolated CD24+ cells from E15 kidneys of mice with the canonical WNT signaling reporter. 40% of cells responded to WNT3a in vitro and when infused into glycerol-injured adult, the cells exhibited β-catenin/TCF reporter activity when integrated into damaged tubules. When embryonic CD24+ cells were treated with a β-catenin/TCF pathway inhibitor (IWR-1 prior to infusion into glycerol-injured mice, tubular integration of cells was sharply reduced. Thus, the endogenous canonical β-catenin/TCF pathway is reactivated during recovery from AKI and is required for integration of exogenous embryonic renal progenitor cells into damaged tubules. These events appear to recapitulate the WNT-dependent inductive process which drives primary nephrogenesis.

  9. High telomerase is a hallmark of undifferentiated spermatogonia and is required for maintenance of male germline stem cells.

    Science.gov (United States)

    Pech, Matthew F; Garbuzov, Alina; Hasegawa, Kazuteru; Sukhwani, Meena; Zhang, Ruixuan J; Benayoun, Bérénice A; Brockman, Stephanie A; Lin, Shengda; Brunet, Anne; Orwig, Kyle E; Artandi, Steven E

    2015-12-01

    Telomerase inactivation causes loss of the male germline in worms, fish, and mice, indicating a conserved dependence on telomere maintenance in this cell lineage. Here, using telomerase reverse transcriptase (Tert) reporter mice, we found that very high telomerase expression is a hallmark of undifferentiated spermatogonia, the mitotic population where germline stem cells reside. We exploited these high telomerase levels as a basis for purifying undifferentiated spermatogonia using fluorescence-activated cell sorting. Telomerase levels in undifferentiated spermatogonia and embryonic stem cells are comparable and much greater than in somatic progenitor compartments. Within the germline, we uncovered an unanticipated gradient of telomerase activity that also enables isolation of more mature populations. Transcriptomic comparisons of Tert(High) undifferentiated spermatogonia and Tert(Low) differentiated spermatogonia by RNA sequencing reveals marked differences in cell cycle and key molecular features of each compartment. Transplantation studies show that germline stem cell activity is confined to the Tert(High) cKit(-) population. Telomere shortening in telomerase knockout strains causes depletion of undifferentiated spermatogonia and eventual loss of all germ cells after undifferentiated spermatogonia drop below a critical threshold. These data reveal that high telomerase expression is a fundamental characteristic of germline stem cells, thus explaining the broad dependence on telomerase for germline immortality in metazoans. PMID:26584619

  10. Distinct Metabolic Requirements of Exhausted and Functional Virus-Specific CD8 T Cells in the Same Host

    Directory of Open Access Journals (Sweden)

    Anna Schurich

    2016-08-01

    Full Text Available T cells undergo profound metabolic changes to meet the increased energy demands of maintaining an antiviral response. We postulated that differences in metabolic reprogramming would shape the efficacy of CD8 T cells mounted against persistent viral infections. We found that the poorly functional PD-1hi T cell response against hepatitis B virus (HBV had upregulated the glucose transporter, Glut1, an effect recapitulated by oxygen deprivation to mimic the intrahepatic environment. Glut1hi HBV-specific T cells were dependent on glucose supplies, unlike the more functional cytomegalovirus (CMV-specific T cells that could utilize oxidative phosphorylation in the absence of glucose. The inability of HBV-specific T cells to switch to oxidative phosphorylation was accompanied by increased mitochondrial size and lower mitochondrial potential, indicative of mitochondrial dysfunction. Interleukin (IL-12, which recovers HBV-specific T cell effector function, increased their mitochondrial potential and reduced their dependence on glycolysis. Our findings suggest that mitochondrial defects limit the metabolic plasticity of exhausted HBV-specific T cells.

  11. ERRγ Is Required for the Metabolic Maturation of Therapeutically Functional Glucose-Responsive β Cells.

    Science.gov (United States)

    Yoshihara, Eiji; Wei, Zong; Lin, Chun Shi; Fang, Sungsoon; Ahmadian, Maryam; Kida, Yasuyuki; Tseng, Tiffany; Dai, Yang; Yu, Ruth T; Liddle, Christopher; Atkins, Annette R; Downes, Michael; Evans, Ronald M

    2016-04-12

    Pancreatic β cells undergo postnatal maturation to achieve maximal glucose-responsive insulin secretion, an energy intensive process. We identify estrogen-related receptor γ (ERRγ) expression as a hallmark of adult, but not neonatal β cells. Postnatal induction of ERRγ drives a transcriptional network activating mitochondrial oxidative phosphorylation, the electron transport chain, and ATP production needed to drive glucose-responsive insulin secretion. Mice deficient in β cell-specific ERRγ expression are glucose intolerant and fail to secrete insulin in response to a glucose challenge. Notably, forced expression of ERRγ in iPSC-derived β-like cells enables glucose-responsive secretion of human insulin in vitro, obviating in vivo maturation to achieve functionality. Moreover, these cells rapidly rescue diabetes when transplanted into β cell-deficient mice. These results identify a key role for ERRγ in β cell metabolic maturation, and offer a reproducible, quantifiable, and scalable approach for in vitro generation of functional human β cell therapeutics. PMID:27076077

  12. High Specificity in Circulating Tumor Cell Identification Is Required for Accurate Evaluation of Programmed Death-Ligand 1.

    Directory of Open Access Journals (Sweden)

    Jennifer L Schehr

    Full Text Available Expression of programmed-death ligand 1 (PD-L1 in non-small cell lung cancer (NSCLC is typically evaluated through invasive biopsies; however, recent advances in the identification of circulating tumor cells (CTCs may be a less invasive method to assay tumor cells for these purposes. These liquid biopsies rely on accurate identification of CTCs from the diverse populations in the blood, where some tumor cells share characteristics with normal blood cells. While many blood cells can be excluded by their high expression of CD45, neutrophils and other immature myeloid subsets have low to absent expression of CD45 and also express PD-L1. Furthermore, cytokeratin is typically used to identify CTCs, but neutrophils may stain non-specifically for intracellular antibodies, including cytokeratin, thus preventing accurate evaluation of PD-L1 expression on tumor cells. This holds even greater significance when evaluating PD-L1 in epithelial cell adhesion molecule (EpCAM positive and EpCAM negative CTCs (as in epithelial-mesenchymal transition (EMT.To evaluate the impact of CTC misidentification on PD-L1 evaluation, we utilized CD11b to identify myeloid cells. CTCs were isolated from patients with metastatic NSCLC using EpCAM, MUC1 or Vimentin capture antibodies and exclusion-based sample preparation (ESP technology.Large populations of CD11b+CD45lo cells were identified in buffy coats and stained non-specifically for intracellular antibodies including cytokeratin. The amount of CD11b+ cells misidentified as CTCs varied among patients; accounting for 33-100% of traditionally identified CTCs. Cells captured with vimentin had a higher frequency of CD11b+ cells at 41%, compared to 20% and 18% with MUC1 or EpCAM, respectively. Cells misidentified as CTCs ultimately skewed PD-L1 expression to varying degrees across patient samples.Interfering myeloid populations can be differentiated from true CTCs with additional staining criteria, thus improving the

  13. High Specificity in Circulating Tumor Cell Identification Is Required for Accurate Evaluation of Programmed Death-Ligand 1

    Science.gov (United States)

    Schultz, Zachery D.; Warrick, Jay W.; Guckenberger, David J.; Pezzi, Hannah M.; Sperger, Jamie M.; Heninger, Erika; Saeed, Anwaar; Leal, Ticiana; Mattox, Kara; Traynor, Anne M.; Campbell, Toby C.; Berry, Scott M.; Beebe, David J.; Lang, Joshua M.

    2016-01-01

    Background Expression of programmed-death ligand 1 (PD-L1) in non-small cell lung cancer (NSCLC) is typically evaluated through invasive biopsies; however, recent advances in the identification of circulating tumor cells (CTCs) may be a less invasive method to assay tumor cells for these purposes. These liquid biopsies rely on accurate identification of CTCs from the diverse populations in the blood, where some tumor cells share characteristics with normal blood cells. While many blood cells can be excluded by their high expression of CD45, neutrophils and other immature myeloid subsets have low to absent expression of CD45 and also express PD-L1. Furthermore, cytokeratin is typically used to identify CTCs, but neutrophils may stain non-specifically for intracellular antibodies, including cytokeratin, thus preventing accurate evaluation of PD-L1 expression on tumor cells. This holds even greater significance when evaluating PD-L1 in epithelial cell adhesion molecule (EpCAM) positive and EpCAM negative CTCs (as in epithelial-mesenchymal transition (EMT)). Methods To evaluate the impact of CTC misidentification on PD-L1 evaluation, we utilized CD11b to identify myeloid cells. CTCs were isolated from patients with metastatic NSCLC using EpCAM, MUC1 or Vimentin capture antibodies and exclusion-based sample preparation (ESP) technology. Results Large populations of CD11b+CD45lo cells were identified in buffy coats and stained non-specifically for intracellular antibodies including cytokeratin. The amount of CD11b+ cells misidentified as CTCs varied among patients; accounting for 33–100% of traditionally identified CTCs. Cells captured with vimentin had a higher frequency of CD11b+ cells at 41%, compared to 20% and 18% with MUC1 or EpCAM, respectively. Cells misidentified as CTCs ultimately skewed PD-L1 expression to varying degrees across patient samples. Conclusions Interfering myeloid populations can be differentiated from true CTCs with additional staining criteria

  14. Rac function is crucial for cell migration but is not required for spreading and focal adhesion formation

    DEFF Research Database (Denmark)

    Steffen, Anika; Ladwein, Markus; Dimchev, Georgi A;

    2013-01-01

    Cell migration is commonly accompanied by protrusion of membrane ruffles and lamellipodia. In two-dimensional migration, protrusion of these thin sheets of cytoplasm is considered relevant to both exploration of new space and initiation of nascent adhesion to the substratum. Lamellipodium formation...... restored by expression of either Rac subfamily member, but not by Cdc42 or RhoG. Cells deficient in Rac showed strong reduction in wound closure and random cell migration and a notable loss of sensitivity to a chemotactic gradient. Despite these defects, Rac-deficient cells were able to spread, formed...... deficiency increased the mobility of different components in focal adhesions, potentially explaining how Rac - although not essential - can contribute to focal adhesion assembly. Together, our data demonstrate that Rac signaling is essential for lamellipodium protrusion and for efficient cell migration, but...

  15. DNA tumor virus oncoproteins and retinoblastoma gene mutations share the ability to relieve the cell's requirement for cyclin D1 function in G1.

    Science.gov (United States)

    Lukas, J; Müller, H; Bartkova, J; Spitkovsky, D; Kjerulff, A A; Jansen-Dürr, P; Strauss, M; Bartek, J

    1994-05-01

    The retinoblastoma gene product (pRB) participates in the regulation of the cell division cycle through complex formation with numerous cellular regulatory proteins including the potentially oncogenic cyclin D1. Extending the current view of the emerging functional interplay between pRB and D-type cyclins, we now report that cyclin D1 expression is positively regulated by pRB. Cyclin D1 mRNA and protein is specifically downregulated in cells expressing SV40 large T antigen, adenovirus E1A, and papillomavirus E7/E6 oncogene products and this effect requires intact RB-binding, CR2 domain of E1A. Exceptionally low expression of cyclin D1 is also seen in genetically RB-deficient cell lines, in which ectopically expressed wild-type pRB results in specific induction of this G1 cyclin. At the functional level, antibody-mediated cyclin D1 knockout experiments demonstrate that the cyclin D1 protein, normally required for G1 progression, is dispensable for passage through the cell cycle in cell lines whose pRB is inactivated through complex formation with T antigen, E1A, or E7 oncoproteins as well as in cells which have suffered loss-of-function mutations of the RB gene. The requirement for cyclin D1 function is not regained upon experimental elevation of cyclin D1 expression in cells with mutant RB, while reintroduction of wild-type RB into RB-deficient cells leads to restoration of the cyclin D1 checkpoint. These results strongly suggest that pRB serves as a major target of cyclin D1 whose cell cycle regulatory function becomes dispensable in cells lacking functional RB. Based on available data including this study, we propose a model for an autoregulatory feedback loop mechanism that regulates both the expression of the cyclin D1 gene and the activity of pRB, thereby contributing to a G1 phase checkpoint control in cycling mammalian cells. PMID:8175885

  16. Chronic parasitic infection maintains high frequencies of short-lived Ly6C+CD4+ effector T cells that are required for protection against re-infection.

    Directory of Open Access Journals (Sweden)

    Nathan C Peters

    2014-12-01

    Full Text Available In contrast to the ability of long-lived CD8(+ memory T cells to mediate protection against systemic viral infections, the relationship between CD4(+ T cell memory and acquired resistance against infectious pathogens remains poorly defined. This is especially true for T helper 1 (Th1 concomitant immunity, in which protection against reinfection coincides with a persisting primary infection. In these situations, pre-existing effector CD4 T cells generated by ongoing chronic infection, not memory cells, may be essential for protection against reinfection. We present a systematic study of the tissue homing properties, functionality, and life span of subsets of memory and effector CD4 T cells activated in the setting of chronic Leishmania major infection in resistant C57Bl/6 mice. We found that pre-existing, CD44(+CD62L(-T-bet(+Ly6C+ effector (T(EFF cells that are short-lived in the absence of infection and are not derived from memory cells reactivated by secondary challenge, mediate concomitant immunity. Upon adoptive transfer and challenge, non-dividing Ly6C(+ T(EFF cells preferentially homed to the skin, released IFN-γ, and conferred protection as compared to CD44(+CD62L(-Ly6C(- effector memory or CD44(+CD62L(+Ly6C(- central memory cells. During chronic infection, Ly6C(+ T(EFF cells were maintained at high frequencies via reactivation of T(CM and the T(EFF themselves. The lack of effective vaccines for many chronic diseases may be because protection against infectious challenge requires the maintenance of pre-existing T(EFF cells, and is therefore not amenable to conventional, memory inducing, vaccination strategies.

  17. An intrinsic B cell defect is required for the production of autoantibodies in the lpr model of murine systemic autoimmunity

    Energy Technology Data Exchange (ETDEWEB)

    Sobel, E.S.; Katagiri, T.; Katagiri, K.; Morris, S.C.; Cohen, P.L.; Eisenberg, R.A. (Univ. of North Carolina, Chapel Hill (USA))

    1991-06-01

    Mice homozygous for the gene lpr develop marked lymphadenopathy and a spectrum of autoantibodies closely resembling that of human systemic lupus erythematosus. The unusual T cell phenotype of the expanded lymphocyte population and the T-dependence of several antibodies in this strain have suggested that primary T cell abnormalities underlie the autoimmune syndrome. Using double chimeras, we now show that expression of the lpr gene in B cells is absolutely necessary for autoantibody production. Combinations of anti-Thy 1.2 + C' treated bone marrow from congenic strains of C57BL/6 mice, differing only at the immunoglobulin heavy chain (Igh) and lpr loci, were transferred into lethally irradiated B6/lpr mice. Double chimerism was documented by allotype-specific surface IgD and IgM immunofluorescence assay of peripheral blood and by allotype-specific enzyme-linked immunosorbent assay for total IgM in serum. Despite the presence of both +/+ and lpr B cells, IgM and IgG2a anti-chromatin as well as IgM anti-IgG were entirely the products of lpr B cells. Total serum IgG2a and IgG1 were also dominated by the lpr phenotype but not to the same extent. A similar experiment using B6/lpr-Igha recipients confirmed these findings. Additional experiments in which B6/lpr recipients were infused with ratios of donor bone marrow favoring B6.C20 +/+ over B6/lpr showed that even though +/+ B cells were overrepresented, autoantibodies were only of the lpr allotype. In addition, in the presence of lpr B cells, normal B cells showed little response to an exogenous, T cell-dependent antigen. The data thus indicate that lpr B cells manifest an intrinsic abnormality which is essential for autoantibody production in the lpr model.

  18. Why Lead Methylammonium tri-IODIDE perovskite-based solar cells requires a mesoporous electron transporting scaffold (but not necessarily a hole conductor)

    CERN Document Server

    Edri, Eran; Henning, Alex; Mukhopadhyay, Sabyasachi; Gartsman, Konstantin; Rosenwaks, Yossi; Hodes, Gary; Cahen, David

    2014-01-01

    CH3NH3PbI3-based solar cells were characterized with electron beam-induced current (EBIC), and compared to CH3NH3PbI3-xClx ones. A spatial map of charge separation efficiency in working cells shows p-i-n structures for both thin film cells. Effective diffusion lengths, LD, (from EBIC profile) show that holes are extracted significantly more efficiently than electrons in CH3NH3PbI3, explaining why CH3NH3PbI3-based cells require mesoporous electron conductors, while CH3NH3PbI3-xClx ones, where LD values are comparable for both charge types, do not.

  19. JunD Is Required for Proliferation of Prostate Cancer Cells and Plays a Role in Transforming Growth Factor-β (TGF-β)-induced Inhibition of Cell Proliferation.

    Science.gov (United States)

    Millena, Ana Cecilia; Vo, BaoHan T; Khan, Shafiq A

    2016-08-19

    TGF-β inhibits proliferation of prostate epithelial cells. However, prostate cancer cells in advanced stages become resistant to inhibitory effects of TGF-β. The intracellular signaling mechanisms involved in differential effects of TGF-β during different stages are largely unknown. Using cell line models, we have shown that TGF-β inhibits proliferation in normal (RWPE-1) and prostate cancer (DU145) cells but does not have any effect on proliferation of prostate cancer (PC3) cells. We have investigated the role of Jun family proteins (c-Jun, JunB, and JunD) in TGF-β effects on cell proliferation. Jun family members were expressed at different levels and responded differentially to TGF-β treatment. TGF-β effects on JunD protein levels, but not mRNA levels, correlated with its effects on cell proliferation. TGF-β induced significant reduction in JunD protein in RWPE-1 and DU145 cells but not in PC3 cells. Selective knockdown of JunD expression using siRNA in DU145 and PC3 cells resulted in significant reduction in cell proliferation, and forced overexpression of JunD increased the proliferation rate. On the other hand, knockdown of c-Jun or JunB had little, if any, effect on cell proliferation; overexpression of c-Jun and JunB decreased the proliferation rate in DU145 cells. Further studies showed that down-regulation of JunD in response to TGF-β treatment is mediated via the proteasomal degradation pathway. In conclusion, we show that specific Jun family members exert differential effects on proliferation in prostate cancer cells in response to TGF-β, and inhibition of cell proliferation by TGF-β requires degradation of JunD protein.

  20. Hepatocyte Nuclear Factor 1A Is a Cell-Intrinsic Transcription Factor Required for B Cell Differentiation and Development in Mice.

    Science.gov (United States)

    von Wnuck Lipinski, Karin; Sattler, Katherine; Peters, Susann; Weske, Sarah; Keul, Petra; Klump, Hannes; Heusch, Gerd; Göthert, Joachim R; Levkau, Bodo

    2016-02-15

    The hepatocyte NF (HNF) family of transcription factors regulates the complex gene networks involved in lipid, carbohydrate, and protein metabolism. In humans, HNF1A mutations cause maturity onset of diabetes in the young type 3, whereas murine HNF6 participates in fetal liver B lymphopoiesis. In this study, we have identified a crucial role for the prototypical member of the family HNF1A in adult bone marrow B lymphopoiesis. HNF1A(-/-) mice exhibited a clear reduction in total blood and splenic B cells and a further pronounced one in transitional B cells. In HNF1A(-/-) bone marrow, all B cell progenitors-from pre-pro-/early pro-B cells to immature B cells-were dramatically reduced and their proliferation rate suppressed. IL-7 administration in vivo failed to boost B cell development in HNF1A(-/-) mice, whereas IL-7 stimulation of HNF1A(-/-) B cell progenitors in vitro revealed a marked impairment in STAT5 phosphorylation. The B cell differentiation potential of HNF1A(-/-) common lymphoid progenitors was severely impaired in vitro, and the expression of the B lymphopoiesis-promoting transcription factors E2A, EBF1, Pax5, and Bach2 was reduced in B cell progenitors in vivo. HNF1A(-/-) bone marrow chimera featured a dramatic defect in B lymphopoiesis recapitulating that of global HNF1A deficiency. The HNF1A(-/-) lymphopoiesis defect was confined to B cells as T lymphopoiesis was unaffected, and bone marrow common lymphoid progenitors and hematopoietic stem cells were even increased. Our data demonstrate that HNF1A is an important cell-intrinsic transcription factor in adult B lymphopoiesis and suggest the IL-7R/STAT5 module to be causally involved in mediating its function.

  1. Human T cell leukemia virus-I (HTLV-I) Tax-mediated apoptosis in activated T cells requires an enhanced intracellular prooxidant state

    OpenAIRE

    Los, Marek Jan; Khazaie, K; Schulze-Osthoff, Klaus; Baeuerle, P A; Schirrmacher, V.; Chlichlia, K.

    1998-01-01

    We have shown that an estradiol-dependent activation of human T cell leukemia virus-I Tax leads to the inhibition of cell proliferation and to the induction of apoptosis, The present study demonstrates that a hormone-dependent activation of Tax promotes an enhanced prooxidant state in stably transfected Jurkat cells as measured by changes in the intracellular levels of glutathione and H2O2; these changes are followed by apoptotic cell death. Additional stimulation of the CD3/TCR pathway enhan...

  2. The production of antibody by invading B cells is required for the clearance of rabies virus from the central nervous system.

    Directory of Open Access Journals (Sweden)

    D Craig Hooper

    Full Text Available BACKGROUND: The pathogenesis of rabies is associated with the inability to deliver immune effectors across the blood-brain barrier and to clear virulent rabies virus from CNS tissues. However, the mechanisms that facilitate immune effector entry into CNS tissues are induced by infection with attenuated rabies virus. METHODOLOGY/PRINCIPAL FINDINGS: Infection of normal mice with attenuated rabies virus but not immunization with killed virus can promote the clearance of pathogenic rabies virus from the CNS. T cell activity in B cell-deficient mice can control the replication of attenuated virus in the CNS, but viral mRNA persists. Low levels of passively administered rabies virus-neutralizing antibody reach infected cells in the cerebellum of B cell-deficient mice but are not sufficient to mediate virus clearance. Production of rabies virus-specific antibody by B cells invading CNS tissues is required for this process, and a substantial proportion of the B cells that accumulate in the CNS of mice infected with attenuated rabies virus produce virus-specific antibodies. CONCLUSIONS/SIGNIFICANCE: The mechanisms required for immune effectors to enter rabies virus-infected tissues are induced by infection with attenuated rabies virus but not by infection with pathogenic rabies viruses or immunization with killed virus. T cell activities can inhibit rabies virus replication, but the production of rabies virus-specific antibodies by infiltrating B cells, as opposed to the leakage of circulating antibody across the BBB, is critical to elimination of the virus. These findings suggest that a pathogenic rabies virus infection may be treatable after the virus has reached the CNS tissues, providing that the appropriate immune effectors can be targeted to the infected tissues.

  3. Idas, a novel phylogenetically conserved geminin-related protein, binds to geminin and is required for cell cycle progression.

    Science.gov (United States)

    Pefani, Dafni-Eleutheria; Dimaki, Maria; Spella, Magda; Karantzelis, Nickolas; Mitsiki, Eirini; Kyrousi, Christina; Symeonidou, Ioanna-Eleni; Perrakis, Anastassis; Taraviras, Stavros; Lygerou, Zoi

    2011-07-01

    Development and homeostasis of multicellular organisms relies on an intricate balance between cell proliferation and differentiation. Geminin regulates the cell cycle by directly binding and inhibiting the DNA replication licensing factor Cdt1. Geminin also interacts with transcriptional regulators of differentiation and chromatin remodelling factors, and its balanced interactions are implicated in proliferation-differentiation decisions during development. Here, we describe Idas (Idas being a cousin of the Gemini in Ancient Greek Mythology), a previously uncharacterised coiled-coil protein related to Geminin. We show that human Idas localizes to the nucleus, forms a complex with Geminin both in cells and in vitro through coiled-coil mediated interactions, and can change Geminin subcellular localization. Idas does not associate with Cdt1 and prevents Geminin from binding to Cdt1 in vitro. Idas depletion from cells affects cell cycle progression; cells accumulate in S phase and are unable to efficiently progress to mitosis. Idas protein levels decrease in anaphase, whereas its overexpression causes mitotic defects. During development, we show that Idas exhibits high level expression in the choroid plexus and the cortical hem of the mouse telencephalon. Our data highlight Idas as a novel Geminin binding partner, implicated in cell cycle progression, and a putative regulator of proliferation-differentiation decisions during development. PMID:21543332

  4. Rice Stomatal Closure Requires Guard Cell Plasma Membrane ATP-Binding Cassette Transporter RCN1/OsABCG5.

    Science.gov (United States)

    Matsuda, Shuichi; Takano, Sho; Sato, Moeko; Furukawa, Kaoru; Nagasawa, Hidetaka; Yoshikawa, Shoko; Kasuga, Jun; Tokuji, Yoshihiko; Yazaki, Kazufumi; Nakazono, Mikio; Takamure, Itsuro; Kato, Kiyoaki

    2016-03-01

    Water stress is one of the major environmental stresses that affect agricultural production worldwide. Water loss from plants occurs primarily through stomatal pores. Here, we report that an Oryza sativa half-size ATP-binding cassette (ABC) subfamily G protein, RCN1/OsABCG5, is involved in stomatal closure mediated by phytohormone abscisic acid (ABA) accumulation in guard cells. We found that the GFP-RCN1/OsABCG5-fusion protein was localized at the plasma membrane in guard cells. The percentage of guard cell pairs containing both ABA and GFP-RCN1/OsABCG5 increased after exogenous ABA treatment, whereas they were co-localized in guard cell pairs regardless of whether exogenous ABA was applied. ABA application resulted in a smaller increase in the percentage of guard cell pairs containing ABA in rcn1 mutant (A684P) and RCN1-RNAi than in wild-type plants. Furthermore, polyethylene glycol (drought stress)-inducible ABA accumulation in guard cells did not occur in rcn1 mutants. Stomata closure mediated by exogenous ABA application was strongly reduced in rcn1 mutants. Finally, rcn1 mutant plants had more rapid water loss from detached leaves than the wild-type plants. These results indicate that in response to drought stress, RCN1/OsABCG5 is involved in accumulation of ABA in guard cells, which is indispensable for stomatal closure. PMID:26708605

  5. A specific sorting signal is not required for the polarized secretion of newly synthesized proteins from cultured intestinal epithelial cells.

    Science.gov (United States)

    Rindler, M J; Traber, M G

    1988-08-01

    Caco-2 cells, derived from human colon, have the morphological, functional, and biochemical properties of small intestinal epithelial cells. After infection with enveloped viruses, influenza virions assembled at the apical plasma membrane while vesicular stomatitis virus (VSV) particles appeared exclusively at the basolateral membrane, similar to the pattern observed in virus-infected Madin-Darby canine kidney (MDCK). When grown in Millicell filter chamber devices and labeled with [35S]methionine, Caco-2 monolayers released all of their radiolabeled secretory products preferentially into the basal chamber. Among the proteins identified were apolipoproteins AI and E, transferrin, and alpha-fetoprotein. No proteins were observed to be secreted preferentially from the apical cell surface. The lysosomal enzyme beta-hexosaminidase was also secreted primarily from the basolateral surface of the cells in the presence or absence of lysosomotropic drugs or tunicamycin, which inhibit the targetting of lysosomal enzymes to lysosomes. Neither of these drug treatments significantly affected the polarized secretion of other nonlysosomal proteins. In addition, growth hormone (GH), which is released in a nonpolar fashion from MDCK cells, was secreted exclusively from the basolateral membrane after transfection of Caco-2 cells with GH cDNA in a pSV2-based expression vector. Similar results were obtained in transient expression experiments and after selection of permanently transformed Caco-2 cells expressing GH. Since both beta-hexosaminidase and GH would be expected to lack sorting signals for polarized exocytosis in epithelial cells, these results indicate that in intestinal cells, proteins transported via the basolateral secretory pathway need not have specific sorting signals.

  6. Hepatocyte nuclear factor 4α is required for cell differentiation and homeostasis in the adult mouse gastric epithelium.

    Science.gov (United States)

    Moore, Benjamin D; Khurana, Shradha S; Huh, Won Jae; Mills, Jason C

    2016-08-01

    We have previously shown that the sequential transcription factors Xbp1→Mist1 (Bhlha15) govern the ultrastructural maturation of the secretory apparatus in enzyme-secreting zymogenic chief cells (ZCs) in the gastric unit. Here we sought to identify transcriptional regulators upstream of X-box binding protein 1 (XBP1) and MIST1. We used immunohistochemistry to characterize Hnf4α(flox/flox) adult mouse stomachs after tamoxifen-induced deletion of Hnf4α We used qRT-PCR, Western blotting, and chromatin immunoprecipitation to define the molecular interaction between hepatocyte nuclear factor 4 alpha (HNF4α) and Xbp1 in mouse stomach and human gastric cells. We show that HNF4α protein is expressed in pit (foveolar) cells, mucous neck cells, and zymogenic chief cells (ZCs) of the corpus gastric unit. Loss of HNF4α in adult mouse stomach led to reduced ZC size and ER content, phenocopying previously characterized effects of Xbp1 deletion. However, HNF4α(Δ/Δ) stomachs also exhibited additional phenotypes including increased proliferation in the isthmal stem cell zone and altered mucous neck cell migration, indicating a role of HNF4α in progenitor cells as well as in ZCs. HNF4α directly occupies the Xbp1 promoter locus in mouse stomach, and forced HNF4α expression increased abundance of XBP1 mRNA in human gastric cancer cells. Finally, as expected, loss of HNF4α caused decreased Xbp1 and Mist1 expression in mouse stomachs. We show that HNF4α regulates homeostatic proliferation in the gastric epithelium and is both necessary and sufficient for the upstream regulation of the Xbp1→Mist1 axis in maintenance of ZC secretory architecture. PMID:27340127

  7. Lipopolysaccharide-Mediated Induction of Concurrent IL-1β and IL-23 Expression in THP-1 Cells Exhibits Differential Requirements for Caspase-1 and Cathepsin B Activity.

    Science.gov (United States)

    Wynick, Christopher; Petes, Carlene; Tigert, Alexander; Gee, Katrina

    2016-08-01

    The inflammasome is a multimeric protein complex required for interleukin (IL)-1β production. Upon lipopolysaccharide (LPS) triggering of toll-like receptor (TLR)-4 and subsequent ATP signaling, the NOD-like receptor containing-pyrin domain 3 (NLRP3) inflammasome is activated to cleave pro-caspase-1 into caspase-1, allowing the secretion of IL-1β. IL-1β is known to function with IL-23 in the regulation of IL-17-producing CD4(+) T cells, Th17 cells, in adaptive immunity. Recently, studies have shown that IL-1β and IL-23 together activate IL-17-producing innate lymphoid cells, demonstrating that the pair may exhibit additional effects on cell differentiation. Using an in vitro model of bacterial infection, LPS treatment of human monocytic cells, we investigated the molecular mechanisms involved in the co-expression of IL-1β and IL-23. We found that IL-1β is partially required for optimal LPS-induced IL-23 production. We also found that IL-23 production was partially dependent on ATP signaling via the P2X7 receptor, whereas IL-1β production required this signaling. Furthermore, we identified a novel role for cathepsin B activity in IL-23 production. Taken together, this study identifies differential requirements for the co-expression of IL-1β and IL-23. Due to their similar roles in Th17 differentiation, characterization of the regulatory mechanisms for LPS-induced IL-1β and IL-23 may reveal novel information into the pathology of the inflammatory response particularly during bacterial infection. PMID:27096899

  8. Transiently Active Wnt/β-Catenin Signaling Is Not Required but Must Be Silenced for Stem Cell Function during Muscle Regeneration

    Directory of Open Access Journals (Sweden)

    Malea M. Murphy

    2014-09-01

    Full Text Available Adult muscle’s exceptional capacity for regeneration is mediated by muscle stem cells, termed satellite cells. As with many stem cells, Wnt/β-catenin signaling has been proposed to be critical in satellite cells during regeneration. Using new genetic reagents, we explicitly test in vivo whether Wnt/β-catenin signaling is necessary and sufficient within satellite cells and their derivatives for regeneration. We find that signaling is transiently active in transit-amplifying myoblasts, but is not required for regeneration or satellite cell self-renewal. Instead, downregulation of transiently activated β-catenin is important to limit the regenerative response, as continuous regeneration is deleterious. Wnt/β-catenin activation in adult satellite cells may simply be a vestige of their developmental lineage, in which β-catenin signaling is critical for fetal myogenesis. In the adult, surprisingly, we show that it is not activation but rather silencing of Wnt/β-catenin signaling that is important for muscle regeneration.

  9. Double-stranded RNA-dependent protein kinase is required for bone calcification in MC3T3-E1 cells in vitro.

    Science.gov (United States)

    Yoshida, Kaya; Okamura, Hirohiko; Amorim, Bruna Rabelo; Ozaki, Akiko; Tanaka, Hiroaki; Morimoto, Hiroyuki; Haneji, Tatsuji

    2005-11-15

    In this study, we demonstrated that double-stranded RNA-dependent protein kinase (PKR) is required for the calcification of osteoblasts via the signal transducers and activators of transcription 1alpha (STAT1alpha) signaling in vitro. A dominant-negative mutant PKR cDNA, in which the amino acid lysine at 296 was replaced with arginine and which does not have catalytic activity, was transfected into mouse osteoblastic MC3T3-E1 cells; thereby, we established cells that stably expressed the PKR mutant gene (PKR-K/R). Phosphorylation of PKR was not stimulated by polyinosic-polycytidylic acid in the mutant cells. The PKR-K/R mutant cells exhibited up-regulated cell growth and had low alkaline phosphatase (ALP) activity. The PKR-K/R mutant cells were not able to form bone nodules in vitro. In the PKR-K/R mutant cells, runt-related gene 2 (Runx2)-mediated transcription decreased compared with the levels in the control cells. The expression of STAT1alpha protein increased and the protein was translocated to the nucleus in the PKR-K/R mutant cells. When the expression of STAT1alpha protein in PKR mutant cells was suppressed using RNAi, the activity of Runx2-mediated transcription recovered to the control level. Our results indicate that PKR is a stimulator of Runx2 transcription and is a negative modulator of STAT1alpha expression. Our findings also suggest that PKR plays important roles in the differentiation and calcification of osteoblasts by modulating STAT1alpha and/or Runx2 expression. PMID:16216244

  10. Analysis of Phosphorylation-dependent Protein Interactions of Adhesion and Degranulation Promoting Adaptor Protein (ADAP) Reveals Novel Interaction Partners Required for Chemokine-directed T cell Migration.

    Science.gov (United States)

    Kuropka, Benno; Witte, Amelie; Sticht, Jana; Waldt, Natalie; Majkut, Paul; Hackenberger, Christian P R; Schraven, Burkhart; Krause, Eberhard; Kliche, Stefanie; Freund, Christian

    2015-11-01

    Stimulation of T cells leads to distinct changes of their adhesive and migratory properties. Signal propagation from activated receptors to integrins depends on scaffolding proteins such as the adhesion and degranulation promoting adaptor protein (ADAP)(1). Here we have comprehensively investigated the phosphotyrosine interactome of ADAP in T cells and define known and novel interaction partners of functional relevance. While most phosphosites reside in unstructured regions of the protein, thereby defining classical SH2 domain interaction sites for master regulators of T cell signaling such as SLP76, Fyn-kinase, and NCK, other binding events depend on structural context. Interaction proteomics using different ADAP constructs comprising most of the known phosphotyrosine motifs as well as the structured domains confirm that a distinct set of proteins is attracted by pY571 of ADAP, including the ζ-chain-associated protein kinase of 70 kDa (ZAP70). The interaction of ADAP and ZAP70 is inducible upon stimulation either of the T cell receptor (TCR) or by chemokine. NMR spectroscopy reveals that the N-terminal SH2 domains within a ZAP70-tandem-SH2 construct is the major site of interaction with phosphorylated ADAP-hSH3(N) and microscale thermophoresis (MST) indicates an intermediate binding affinity (Kd = 2.3 μm). Interestingly, although T cell receptor dependent events such as T cell/antigen presenting cell (APC) conjugate formation and adhesion are not affected by mutation of Y571, migration of T cells along a chemokine gradient is compromised. Thus, although most phospho-sites in ADAP are linked to T cell receptor related functions we have identified a unique phosphotyrosine that is solely required for chemokine induced T cell behavior.

  11. Girdin/GIV is upregulated by cyclic tension, propagates mechanical signal transduction, and is required for the cellular proliferation and migration of MG-63 cells

    International Nuclear Information System (INIS)

    To explore how Girdin/GIV is regulated by cyclic tension and propagates downstream signals to affect cell proliferation and migration. Human osteoblast-like MG-63 cells were exposed to cyclic tension force at 4000 μstrain and 0.5 Hz for 6 h, produced by a four-point bending system. Cyclic tension force upregulated Girdin and Akt expression and phosphorylation in cultured MG-63 cells. Girdin and Akt each promoted the phosphorylation of the other under stimulated tension. In vitro MTT and transwell assays showed that Girdin and Akt are required for cell proliferation and migration during cellular quiescence. Moreover, STAT3 was determined to be essential for Girdin expression under stimulated tension force in the physiological condition, as well as for osteoblast proliferation and migration during quiescence. These findings suggest that the STAT3/Girdin/Akt pathway activates in osteoblasts in response to mechanical stimulation and may play a significant role in triggering osteoblast proliferation and migration during orthodontic treatment. - Highlights: • Tension force upregulates Girdin and Akt expression and phosphorylation. • Girdin and Akt promotes the phosphorylation of each other under tension stimulation. • Girdin and Akt are required for MG-63 cell proliferation and migration. • STAT3 is essential for Girdin expression after application of the tension forces

  12. Girdin/GIV is upregulated by cyclic tension, propagates mechanical signal transduction, and is required for the cellular proliferation and migration of MG-63 cells

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Jiang-Tian; Li, Yan; Yu, Bing; Gao, Guo-Jie; Zhou, Ting; Li, Song, E-mail: song_li59@126.com

    2015-08-21

    To explore how Girdin/GIV is regulated by cyclic tension and propagates downstream signals to affect cell proliferation and migration. Human osteoblast-like MG-63 cells were exposed to cyclic tension force at 4000 μstrain and 0.5 Hz for 6 h, produced by a four-point bending system. Cyclic tension force upregulated Girdin and Akt expression and phosphorylation in cultured MG-63 cells. Girdin and Akt each promoted the phosphorylation of the other under stimulated tension. In vitro MTT and transwell assays showed that Girdin and Akt are required for cell proliferation and migration during cellular quiescence. Moreover, STAT3 was determined to be essential for Girdin expression under stimulated tension force in the physiological condition, as well as for osteoblast proliferation and migration during quiescence. These findings suggest that the STAT3/Girdin/Akt pathway activates in osteoblasts in response to mechanical stimulation and may play a significant role in triggering osteoblast proliferation and migration during orthodontic treatment. - Highlights: • Tension force upregulates Girdin and Akt expression and phosphorylation. • Girdin and Akt promotes the phosphorylation of each other under tension stimulation. • Girdin and Akt are required for MG-63 cell proliferation and migration. • STAT3 is essential for Girdin expression after application of the tension forces.

  13. Ankyrin-B directs membrane tethering of periaxin and is required for maintenance of lens fiber cell hexagonal shape and mechanics.

    Science.gov (United States)

    Maddala, Rupalatha; Walters, Mark; Brophy, Peter J; Bennett, Vann; Rao, Ponugoti V

    2016-01-15

    Periaxin (Prx), a PDZ domain protein expressed preferentially in myelinating Schwann cells and lens fibers, plays a key role in membrane scaffolding and cytoarchitecture. Little is known, however, about how Prx is anchored to the plasma membrane. Here we report that ankyrin-B (AnkB), a well-characterized adaptor protein involved in linking the spectrin-actin cytoskeleton to integral membrane proteins, is required for membrane association of Prx in lens fibers and colocalizes with Prx in hexagonal fiber cells. Under AnkB haploinsufficiency, Prx accumulates in the soluble fraction with a concomitant loss from the membrane-enriched fraction of mouse lenses. Moreover, AnkB haploinsufficiency induced age-dependent disruptions in fiber cell hexagonal geometry and radial alignment and decreased compressive stiffness in mouse lenses parallel to the changes observed in Prx null mouse lens. Both AnkB- and Prx-deficient mice exhibit disruptions in membrane organization of the spectrin-actin network and the dystrophin-glycoprotein complex in lens fiber cells. Taken together, these observations reveal that AnkB is required for Prx membrane anchoring and for maintenance of lens fiber cell hexagonal geometry, membrane skeleton organization, and biomechanics. PMID:26538089

  14. Orphan nuclear receptor Nur77 is required for the differentiation of C6 glioma cells induced by cholera toxin

    Institute of Scientific and Technical Information of China (English)

    Dong XU; Yi-jun HUANG; Yan LI; Wei YIN; Guang-mei YAN

    2009-01-01

    Aim: To investigate a possible regulator gene involved in the cholera toxin-induced differentiation of rat C6 glioma cells. Methods: The global changes in the mRNA expression pattern induced by cholera toxin were analyzed using gene chip microarray. The selected gene was then silenced by RNA interference or overexpressed with an ORF plasmid to determine its necessity in this process. Results: Nur77, a member of the orphan nuclear receptor family (NR4A), was markedly up-regulated during the process of differentiation. Furthermore, RNAi of nur77 attenuated the induction effect of cholera toxin on C6 cells, whereas overexpression of nur77 led to similarly differentiated behavior, including morphologic and biomarker changes, as well as cell cycle arrest. Conclusion: Nur77 participated actively and essentially as an important regulator in the cholera toxin-induced differentiation of C6 cells.

  15. Requirement of Fra proteins for communication channels between cells in the filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120.

    Science.gov (United States)

    Omairi-Nasser, Amin; Mariscal, Vicente; Austin, Jotham R; Haselkorn, Robert

    2015-08-11

    The filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 differentiates specialized cells, heterocysts, that fix atmospheric nitrogen and transfer the fixed nitrogen to adjacent vegetative cells. Reciprocally, vegetative cells transfer fixed carbon to heterocysts. Several routes have been described for metabolite exchange within the filament, one of which involves communicating channels that penetrate the septum between adjacent cells. Several fra gene mutants were isolated 25 y ago on the basis of their phenotypes: inability to fix nitrogen and fragmentation of filaments upon transfer from N+ to N- media. Cryopreservation combined with electron tomography were used to investigate the role of three fra gene products in channel formation. FraC and FraG are clearly involved in channel formation, whereas FraD has a minor part. Additionally, FraG was located close to the cytoplasmic membrane and in the heterocyst neck, using immunogold labeling with antibody raised to the N-terminal domain of the FraG protein.

  16. WASH is required for the differentiation commitment of hematopoietic stem cells in a c-Myc–dependent manner

    OpenAIRE

    Xia, Pengyan; Wang, Shuo; Huang, Guanling; Zhu, Pingping; Li, Man; Ye, Buqing; Du, Ying; Fan, Zusen

    2014-01-01

    Hematopoiesis is fully dependent on hematopoietic stem cells (HSCs) that possess the capacity to self-renew and differentiate into all blood cell lineages. WASH, Wiskott–Aldrich syndrome protein (WASP) and SCAR homologue (WASH) is involved in endosomal sorting as an actin-nucleating protein. Here, we show that conditional WASH deletion in the hematopoietic system causes defective blood production of the host, leading to severe cytopenia and rapid anemia. WASH deficiency causes the accumulatio...

  17. Vaccine-induced protection against 3 systemic mycoses endemic to North America requires Th17 cells in mice

    OpenAIRE

    Wüthrich, Marcel; Gern, Benjamin; Hung, Chiung Yu; Ersland, Karen; Rocco, Nicole; Pick-Jacobs, John; Galles, Kevin; Filutowicz, Hanna; Warner, Thomas; Evans, Michael; Cole, Garry; Klein, Bruce

    2016-01-01

    Worldwide rates of systemic fungal infections, including three of the major pathogens responsible for such infections in North America (Coccidioides posadasii, Histoplasma capsulatum, and Blastomyces dermatitidis), have soared recently, spurring interest in developing vaccines. The development of Th1 cells is believed to be crucial for protective immunity against pathogenic fungi, whereas the role of Th17 cells is vigorously debated. In models of primary fungal infection, some studies have sh...

  18. Requirements for CD4+ cells and gamma interferon in resolution of established Cryptosporidium parvum infection in mice.

    OpenAIRE

    W. Chen; Harp, J A; Harmsen, A G

    1993-01-01

    The importance of CD4+ cells and gamma interferon (IFN-gamma) in the resolution of established Cryptosporidium parvum infection was investigated with a murine model of cryptosporidiosis in severe combined immunodeficient (SCID) mice. C. parvum-infected SCID mice were reconstituted with spleen cells from immunocompetent donors. The recipients were able to resolve their C. parvum infection by 17 days postreconstitution. Treatment of reconstituted SCID mice with either anti-CD4 monoclonal antibo...

  19. An early and massive wave of germinal cell apoptosis is required for the development of functional spermatogenesis.

    OpenAIRE

    Rodriguez, I; Ody, C; Araki, K; Garcia, I.; Vassalli, P.

    1997-01-01

    Transgenic mice expressing high levels of the BclxL or Bcl2 proteins in the male germinal cells show a highly abnormal adult spermatogenesis accompanied by sterility. This appears to result from the prevention of an early and massive wave of apoptosis in the testis, which occurs among germinal cells during the first round of spermatogenesis. In contrast, sporadic apoptosis among spermatogonia, which occurs in normal adult testis, is not prevented in adult transgenic mice. The physiological ea...

  20. Killing of p53-deficient hepatoma cells by parvovirus H-1 and chemotherapeutics requires promyelocytic leukemia protein

    Institute of Scientific and Technical Information of China (English)

    Maike Sieben; Markus Moehler; Kerstin Herzer; Maja Zeidler; Vera Heinrichs; Barbara Leuchs; Martin Schuler; Jan J Cornelis; Peter R Galle; Jean Rommelaere

    2008-01-01

    AIM: To evaluate the synergistic targeting and killing of human hepatocellular carcinoma (HCC) cells lacking p53 by the oncolytic autonomous parvovirus (PV) H-1 and chemotherapeutic agents and its dependence on functional promyelocytic leukemia protein (PML).METHODS: The role of p53 and PML in regulating cytotoxicity and gene transfer mediated by wild-type (wt)PV H-1 were explored in two pairs of isogenic human hepatoma cell lines with different p53 status.Furthermore,H-1 PV infection was combined with cytostatic drug treatment.RESULTS: While the HCC cells with different p53 status studied were all susceptible to H-1 PV-induced apoptosis,the cytotoxicity of H-1 PV was more pronounced in p53-negative than in p53-positive cells.Apoptosis rates in p53-negative cell lines treated by genotoxic drugs were further enhanced by a treatment with H-1 PV.In flow cytometric analyses,H-1 PV infection resulted in a reduction of the mitochondrial transmembrane potential.In addition,H-1 PV cells showed a significant increase in PML expression.Knocking down PML expression resulted in a striking reduction of the level of H-1 PV infected tumor cell death.CONCLUSION: H-1 PV is a suitable agent to circumvent the resistance of p53-negative HCC cells to genotoxic agents,and it enhances the apoptotic process which is dependent on functional PML.Thus,H-1 PV and its oncolytic vector derivatives may be considered as therapeutic options for HCC,particularly for p53-negative tumors.

  1. Oxidation reactions are required to produce atranorin from acetate by alginate-immobilized cells of Cladonia verticillaris.

    OpenAIRE

    Sebastián, B.; Fontaniella, B.; E. C. Pereira; Vicente, C

    2000-01-01

    Atranorin, a p-depside of the b-orcinol series, is produced by several Cladonia species. Immobilized cells of Cladonia verticillaris in calcium alginate are able to produce atranorin when they are supplied with 1.0 mM acetate as a precursor. Production of the depside is enhanced by adding an oxidant agent (NAD+ or FMN) to the incubation media and its secretion to these media is facilitated by permeabilizing the immobilized cells with 2 % iso-propanol.

  2. Expression of ceramide glucosyltransferases, which are essential for glycosphingolipid synthesis, is only required in a small subset of C. elegans cells

    DEFF Research Database (Denmark)

    Marza, Esther; Simonsen, Karina T; Færgeman, Nils J;

    2009-01-01

    Glycosphingolipids (GSLs) are glycosylated derivatives of ceramide in the lipid bilayer. Their ubiquitous distribution and complexity suggest that they have important functions, but what these are in vivo is still poorly understood. Here, we characterize the phenotype of Caenorhabditis elegans...... mutants with essentially no GSLs. The C. elegans genome encodes three ceramide glucosyltransferase (CGT) genes, which encode enzymes required for GSL biosynthesis. Animals lacking CGT do not synthesize GSLs, arrest growth at the first larval stage, and display defects in a subset of cells...... suggest that GSLs are dispensable in most C. elegans cells, including those of the nervous system....

  3. A novel mechanism for direct real-time polymerase chain reaction that does not require DNA isolation from prokaryotic cells.

    Science.gov (United States)

    Soejima, Takashi; Xiao, Jin-Zhong; Abe, Fumiaki

    2016-01-01

    Typically, polymerase chain reaction (PCR) is performed after DNA isolation. Real-time PCR (qPCR), also known as direct qPCR in mammalian cells with weak membranes, is a common technique using crude samples subjected to preliminary boiling to elute DNA. However, applying this methodology to prokaryotic cells, which have solid cell walls, in contrast to mammalian cells which immediately burst in water, can result in poor detection. We successfully achieved PCR elongation with the addition of 1.3 cfu of Cronobacter muytjensii to a newly developed direct qPCR master mix without performing any crude DNA extraction (detection limit of 1.6 × 10(0) cfu/ml for the test sample compared with a detection limit of 1.6 × 10(3) cfu/ml primarily for crude (boiling) or classical DNA isolation). We revealed that the chromosomal DNA retained in prokaryotic cells can function as a PCR template, similarly to the mechanism in in situ PCR. Elucidating this reaction mechanism may contribute to the development of an innovative master mix for direct qPCR to detect genes in a single bacterium with solid cell walls and might lead to numerous novel findings in prokaryotic genomics research. PMID:27334801

  4. Bst1 is required for Candida albicans infecting host via facilitating cell wall anchorage of Glycosylphosphatidyl inositol anchored proteins

    Science.gov (United States)

    Liu, Wei; Zou, Zui; Huang, Xin; Shen, Hui; He, Li Juan; Chen, Si Min; Li, Li Ping; Yan, Lan; Zhang, Shi Qun; Zhang, Jun Dong; Xu, Zheng; Xu, Guo Tong; An, Mao Mao; Jiang, Yuan Ying

    2016-01-01

    Glycosylphosphatidyl inositol anchored proteins (GPI-APs) on fungal cell wall are essential for invasive infections. While the function of inositol deacylation of GPI-APs in mammalian cells has been previously characterized the impact of inositol deacylation in fungi and implications to host infection remains largely unexplored. Herein we describe our identification of BST1, an inositol deacylase of GPI-Aps in Candida albicans, was critical for GPI-APs cell wall attachment and host infection. BST1-deficient C. albicans (bst1Δ/Δ) was associated with severely impaired cell wall anchorage of GPI-APs and subsequen unmasked β-(1,3)-glucan. Consistent with the aberrant cell wall structures, bst1Δ/Δ strain did not display an invasive ability and could be recognized more efficiently by host immune systems. Moreover, BST1 null mutants or those expressing Bst1 variants did not display inositol deacylation activity and exhibited severely attenuated virulence and reduced organic colonization in a murine systemic candidiasis model. Thus, Bst1 can facilitate cell wall anchorage of GPI-APs in C. albicans by inositol deacylation, and is critical for host invasion and immune escape. PMID:27708385

  5. Pep1, a secreted effector protein of Ustilago maydis, is required for successful invasion of plant cells.

    Directory of Open Access Journals (Sweden)

    Gunther Doehlemann

    2009-02-01

    Full Text Available The basidiomycete Ustilago maydis causes smut disease in maize. Colonization of the host plant is initiated by direct penetration of cuticle and cell wall of maize epidermis cells. The invading hyphae are surrounded by the plant plasma membrane and proliferate within the plant tissue. We identified a novel secreted protein, termed Pep1, that is essential for penetration. Disruption mutants of pep1 are not affected in saprophytic growth and develop normal infection structures. However, Deltapep1 mutants arrest during penetration of the epidermal cell and elicit a strong plant defense response. Using Affymetrix maize arrays, we identified 116 plant genes which are differentially regulated in Deltapep1 compared to wild type infections. Most of these genes are related to plant defense. By in vivo immunolocalization, live-cell imaging and plasmolysis approaches, we detected Pep1 in the apoplastic space as well as its accumulation at sites of cell-to-cell passages. Site-directed mutagenesis identified two of the four cysteine residues in Pep1 as essential for function, suggesting that the formation of disulfide bridges is crucial for proper protein folding. The barley covered smut fungus Ustilago hordei contains an ortholog of pep1 which is needed for penetration of barley and which is able to complement the U. maydis Deltapep1 mutant. Based on these results, we conclude that Pep1 has a conserved function essential for establishing compatibility that is not restricted to the U. maydis / maize interaction.

  6. Cutting Edge: B Cell-Intrinsic T-bet Expression Is Required To Control Chronic Viral Infection.

    Science.gov (United States)

    Barnett, Burton E; Staupe, Ryan P; Odorizzi, Pamela M; Palko, Olesya; Tomov, Vesselin T; Mahan, Alison E; Gunn, Bronwyn; Chen, Diana; Paley, Michael A; Alter, Galit; Reiner, Steven L; Lauer, Georg M; Teijaro, John R; Wherry, E John

    2016-08-15

    The role of Ab and B cells in preventing infection is established. In contrast, the role of B cell responses in containing chronic infections remains poorly understood. IgG2a (IgG1 in humans) can prevent acute infections, and T-bet promotes IgG2a isotype switching. However, whether IgG2a and B cell-expressed T-bet influence the host-pathogen balance during persisting infections is unclear. We demonstrate that B cell-specific loss of T-bet prevents control of persisting viral infection. T-bet in B cells controlled IgG2a production, as well as mucosal localization, proliferation, glycosylation, and a broad transcriptional program. T-bet controlled a broad antiviral program in addition to IgG2a because T-bet in B cells was important, even in the presence of virus-specific IgG2a. Our data support a model in which T-bet is a universal controller of antiviral immunity across multiple immune lineages. PMID:27430722

  7. The actin-related protein Sac1 is required for morphogenesis and cell wall integrity in Candida albicans.</