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Sample records for cell fusion induced

  1. Studies on virus-induced cell fusion. Progress report, August 1, 1975--April 30, 1976. [Herpes simplex

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    Person, S.

    1976-01-01

    Progress is reported on the following research projects: mechanism of cell fusion induced by fusion-causing mutants of herpes simplex virus type I; quantitative assays for kinetics of cell fusion; neutral sphingoglycolipids in wild type and mutant infected cells; effects of alteration in oligosaccharide metabolism on cell fusion; and blocking of fusion by ..beta..-galactosidase and NH/sub 4/Cl. (HLW)

  2. Laser-induced fusion of human embryonic stem cells with optical tweezers

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    Chen, Shuxun; Cheng, Jinping; Kong, Chi-Wing; Wang, Xiaolin; Han Cheng, Shuk; Li, Ronald A.; Sun, Dong

    2013-07-01

    We report a study on the laser-induced fusion of human embryonic stem cells (hESCs) at the single-cell level. Cells were manipulated by optical tweezers and fused under irradiation with pulsed UV laser at 355 nm. Successful fusion was indicated by green fluorescence protein transfer. The influence of laser pulse energy on the fusion efficiency was investigated. The fused products were viable as gauged by live cell staining. Successful fusion of hESCs with somatic cells was also demonstrated. The reported fusion outcome may facilitate studies of cell differentiation, maturation, and reprogramming.

  3. Laser-induced fusion of human embryonic stem cells with optical tweezers

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    Chen Shuxun; Wang Xiaolin; Sun Dong [Department of Mechanical and Biomedical Engineering, City University of Hong Kong (Hong Kong); Cheng Jinping; Han Cheng, Shuk [Department of Biology and Chemistry, City University of Hong Kong (Hong Kong); Kong, Chi-Wing [Stem Cell and Regenerative Medicine Consortium, and Departments of Medicine and Physiology, LKS Faculty of Medicine, University of Hong Kong (Hong Kong); Li, Ronald A. [Stem Cell and Regenerative Medicine Consortium, and Departments of Medicine and Physiology, LKS Faculty of Medicine, University of Hong Kong (Hong Kong); Center of Cardiovascular Research, Mount Sinai School of Medicine, New York, New York 10029 (United States)

    2013-07-15

    We report a study on the laser-induced fusion of human embryonic stem cells (hESCs) at the single-cell level. Cells were manipulated by optical tweezers and fused under irradiation with pulsed UV laser at 355 nm. Successful fusion was indicated by green fluorescence protein transfer. The influence of laser pulse energy on the fusion efficiency was investigated. The fused products were viable as gauged by live cell staining. Successful fusion of hESCs with somatic cells was also demonstrated. The reported fusion outcome may facilitate studies of cell differentiation, maturation, and reprogramming.

  4. Semiallogenic fusions of MSI+ tumor cells and activated B cells induce MSI-specific T cell responses

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    Klier Ulrike

    2011-09-01

    Full Text Available Abstract Background Various strategies have been developed to transfer tumor-specific antigens into antigen presenting cells in order to induce cytotoxic T cell responses against tumor cells. One approach uses cellular vaccines based on fusions of autologous antigen presenting cells and allogeneic tumor cells. The fusion cells combine antigenicity of the tumor cell with optimal immunostimulatory capacity of the antigen presenting cells. Microsatellite instability caused by mutational inactivation of DNA mismatch repair genes results in translational frameshifts when affecting coding regions. It has been shown by us and others that these mutant proteins lead to the presentation of immunogenic frameshift peptides that are - in principle - recognized by a multiplicity of effector T cells. Methods We chose microsatellite instability-induced frameshift antigens as ideal to test for induction of tumor specific T cell responses by semiallogenic fusions of microsatellite instable carcinoma cells with CD40-activated B cells. Two fusion clones of HCT116 with activated B cells were selected for stimulation of T cells autologous to the B cell fusion partner. Outgrowing T cells were phenotyped and tested in functional assays. Results The fusion clones expressed frameshift antigens as well as high amounts of MHC and costimulatory molecules. Autologous T cells stimulated with these fusions were predominantly CD4+, activated, and reacted specifically against the fusion clones and also against the tumor cell fusion partner. Interestingly, a response toward 6 frameshift-derived peptides (of 14 tested could be observed. Conclusion Cellular fusions of MSI+ carcinoma cells and activated B cells combine the antigen-presenting capacity of the B cell with the antigenic repertoire of the carcinoma cell. They present frameshift-derived peptides and can induce specific and fully functional T cells recognizing not only fusion cells but also the carcinoma cells. These

  5. Semiallogenic fusions of MSI+ tumor cells and activated B cells induce MSI-specific T cell responses

    International Nuclear Information System (INIS)

    Garbe, Yvette; Klier, Ulrike; Linnebacher, Michael

    2011-01-01

    Various strategies have been developed to transfer tumor-specific antigens into antigen presenting cells in order to induce cytotoxic T cell responses against tumor cells. One approach uses cellular vaccines based on fusions of autologous antigen presenting cells and allogeneic tumor cells. The fusion cells combine antigenicity of the tumor cell with optimal immunostimulatory capacity of the antigen presenting cells. Microsatellite instability caused by mutational inactivation of DNA mismatch repair genes results in translational frameshifts when affecting coding regions. It has been shown by us and others that these mutant proteins lead to the presentation of immunogenic frameshift peptides that are - in principle - recognized by a multiplicity of effector T cells. We chose microsatellite instability-induced frameshift antigens as ideal to test for induction of tumor specific T cell responses by semiallogenic fusions of microsatellite instable carcinoma cells with CD40-activated B cells. Two fusion clones of HCT116 with activated B cells were selected for stimulation of T cells autologous to the B cell fusion partner. Outgrowing T cells were phenotyped and tested in functional assays. The fusion clones expressed frameshift antigens as well as high amounts of MHC and costimulatory molecules. Autologous T cells stimulated with these fusions were predominantly CD4 + , activated, and reacted specifically against the fusion clones and also against the tumor cell fusion partner. Interestingly, a response toward 6 frameshift-derived peptides (of 14 tested) could be observed. Cellular fusions of MSI + carcinoma cells and activated B cells combine the antigen-presenting capacity of the B cell with the antigenic repertoire of the carcinoma cell. They present frameshift-derived peptides and can induce specific and fully functional T cells recognizing not only fusion cells but also the carcinoma cells. These hybrid cells may have great potential for cellular immunotherapy and

  6. Lipopolysaccharide-induced multinuclear cells: Increased internalization of polystyrene beads and possible signals for cell fusion

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    Nakanishi-Matsui, Mayumi, E-mail: nakanim@iwate-med.ac.jp; Yano, Shio; Futai, Masamitsu

    2013-11-01

    Highlights: •LPS induces multinuclear cells from murine macrophage-derived RAW264.7 cells. •Large beads are internalized by cells actively fusing to become multinuclear. •The multinuclear cell formation is inhibited by anti-inflammatory cytokine, IL10. •Signal transduction for cell fusion is different from that for inflammation. -- Abstract: A murine macrophage-derived line, RAW264.7, becomes multinuclear on stimulation with lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria. These multinuclear cells internalized more polystyrene beads than mononuclear cells or osteoclasts (Nakanishi-Matsui, M., Yano, S., Matsumoto, N., and Futai, M., 2012). In this study, we analyzed the time courses of cell fusion in the presence of large beads. They were internalized into cells actively fusing to become multinuclear. However, the multinuclear cells once formed showed only low phagocytosis activity. These results suggest that formation of the multinuclear cells and bead internalization took place simultaneously. The formation of multinuclear cells was blocked by inhibitors for phosphoinositide 3-kinase, phospholipase C, calcineurin, and c-Jun N-terminal kinase. In addition, interleukin 6 and 10 also exhibited inhibitory effects. These signaling molecules and cytokines may play a crucial role in the LPS-induced multinuclear cell formation.

  7. PKC-Mediated ZYG1 Phosphorylation Induces Fusion of Myoblasts as well as of Dictyostelium Cells

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    Aiko Amagai

    2012-01-01

    Full Text Available We have previously demonstrated that a novel protein ZYG1 induces sexual cell fusion (zygote formation of Dictyostelium cells. In the process of cell fusion, involvements of signal transduction pathways via Ca2+ and PKC (protein kinase C have been suggested because zygote formation is greatly enhanced by PKC activators. In fact, there are several deduced sites phosphorylated by PKC in ZYG1 protein. Thereupon, we designed the present work to examine whether or not ZYG1 is actually phosphorylated by PKC and localized at the regions of cell-cell contacts where cell fusion occurs. These were ascertained, suggesting that ZYG1 might be the target protein for PKC. A humanized version of zyg1 cDNA (mzyg1 was introduced into myoblasts to know if ZYG1 is also effective in cell fusion of myoblasts. Quite interestingly, enforced expression of ZYG1 in myoblasts was found to induce markedly their cell fusion, thus strongly suggesting the existence of a common signaling pathway for cell fusion beyond the difference of species.

  8. An unusual dependence of human herpesvirus-8 glycoproteins-induced cell-to-cell fusion on heparan sulfate

    International Nuclear Information System (INIS)

    Tiwari, Vaibhav; Darmani, Nissar A.; Thrush, Gerald R.; Shukla, Deepak

    2009-01-01

    Human herpesvirus-8 (HHV-8) is known to interact with cell surface heparan sulfate (HS) for entry into a target cell. Here we investigated the role of HS during HHV-8 glycoproteins-induced cell fusion. Interestingly, the observed fusion demonstrated an unusual dependence on HS as evident from following lines of evidence: (1) a significant reduction in cell-to-cell fusion occurred when target cells were treated with heparinase; (2) in a competition assay, when the effector cells expressing HHV-8 glycoproteins were challenged with soluble HS, cell-to-cell fusion was reduced; and, (3) co-expression of HHV-8 glycoproteins gH-gL on target cells resulted in inhibition of cell surface HS expression. Taken together, our results indicate that cell surface HS can play an additional role during HHV-8 pathogenesis.

  9. Macrophage traits in cancer cells are induced by macrophage-cancer cell fusion and cannot be explained by cellular interaction.

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    Shabo, Ivan; Midtbö, Kristine; Andersson, Henrik; Åkerlund, Emma; Olsson, Hans; Wegman, Pia; Gunnarsson, Cecilia; Lindström, Annelie

    2015-11-20

    Cell fusion is a natural process in normal development and tissue regeneration. Fusion between cancer cells and macrophages generates metastatic hybrids with genetic and phenotypic characteristics from both maternal cells. However, there are no clinical markers for detecting cell fusion in clinical context. Macrophage-specific antigen CD163 expression in tumor cells is reported in breast and colorectal cancers and proposed being caused by macrophages-cancer cell fusion in tumor stroma. The purpose of this study is to examine the cell fusion process as a biological explanation for macrophage phenotype in breast. Monocytes, harvested from male blood donor, were activated to M2 macrophages and co-cultured in ThinCert transwell system with GFP-labeled MCF-7 cancer cells. MCF7/macrophage hybrids were generated by spontaneous cell fusion, isolated by fluorescence-activated cell sorting and confirmed by fluorescence microscopy, short tandem repeats analysis and flow cytometry. CD163 expression was evaluated in breast tumor samples material from 127 women by immunohistochemistry. MCF-7/macrophage hybrids were generated spontaneously at average rate of 2 % and showed phenotypic and genetic traits from both maternal cells. CD163 expression in MCF-7 cells could not be induced by paracrine interaction with M2-activated macrophages. CD163 positive cancer cells in tumor sections grew in clonal collection and a cutoff point >25 % of positive cancer cells was significantly correlated to disease free and overall survival. In conclusion, macrophage traits in breast cancer might be caused by cell fusion rather than explained by paracrine cellular interaction. These data provide new insights into the role of cell fusion in breast cancer and contributes to the development of clinical markers to identify cell fusion.

  10. Myeloid and lymphoid contribution to non-haematopoietic lineages through irradiation-induced heterotypic cell fusion

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    Nygren, J.M.; Liuba, K.; Breitbach, M.

    2008-01-01

    is induced by organ-specific injuries or whole-body irradiation, which has been used in previous studies to condition recipients of bone marrow transplants. Our findings demonstrate that blood cells of the lymphoid and myeloid lineages contribute to various non-haematopoietic tissues by forming rare fusion...

  11. A Double-Switch Cell Fusion-Inducible Transgene Expression System for Neural Stem Cell-Based Antiglioma Gene Therapy

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    Yumei Luo

    2015-01-01

    Full Text Available Recent progress in neural stem cell- (NSC- based tumor-targeted gene therapy showed that NSC vectors expressing an artificially engineered viral fusogenic protein, VSV-G H162R, could cause tumor cell death specifically under acidic tumor microenvironment by syncytia formation; however, the killing efficiency still had much room to improve. In the view that coexpression of another antitumoral gene with VSV-G can augment the bystander effect, a synthetic regulatory system that triggers transgene expression in a cell fusion-inducible manner has been proposed. Here we have developed a double-switch cell fusion-inducible transgene expression system (DoFIT to drive transgene expression upon VSV-G-mediated NSC-glioma cell fusion. In this binary system, transgene expression is coregulated by a glioma-specific promoter and targeting sequences of a microRNA (miR that is highly expressed in NSCs but lowly expressed in glioma cells. Thus, transgene expression is “switched off” by the miR in NSC vectors, but after cell fusion with glioma cells, the miR is diluted and loses its suppressive effect. Meanwhile, in the syncytia, transgene expression is “switched on” by the glioma-specific promoter. Our in vitro and in vivo experimental data show that DoFIT successfully abolishes luciferase reporter gene expression in NSC vectors but activates it specifically after VSV-G-mediated NSC-glioma cell fusion.

  12. Cell fusion by ionizing radiation

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    Khair, M.B.

    1993-08-01

    The relevance and importance of cell fusion are illustrated by the notion that current interest in this phenomenon is shared by scientists in quite varied disciplines. The diversity of cellular membrane fusion phenomena could provoke one to think that there must be a multitude of mechanisms that can account for such diversity. But, in general, the mechanism for the fusion reaction itself could be very similar in many, or even all, cases. Cell fusion can be induced by several factors such as virus Sendai, polyethylene glycol, electric current and ionizing radiation. This article provides the reader with short view of recent progress in research on cell fusion and gives some explanations about fusion mechanisms. This study shows for the first time, the results of the cell fusion induced by ionizing radiations that we have obtained in our researches and the work performed by other groups. (author). 44 refs

  13. Unraveling a three-step spatiotemporal mechanism of triggering of receptor-induced Nipah virus fusion and cell entry.

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    Qian Liu

    Full Text Available Membrane fusion is essential for entry of the biomedically-important paramyxoviruses into their host cells (viral-cell fusion, and for syncytia formation (cell-cell fusion, often induced by paramyxoviral infections [e.g. those of the deadly Nipah virus (NiV]. For most paramyxoviruses, membrane fusion requires two viral glycoproteins. Upon receptor binding, the attachment glycoprotein (HN/H/G triggers the fusion glycoprotein (F to undergo conformational changes that merge viral and/or cell membranes. However, a significant knowledge gap remains on how HN/H/G couples cell receptor binding to F-triggering. Via interdisciplinary approaches we report the first comprehensive mechanism of NiV membrane fusion triggering, involving three spatiotemporally sequential cell receptor-induced conformational steps in NiV-G: two in the head and one in the stalk. Interestingly, a headless NiV-G mutant was able to trigger NiV-F, and the two head conformational steps were required for the exposure of the stalk domain. Moreover, the headless NiV-G prematurely triggered NiV-F on virions, indicating that the NiV-G head prevents premature triggering of NiV-F on virions by concealing a F-triggering stalk domain until the correct time and place: receptor-binding. Based on these and recent paramyxovirus findings, we present a comprehensive and fundamentally conserved mechanistic model of paramyxovirus membrane fusion triggering and cell entry.

  14. Macrophage traits in cancer cells are induced by macrophage-cancer cell fusion and cannot be explained by cellular interaction

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    Shabo, Ivan; Midtb?, Kristine; Andersson, Henrik; ?kerlund, Emma; Olsson, Hans; Wegman, Pia; Gunnarsson, Cecilia; Lindstr?m, Annelie

    2015-01-01

    Background: Cell fusion is a natural process in normal development and tissue regeneration. Fusion between cancer cells and macrophages generates metastatic hybrids with genetic and phenotypic characteristics from both maternal cells. However, there are no clinical markers for detecting cell fusion in clinical context. Macrophage-specific antigen CD163 expression in tumor cells is reported in breast and colorectal cancers and proposed being caused by macrophages-cancer cell fusion in tumor st...

  15. Studies on virus-induced cell fusion. Progress report, August 1, 1977--June 30, 1978

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    Person, S.

    1978-07-01

    We have previously postulated that wild-type Herpes Simplex Virus type I (HSV-1) infections are characterized by the presence of a fusion factor and a fusion inhibitor activity. The fusion inhibitor presumably is dominant so that a small fraction of cells fuse in a typical wild-type infection. Furthermore, the syn mutants isolated in our laboratory are thought to cause extensive cell fusion because the production of active fusion inhibitor in cell membranes is delayed. If mutations existed that altered both the fusion factor and fusion inhibitor activity then separate viruses containing these two mutations might be able to complement each other, each supplying the defective gene product missing in the other virus. This would produce a wild type and not a syncytial mutant response. Complementation tests between two viruses, tsB5 and syn 20, which are thought to contain defects in the production of active fusion factor and fusion inhibitor activity, respectively, were done. A wild-type response was observed indicating that the mutations affecting fusion were in two separate genes.

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

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

    2005-01-01

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

  17. Reprogramming of somatic cells induced by fusion of embryonic stem cells using hemagglutinating virus of Japan envelope (HVJ-E)

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    Yue, Xiao-shan [Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan); Department of Biomolecular Engineering, Graduate School of Bioscience and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501 (Japan); Fujishiro, Masako [Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan); Toyoda, Masashi [Department of Reproductive Biology, National Institute for Child Health and Development, 2-10-1, Okura, Setagaya-ku, Tokyo 157-8535 (Japan); Akaike, Toshihiro [Department of Biomolecular Engineering, Graduate School of Bioscience and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501 (Japan); Ito, Yoshihiro, E-mail: y-ito@riken.jp [Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan); Department of Biomolecular Engineering, Graduate School of Bioscience and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501 (Japan)

    2010-04-16

    In this research, hemagglutinating virus of Japan envelope (HVJ-E) was used to reprogram somatic cells by fusion with mouse embryonic stem (ES) cells. Neomycin-resistant mouse embryonic fibroblasts (MEFs) were used as somatic cells. Nanog-overexpressing puromycin-resistant EB3 cells were used as mouse ES cells. These two cells were fused by exposing to HVJ-E and the generated fusion cells were selected by puromycin and G418 to get the stable fusion cell line. The fusion cells form colonies in feeder-free culture system. Microsatellite analysis of the fusion cells showed that they possessed genes from both ES cells and fibroblasts. The fusion cells were tetraploid, had alkali phosphatase activity, and expressed stem cell marker genes such as Pou5f1, Nanog, and Sox2, but not the fibroblast cell marker genes such as Col1a1 and Col1a2. The pluripotency of fusion cells was confirmed by their expression of marker genes for all the three germ layers after differentiation induction, and by their ability to form teratoma which contained all the three primary layers. Our results show that HVJ-E can be used as a fusion reagent for reprogramming of somatic cells.

  18. Cell fusions in mammals

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    Larsson, Lars-Inge; Bjerregaard, Bolette; Talts, Jan Fredrik

    2008-01-01

    Cell fusions are important to fertilization, placentation, development of skeletal muscle and bone, calcium homeostasis and the immune defense system. Additionally, cell fusions participate in tissue repair and may be important to cancer development and progression. A large number of factors appear...... to regulate cell fusions, including receptors and ligands, membrane domain organizing proteins, proteases, signaling molecules and fusogenic proteins forming alpha-helical bundles that bring membranes close together. The syncytin family of proteins represent true fusogens and the founding member, syncytin-1......, has been documented to be involved in fusions between placental trophoblasts, between cancer cells and between cancer cells and host ells. We review the literature with emphasis on the syncytin family and propose that syncytins may represent universal fusogens in primates and rodents, which work...

  19. Translocation of cell penetrating peptides and calcium-induced membrane fusion share same mechanism

    Czech Academy of Sciences Publication Activity Database

    Magarkar, Aniket; Allolio, Christoph; Jurkiewicz, Piotr; Baxová, Katarína; Šachl, Radek; Horinek, D.; Heinz, V.; Rachel, R.; Ziegler, C.; Jungwirth, Pavel

    2017-01-01

    Roč. 46, Suppl 1 (2017), S386 ISSN 0175-7571. [IUPAB congress /19./ and EBSA congress /11./. 16.07.2017-20.07.2017, Edinburgh] Institutional support: RVO:61388963 ; RVO:61388955 Keywords : membrane interactions * membrane fusion * cell penetration Subject RIV: BO - Biophysics

  20. Cell fusion and nuclear fusion in plants.

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    Maruyama, Daisuke; Ohtsu, Mina; Higashiyama, Tetsuya

    2016-12-01

    Eukaryotic cells are surrounded by a plasma membrane and have a large nucleus containing the genomic DNA, which is enclosed by a nuclear envelope consisting of the outer and inner nuclear membranes. Although these membranes maintain the identity of cells, they sometimes fuse to each other, such as to produce a zygote during sexual reproduction or to give rise to other characteristically polyploid tissues. Recent studies have demonstrated that the mechanisms of plasma membrane or nuclear membrane fusion in plants are shared to some extent with those of yeasts and animals, despite the unique features of plant cells including thick cell walls and intercellular connections. Here, we summarize the key factors in the fusion of these membranes during plant reproduction, and also focus on "non-gametic cell fusion," which was thought to be rare in plant tissue, in which each cell is separated by a cell wall. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Andrographolide sensitizes cisplatin-induced apoptosis via suppression of autophagosome-lysosome fusion in human cancer cells.

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    Zhou, Jing; Hu, Shuai-Er; Tan, Shi-Hao; Cao, Ruoxi; Chen, Yiyang; Xia, Dajing; Zhu, Xinqiang; Yang, Xing-Fen; Ong, Choon-Nam; Shen, Han-Ming

    2012-03-01

    Suppression of autophagy has been increasingly recognized as a novel cancer therapeutic approach. Andrographolide (Andro), a diterpenoid lactone isolated from an herbal plant Andrographis paniculata, is known to possess anti-inflammatory and anticancer activity. In this study, we sought to examine the effect of Andro on autophagy, and to evaluate whether such effect is relevant to the sensitization effect of Andro on apoptosis induced by DNA damage agents in cancer cells. First, we found that Andro is able to significantly enhance autophagic markers in various cancer cell lines, including GFP-LC3 puncta and LC3-II level. Interestingly, Andro treatment also led to marked increase of p62 protein level and addition of chloroquine (CQ) failed to further enhance either LC3-II or p62 level, indicating that Andro is likely to suppress autophagic flux at the maturation and degradation stage. Next, we provided evidence that Andro inhibits autophagosome maturation not by affecting the lysosomal function, but by impairing autophagosome-lysosome fusion. Lastly, we demonstrated that treatment with cisplatin, a DNA damage agent, induces autophagy in cancer cells. Importantly, Andro is capable of sensitizing cisplatin-induced cell killing determined with both short-term apoptosis assays and long-term clonogenic test, via suppression of autophagy, a process independent of p53. In summary, these observations collectively suggest that Andro could be a promising anti-cancer agent in combination therapy via its potent inhibitory effect on autophagy by disrupting autophagosome-lysosome fusion.

  2. [Construction of genetically modified dendritic cell vaccine expressing bcr/abl fusion gene and inducing specific cytotoxic T lymphocytes to kill K562 cells in vitro].

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    Wang, Wen-Wen; Huang, Ren-Wei; Hu, Yuan; Li, Xu-Dong; Wang, Dong-Ning; He, Yi; Liu, Jia-Jun

    2009-06-01

    Specific immunological effect mediated by T lymphocytes plays an important role in treating chronic myelocytic leukemia (CML). Dendritic cells (DCs)-based immunotherapy has become popular in treating tumors. This study was to construct DC vaccines by transducing with replication-defective recombinant adenoviruses expressing bcr/abl fusion gene of CML, observe the lethal effects of specific cytotoxic T lymphocytes (CTLs) triggered by genetically modified DC vaccines expressing bcr/abl fusion gene against K562 cells in vitro. DNA fragment of bcr/abl fusion gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) to construct a recombinant adenovirus vector and produce recombinant adenoviruses. DCs were induced from peripheral blood monocytes in vitro, and transfected with recombinant adenoviruses or pulsed with peptide to induce specific CTLs. The lethal effect of CTLs against leukemic K562 cells in vitro was observed. We successfully constructed the replication-defective recombinant adenoviral vector expressing bcr/abl fusion gene. The recombinant adenoviruses we produced had a high virus titer of 2.0 x 10(10) pfu/mL. Transfection efficiency of DCs in vitro was 50%-60%. DC vaccines expressing bcr/abl fusion gene were successfully prepared and used to induce specific CTLs. With effector:target cell ratios of 40:1 and 20:1, the killing rates of K562 cells by CTLs were (47.6+/-4.7)% and (47.5+/-1.6)% in genetically modified DCs group, (25.8+/-4.4)% and (24.6+/-6.3)% in peptide-pulsed DCs group, and were (5.7+/-1.3)% and (4.5+/-1.6)% in control DCs group. The differences between every two groups were significant (all Pfusion gene has a stronger contribution than peptide-pulsed DCs in triggering specific CTLs against K562 cells.

  3. Asymmetric constriction of dividing Escherichia coli cells induced by expression of a fusion between two min proteins.

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    Rowlett, Veronica Wells; Margolin, William

    2014-06-01

    The Min system, consisting of MinC, MinD, and MinE, plays an important role in localizing the Escherichia coli cell division machinery to midcell by preventing FtsZ ring (Z ring) formation at cell poles. MinC has two domains, MinCn and MinCc, which both bind to FtsZ and act synergistically to inhibit FtsZ polymerization. Binary fission of E. coli usually proceeds symmetrically, with daughter cells at roughly 180° to each other. In contrast, we discovered that overproduction of an artificial MinCc-MinD fusion protein in the absence of other Min proteins induced frequent and dramatic jackknife-like bending of cells at division septa, with cell constriction predominantly on the outside of the bend. Mutations in the fusion known to disrupt MinCc-FtsZ, MinCc-MinD, or MinD-membrane interactions largely suppressed bending division. Imaging of FtsZ-green fluorescent protein (GFP) showed no obvious asymmetric localization of FtsZ during MinCc-MinD overproduction, suggesting that a downstream activity of the Z ring was inhibited asymmetrically. Consistent with this, MinCc-MinD fusions localized predominantly to segments of the Z ring at the inside of developing cell bends, while FtsA (but not ZipA) tended to localize to the outside. As FtsA is required for ring constriction, we propose that this asymmetric localization pattern blocks constriction of the inside of the septal ring while permitting continued constriction of the outside portion.

  4. Cell fusion in tumor progression: the isolation of cell fusion products by physical methods

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    Vincitorio Massimo

    2011-09-01

    Full Text Available Abstract Background Cell fusion induced by polyethylene glycol (PEG is an efficient but poorly controlled procedure for obtaining somatic cell hybrids used in gene mapping, monoclonal antibody production, and tumour immunotherapy. Genetic selection techniques and fluorescent cell sorting are usually employed to isolate cell fusion products, but both procedures have several drawbacks. Results Here we describe a simple improvement in PEG-mediated cell fusion that was obtained by modifying the standard single-step procedure. We found that the use of two PEG undertreatments obtains a better yield of cell fusion products than the standard method, and most of these products are bi- or trinucleated polykaryocytes. Fusion rate was quantified using fluorescent cell staining microscopy. We used this improved cell fusion and cell isolation method to compare giant cells obtained in vitro and giant cells obtained in vivo from patients with Hodgkin's disease and erythroleukemia. Conclusions In the present study we show how to improve PEG-mediated cell fusion and that cell separation by velocity sedimentation offers a simple alternative for the efficient purification of cell fusion products and to investigate giant cell formation in tumor development.

  5. Ubiquitin-fusion degradation pathway: A new strategy for inducing CD8 cells specific for mycobacterial HSP65

    International Nuclear Information System (INIS)

    Shen Jianying; Hisaeda, Hajime; Chou Bin; Yu Qingsheng; Tu Liping; Himeno, Kunisuke

    2008-01-01

    The ubiquitin-proteasome system (UPS) plays an indispensable role in inducing MHC class I-restricted CD8 + T cells. In this study, we exploited UPS to induce CD8 + T cells specific for mycobacterial HSP65 (mHSP65), one of the leading vaccine candidates against infection with Mycobacterium tuberculosis. A chimeric DNA termed pU-HSP65 encoding a fusion protein between murine ubiquitin and mHSP65 was constructed, and C57BL/6 (B6) mice were immunized with the DNA using gene gun bombardment. Mice immunized with the chimeric DNA acquired potent resistance against challenge with the syngeneic B16F1 melanoma cells transfected with the mHSP65 gene (HSP65/B16F1), compared with those immunized with DNA encoding only mHSP65. Splenocytes from the former group of mice showed a higher grade of cytotoxic activity against HSP65/B16F1 cells and contained a larger number of granzyme B- or IFN-γ-producing CD8 + T cells compared with those from the latter group of mice

  6. Copper deficiency alters cell bioenergetics and induces mitochondrial fusion through up-regulation of MFN2 and OPA1 in erythropoietic cells

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    Bustos, Rodrigo I.; Jensen, Erik L.; Ruiz, Lina M.; Rivera, Salvador; Ruiz, Sebastián [Center for Biomedical Research, Faculty of Biological Sciences and Faculty of Medicine, Universidad Andres Bello, Santiago (Chile); Simon, Felipe; Riedel, Claudia [Center for Biomedical Research, Faculty of Biological Sciences and Faculty of Medicine, Universidad Andres Bello, Santiago (Chile); Millennium Institute of Immunology and Immunotherapy, Santiago (Chile); Ferrick, David [Seahorse Bioscience, Billerica, MA (United States); Elorza, Alvaro A., E-mail: aelorza@unab.cl [Center for Biomedical Research, Faculty of Biological Sciences and Faculty of Medicine, Universidad Andres Bello, Santiago (Chile); Millennium Institute of Immunology and Immunotherapy, Santiago (Chile)

    2013-08-02

    Highlights: •In copper deficiency, cell proliferation is not affected. In turn, cell differentiation is impaired. •Enlarged mitochondria are due to up-regulation of MNF2 and OPA1. •Mitochondria turn off respiratory chain and ROS production. •Energy metabolism switch from mitochondria to glycolysis. -- Abstract: Copper is essential in cell physiology, participating in numerous enzyme reactions. In mitochondria, copper is a cofactor for respiratory complex IV, the cytochrome c oxidase. Low copper content is associated with anemia and the appearance of enlarged mitochondria in erythropoietic cells. These findings suggest a connection between copper metabolism and bioenergetics, mitochondrial dynamics and erythropoiesis, which has not been explored so far. Here, we describe that bathocuproine disulfonate-induced copper deficiency does not alter erythropoietic cell proliferation nor induce apoptosis. However it does impair erythroid differentiation, which is associated with a metabolic switch between the two main energy-generating pathways. That is, from mitochondrial function to glycolysis. Switching off mitochondria implies a reduction in oxygen consumption and ROS generation along with an increase in mitochondrial membrane potential. Mitochondrial fusion proteins MFN2 and OPA1 were up-regulated along with the ability of mitochondria to fuse. Morphometric analysis of mitochondria did not show changes in total mitochondrial biomass but rather bigger mitochondria because of increased fusion. Similar results were also obtained with human CD34+, which were induced to differentiate into red blood cells. In all, we have shown that adequate copper levels are important for maintaining proper mitochondrial function and for erythroid differentiation where the energy metabolic switch plus the up-regulation of fusion proteins define an adaptive response to copper deprivation to keep cells alive.

  7. Copper deficiency alters cell bioenergetics and induces mitochondrial fusion through up-regulation of MFN2 and OPA1 in erythropoietic cells

    International Nuclear Information System (INIS)

    Bustos, Rodrigo I.; Jensen, Erik L.; Ruiz, Lina M.; Rivera, Salvador; Ruiz, Sebastián; Simon, Felipe; Riedel, Claudia; Ferrick, David; Elorza, Alvaro A.

    2013-01-01

    Highlights: •In copper deficiency, cell proliferation is not affected. In turn, cell differentiation is impaired. •Enlarged mitochondria are due to up-regulation of MNF2 and OPA1. •Mitochondria turn off respiratory chain and ROS production. •Energy metabolism switch from mitochondria to glycolysis. -- Abstract: Copper is essential in cell physiology, participating in numerous enzyme reactions. In mitochondria, copper is a cofactor for respiratory complex IV, the cytochrome c oxidase. Low copper content is associated with anemia and the appearance of enlarged mitochondria in erythropoietic cells. These findings suggest a connection between copper metabolism and bioenergetics, mitochondrial dynamics and erythropoiesis, which has not been explored so far. Here, we describe that bathocuproine disulfonate-induced copper deficiency does not alter erythropoietic cell proliferation nor induce apoptosis. However it does impair erythroid differentiation, which is associated with a metabolic switch between the two main energy-generating pathways. That is, from mitochondrial function to glycolysis. Switching off mitochondria implies a reduction in oxygen consumption and ROS generation along with an increase in mitochondrial membrane potential. Mitochondrial fusion proteins MFN2 and OPA1 were up-regulated along with the ability of mitochondria to fuse. Morphometric analysis of mitochondria did not show changes in total mitochondrial biomass but rather bigger mitochondria because of increased fusion. Similar results were also obtained with human CD34+, which were induced to differentiate into red blood cells. In all, we have shown that adequate copper levels are important for maintaining proper mitochondrial function and for erythroid differentiation where the energy metabolic switch plus the up-regulation of fusion proteins define an adaptive response to copper deprivation to keep cells alive

  8. A Cell-Cell Fusion Assay to Assess Arenavirus Envelope Glycoprotein Membrane-Fusion Activity.

    Science.gov (United States)

    York, Joanne; Nunberg, Jack H

    2018-01-01

    For many viruses that enter their target cells through pH-dependent fusion of the viral and endosomal membranes, cell-cell fusion assays can provide an experimental platform for investigating the structure-function relationships that promote envelope glycoprotein membrane-fusion activity. Typically, these assays employ effector cells expressing the recombinant envelope glycoprotein on the cell surface and target cells engineered to quantitatively report fusion with the effector cell. In the protocol described here, Vero cells are transfected with a plasmid encoding the arenavirus envelope glycoprotein complex GPC and infected with the vTF7-3 vaccinia virus expressing the bacteriophage T7 RNA polymerase. These effector cells are mixed with target cells infected with the vCB21R-lacZ vaccinia virus encoding a β-galactosidase reporter under the control of the T7 promoter. Cell-cell fusion is induced upon exposure to low-pH medium (pH 5.0), and the resultant expression of the β-galactosidase reporter is quantitated using a chemiluminescent substrate. We have utilized this robust microplate cell-cell fusion assay extensively to study arenavirus entry and its inhibition by small-molecule fusion inhibitors.

  9. Induction of cell-cell fusion by ectromelia virus is not inhibited by its fusion inhibitory complex

    Directory of Open Access Journals (Sweden)

    Fuchs Pinhas

    2009-09-01

    Full Text Available Abstract Background Ectromelia virus, a member of the Orthopox genus, is the causative agent of the highly infectious mousepox disease. Previous studies have shown that different poxviruses induce cell-cell fusion which is manifested by the formation of multinucleated-giant cells (polykaryocytes. This phenomenon has been widely studied with vaccinia virus in conditions which require artificial acidification of the medium. Results We show that Ectromelia virus induces cell-cell fusion under neutral pH conditions and requires the presence of a sufficient amount of viral particles on the plasma membrane of infected cells. This could be achieved by infection with a replicating virus and its propagation in infected cells (fusion "from within" or by infection with a high amount of virus particles per cell (fusion "from without". Inhibition of virus maturation or inhibition of virus transport on microtubules towards the plasma membrane resulted in a complete inhibition of syncytia formation. We show that in contrast to vaccinia virus, Ectromelia virus induces cell-cell fusion irrespectively of its hemagglutination properties and cell-surface expression of the orthologs of the fusion inhibitory complex, A56 and K2. Additionally, cell-cell fusion was also detected in mice lungs following lethal respiratory infection. Conclusion Ectromelia virus induces spontaneous cell-cell fusion in-vitro and in-vivo although expressing an A56/K2 fusion inhibitory complex. This syncytia formation property cannot be attributed to the 37 amino acid deletion in ECTV A56.

  10. [Tat-based cell-cell fusion method for screening HIV-1 fusion inhibitors].

    Science.gov (United States)

    Wang, Xiaoli; Yang, Yishu; Shen, Sisi; Wang, Xianliang; Feng, Tian; Hu, Qin; Zeng, Yi

    2018-03-25

    An HIV-1 cell-cell fusion system was developed to screen HIV-1 entry inhibitors that block cell-cell fusion. In this system, the pEGFP-Tat plasmid was constructed and cotransfected into effector cells (HEK-293T) with HIV-1 envelope plasmid. TZM-bl cell, a genetically engineered cell line that expresses CD4, CXCR4, CCR5 as well as Tat-inducible β-galactosidase and luciferase reporter gene, was used as target cell. Thus, the co-culture of target cells and effector cells allows the cell fusion via Env and the activity of the fusion inhibitor can be quantified by measuring the reporter protein expression. The experimental parameters were optimized and 11 anti-HIV-1 agents including CCR5 antagonist maraviroc, reverse transcription inhibitor zidovudine (AZT) and integrase inhibitor raltegravir were tested. The result showed that the system exhibited high specificity and sensitivity. Two of eight tested anti-HIV-1 agents were found to block the cell-cell fusion. The system is suitable for efficient screening of HIV-1 cell-cell fusion inhibitors.

  11. Enhanced vaccine-induced CD8+ T cell responses to malaria antigen ME-TRAP by fusion to MHC class ii invariant chain.

    Directory of Open Access Journals (Sweden)

    Alexandra J Spencer

    Full Text Available The orthodox role of the invariant chain (CD74; Ii is in antigen presentation to CD4+ T cells, but enhanced CD8+ T cells responses have been reported after vaccination with vectored viral vaccines encoding a fusion of Ii to the antigen of interest. In this study we assessed whether fusion of the malarial antigen, ME-TRAP, to Ii could increase the vaccine-induced CD8+ T cell response. Following single or heterologous prime-boost vaccination of mice with a recombinant chimpanzee adenovirus vector, ChAd63, or recombinant modified vaccinia virus Ankara (MVA, higher frequencies of antigen-specific CD4+ and CD8+ T cells were observed, with the largest increases observed following a ChAd63-MVA heterologous prime-boost regimen. Studies in non-human primates confirmed the ability of Ii-fusion to augment the T cell response, where a 4-fold increase was maintained up to 11 weeks after the MVA boost. Of the numerous different approaches explored to increase vectored vaccine induced immunogenicity over the years, fusion to the invariant chain showed a consistent enhancement in CD8+ T cell responses across different animal species and may therefore find application in the development of vaccines against human malaria and other diseases where high levels of cell-mediated immunity are required.

  12. Respiratory syncytial virus fusion glycoprotein expressed in insect cells form protein nanoparticles that induce protective immunity in cotton rats.

    Directory of Open Access Journals (Sweden)

    Gale Smith

    Full Text Available Respiratory Syncytial Virus (RSV is an important viral agent causing severe respiratory tract disease in infants and children as well as in the elderly and immunocompromised individuals. The lack of a safe and effective RSV vaccine represents a major unmet medical need. RSV fusion (F surface glycoprotein was modified and cloned into a baculovirus vector for efficient expression in Sf9 insect cells. Recombinant RSV F was glycosylated and cleaved into covalently linked F2 and F1 polypeptides that formed homotrimers. RSV F extracted and purified from insect cell membranes assembled into 40 nm protein nanoparticles composed of multiple RSV F oligomers arranged in the form of rosettes. The immunogenicity and protective efficacy of purified RSV F nanoparticles was compared to live and formalin inactivated RSV in cotton rats. Immunized animals induced neutralizing serum antibodies, inhibited virus replication in the lungs, and had no signs of disease enhancement in the respiratory track of challenged animals. RSV F nanoparticles also induced IgG competitive for binding of palivizumab neutralizing monoclonal antibody to RSV F antigenic site II. Antibodies to this epitope are known to protect against RSV when passively administered in high risk infants. Together these data provide a rational for continued development a recombinant RSV F nanoparticle vaccine candidate.

  13. Herpes Simplex Virus 1 Glycoprotein M and the Membrane-Associated Protein UL11 Are Required for Virus-Induced Cell Fusion and Efficient Virus Entry

    Science.gov (United States)

    Kim, In-Joong; Chouljenko, Vladimir N.; Walker, Jason D.

    2013-01-01

    Herpes simplex virus 1 (HSV-1) facilitates virus entry into cells and cell-to-cell spread by mediating fusion of the viral envelope with cellular membranes and fusion of adjacent cellular membranes. Although virus strains isolated from herpetic lesions cause limited cell fusion in cell culture, clinical herpetic lesions typically contain large syncytia, underscoring the importance of cell-to-cell fusion in virus spread in infected tissues. Certain mutations in glycoprotein B (gB), gK, UL20, and other viral genes drastically enhance virus-induced cell fusion in vitro and in vivo. Recent work has suggested that gB is the sole fusogenic glycoprotein, regulated by interactions with the viral glycoproteins gD, gH/gL, and gK, membrane protein UL20, and cellular receptors. Recombinant viruses were constructed to abolish either gM or UL11 expression in the presence of strong syncytial mutations in either gB or gK. Virus-induced cell fusion caused by deletion of the carboxyl-terminal 28 amino acids of gB or the dominant syncytial mutation in gK (Ala to Val at amino acid 40) was drastically reduced in the absence of gM. Similarly, syncytial mutations in either gB or gK did not cause cell fusion in the absence of UL11. Neither the gM nor UL11 gene deletion substantially affected gB, gC, gD, gE, and gH glycoprotein synthesis and expression on infected cell surfaces. Two-way immunoprecipitation experiments revealed that the membrane protein UL20, which is found as a protein complex with gK, interacted with gM while gM did not interact with other viral glycoproteins. Viruses produced in the absence of gM or UL11 entered into cells more slowly than their parental wild-type virus strain. Collectively, these results indicate that gM and UL11 are required for efficient membrane fusion events during virus entry and virus spread. PMID:23678175

  14. Stem Cells in Spinal Fusion.

    Science.gov (United States)

    Robbins, Michael A; Haudenschild, Dominik R; Wegner, Adam M; Klineberg, Eric O

    2017-12-01

    Review of literature. This review of literature investigates the application of mesenchymal stem cells (MSCs) in spinal fusion, highlights potential uses in the development of bone grafts, and discusses limitations based on both preclinical and clinical models. A review of literature was conducted looking at current studies using stem cells for augmentation of spinal fusion in both animal and human models. Eleven preclinical studies were found that used various animal models. Average fusion rates across studies were 59.8% for autograft and 73.7% for stem cell-based grafts. Outcomes included manual palpation and stressing of the fusion, radiography, micro-computed tomography (μCT), and histological analysis. Fifteen clinical studies, 7 prospective and 8 retrospective, were found. Fusion rates ranged from 60% to 100%, averaging 87.1% in experimental groups and 87.2% in autograft control groups. It appears that there is minimal clinical difference between commercially available stem cells and bone marrow aspirates indicating that MSCs may be a good choice in a patient with poor marrow quality. Overcoming morbidity and limitations of autograft for spinal fusion, remains a significant problem for spinal surgeons and further studies are needed to determine the efficacy of stem cells in augmenting spinal fusion.

  15. Nuclear fusion-independent smooth muscle differentiation of human adipose-derived stem cells induced by a smooth muscle environment.

    Science.gov (United States)

    Zhang, Rong; Jack, Gregory S; Rao, Nagesh; Zuk, Patricia; Ignarro, Louis J; Wu, Benjamin; Rodríguez, Larissa V

    2012-03-01

    Human adipose-derived stem cells hASC have been isolated and were shown to have multilineage differentiation capacity. Although both plasticity and cell fusion have been suggested as mechanisms for cell differentiation in vivo, the effect of the local in vivo environment on the differentiation of adipose-derived stem cells has not been evaluated. We previously reported the in vitro capacity of smooth muscle differentiation of these cells. In this study, we evaluate the effect of an in vivo smooth muscle environment in the differentiation of hASC. We studied this by two experimental designs: (a) in vivo evaluation of smooth muscle differentiation of hASC injected into a smooth muscle environment and (b) in vitro evaluation of smooth muscle differentiation capacity of hASC exposed to bladder smooth muscle cells. Our results indicate a time-dependent differentiation of hASC into mature smooth muscle cells when these cells are injected into the smooth musculature of the urinary bladder. Similar findings were seen when the cells were cocultured in vitro with primary bladder smooth muscle cells. Chromosomal analysis demonstrated that microenvironment cues rather than nuclear fusion are responsible for this differentiation. We conclude that cell plasticity is present in hASCs, and their differentiation is accomplished in the absence of nuclear fusion. Copyright © 2011 AlphaMed Press.

  16. Matrix metalloproteinase-9 (MMP9) is involved in the TNF-α-induced fusion of human M13SV1-Cre breast epithelial cells and human MDA-MB-435-pFDR1 cancer cells.

    Science.gov (United States)

    Weiler, Julian; Mohr, Marieke; Zänker, Kurt S; Dittmar, Thomas

    2018-04-10

    In addition to physiological events such as fertilisation, placentation, osteoclastogenesis, or tissue regeneration/wound healing, cell fusion is involved in pathophysiological conditions such as cancer. Cell fusion, which applies to both the proteins and conditions that induce the merging of two or more cells, is not a fully understood process. Inflammation/pro-inflammatory cytokines might be a positive trigger for cell fusion. Using a Cre-LoxP-based cell fusion assay we demonstrated that the fusion between human M13SV1-Cre breast epithelial cells and human MDA-MB-435-pFDR1 cancer cells was induced by the pro-inflammatory cytokine tumour necrosis factor-α (TNF-α). The gene expression profile of the cells in the presence of TNF-α and under normoxic and hypoxic conditions was analysed by cDNA microarray analysis. cDNA microarray data were verified by qPCR, PCR, Western blot and zymography. Quantification of cell fusion events was determined by flow cytometry. Proteins of interest were either blocked or knocked-down using a specific inhibitor, siRNA or a blocking antibody. The data showed an up-regulation of various genes, including claudin-1 (CLDN1), ICAM1, CCL2 and MMP9 in M13SV1-Cre and/or MDA-MB-435-pFDR1 cells. Inhibition of these proteins using a blocking ICAM1 antibody, CLDN1 siRNA or an MMP9 inhibitor showed that only the blockage of MMP9 was correlated with a decreased fusion rate of the cells. Likewise, the tetracycline-based antibiotic minocycline, which exhibits anti-inflammatory properties, was also effective in both inhibiting the TNF-α-induced MMP9 expression in M13SV1-Cre cells and blocking the TNF-α-induced fusion frequency of human M13SV1-Cre breast epithelial cells and human MDA-MB-435-pFDR1 cancer cells. The matrix metalloproteinase-9 (MMP9) is most likely involved in the TNF-α-mediated fusion of human M13SV1-Cre breast epithelial cells and human MDA-MB-435-pFDR1 cancer cells. Likewise, our data indicate that the tetracycline

  17. The hallmarks of cell-cell fusion.

    Science.gov (United States)

    Hernández, Javier M; Podbilewicz, Benjamin

    2017-12-15

    Cell-cell fusion is essential for fertilization and organ development. Dedicated proteins known as fusogens are responsible for mediating membrane fusion. However, until recently, these proteins either remained unidentified or were poorly understood at the mechanistic level. Here, we review how fusogens surmount multiple energy barriers to mediate cell-cell fusion. We describe how early preparatory steps bring membranes to a distance of ∼10 nm, while fusogens act in the final approach between membranes. The mechanical force exerted by cell fusogens and the accompanying lipidic rearrangements constitute the hallmarks of cell-cell fusion. Finally, we discuss the relationship between viral and eukaryotic fusogens, highlight a classification scheme regrouping a superfamily of fusogens called Fusexins, and propose new questions and avenues of enquiry. © 2017. Published by The Company of Biologists Ltd.

  18. Laser induced fusion - theoretical aspects

    International Nuclear Information System (INIS)

    Lawande, S.V.; Gunye, M.R.

    1979-01-01

    The theoretical aspects of thermonuclear fusion induced by laser are discussed. After outlining the basic features and the energetics of laser fusion in the chapter 1, various non-linear mechanisms responsible for an enhanced absorption of laser energy into the plasma and the stimulated scattering processes which hinder the absorption are discussed in the second chapter on laser plasma interactions. The third chapter on gas dynamics and the shock phenomena presents the mathematical formulation of the compression to high densities of the core of the pellet for its implosion. A hydrodynamic model developed to stimulate the evolution of laser heated symmetric plasma is outlined in the chapter four on numerichigly relativistic noninteracting particles, regular bouncing states may occur at high densities, or at high temperatures. The latter case is considered in details for the collapse phase of a hot universe; lepton pair creation may completely decelerate the collapse of a hot hadronic plasma, provided the observational parameters, the Hubble constant Hsub(deg), the matter parameter Ωsub(deg) and the deceleration parameter qsub(deg) satisfy certain constraint conditions

  19. Activation of antitumor cytotoxic T lymphocytes by fusions of human dendritic cells and breast carcinoma cells

    Science.gov (United States)

    Gong, Jianlin; Avigan, David; Chen, Dongshu; Wu, Zekui; Koido, Shigeo; Kashiwaba, Masahiro; Kufe, Donald

    2000-03-01

    We have reported that fusions of murine dendritic cells (DCs) and murine carcinoma cells reverse unresponsiveness to tumor-associated antigens and induce the rejection of established metastases. In the present study, fusions were generated with primary human breast carcinoma cells and autologous DCs. Fusion cells coexpressed tumor-associated antigens and DC-derived costimulatory molecules. The fusion cells also retained the functional potency of DCs and stimulated autologous T cell proliferation. Significantly, the results show that autologous T cells are primed by the fusion cells to induce MHC class I-dependent lysis of autologous breast tumor cells. These findings demonstrate that fusions of human breast cancer cells and DCs activate T cell responses against autologous tumors.

  20. Pandemic H1N1 influenza A directly induces a robust and acute inflammatory gene signature in primary human bronchial epithelial cells downstream of membrane fusion

    Energy Technology Data Exchange (ETDEWEB)

    Paquette, Stéphane G. [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); Institute of Medical Science, Faculty of Medicine, University of Toronto, Toronto, Ontario (Canada); Banner, David [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); Chi, Le Thi Bao [Department of Microbiology, Hue University of Medicine and Pharmacy, Thua Thien Hue (Viet Nam); Carlo Urbani Centre, Hue University of Medicine and Pharmacy, Thua Thien Hue (Viet Nam); Leon, Alberto J. [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); International Institute of Infection and Immunity, Shantou University Medical College, Shantou, Guangdong (China); Xu, Luoling; Ran, Longsi [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); Huang, Stephen S.H. [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); Department of Immunology, Faculty of Medicine, University of Toronto, Toronto, Ontario (Canada); Farooqui, Amber [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); International Institute of Infection and Immunity, Shantou University Medical College, Shantou, Guangdong (China); and others

    2014-01-05

    Pandemic H1N1 influenza A (H1N1pdm) elicits stronger pulmonary inflammation than previously circulating seasonal H1N1 influenza A (sH1N1), yet mechanisms of inflammatory activation in respiratory epithelial cells during H1N1pdm infection are unclear. We investigated host responses to H1N1pdm/sH1N1 infection and virus entry mechanisms in primary human bronchial epithelial cells in vitro. H1N1pdm infection rapidly initiated a robust inflammatory gene signature (3 h post-infection) not elicited by sH1N1 infection. Protein secretion inhibition had no effect on gene induction. Infection with membrane fusion deficient H1N1pdm failed to induce robust inflammatory gene expression which was rescued with restoration of fusion ability, suggesting H1N1pdm directly triggered the inflammatory signature downstream of membrane fusion. Investigation of intra-virion components revealed H1N1pdm viral RNA (vRNA) triggered a stronger inflammatory phenotype than sH1N1 vRNA. Thus, our study is first to report H1N1pdm induces greater inflammatory gene expression than sH1N1 in vitro due to direct virus–epithelial cell interaction. - Highlights: • We investigated H1N1pdm/sH1N1 infection in primary epithelial cells. • H1N1pdm directly initiated a robust inflammatory gene signature, sH1N1 did not. • H1N1pdm viral RNA triggered a stronger response than sH1N1. • H1N1pdm induces greater response due to direct virus–cell interaction. • These results have potential to impact vaccine and therapeutic development.

  1. Pandemic H1N1 influenza A directly induces a robust and acute inflammatory gene signature in primary human bronchial epithelial cells downstream of membrane fusion

    International Nuclear Information System (INIS)

    Paquette, Stéphane G.; Banner, David; Chi, Le Thi Bao; Leon, Alberto J.; Xu, Luoling; Ran, Longsi; Huang, Stephen S.H.; Farooqui, Amber

    2014-01-01

    Pandemic H1N1 influenza A (H1N1pdm) elicits stronger pulmonary inflammation than previously circulating seasonal H1N1 influenza A (sH1N1), yet mechanisms of inflammatory activation in respiratory epithelial cells during H1N1pdm infection are unclear. We investigated host responses to H1N1pdm/sH1N1 infection and virus entry mechanisms in primary human bronchial epithelial cells in vitro. H1N1pdm infection rapidly initiated a robust inflammatory gene signature (3 h post-infection) not elicited by sH1N1 infection. Protein secretion inhibition had no effect on gene induction. Infection with membrane fusion deficient H1N1pdm failed to induce robust inflammatory gene expression which was rescued with restoration of fusion ability, suggesting H1N1pdm directly triggered the inflammatory signature downstream of membrane fusion. Investigation of intra-virion components revealed H1N1pdm viral RNA (vRNA) triggered a stronger inflammatory phenotype than sH1N1 vRNA. Thus, our study is first to report H1N1pdm induces greater inflammatory gene expression than sH1N1 in vitro due to direct virus–epithelial cell interaction. - Highlights: • We investigated H1N1pdm/sH1N1 infection in primary epithelial cells. • H1N1pdm directly initiated a robust inflammatory gene signature, sH1N1 did not. • H1N1pdm viral RNA triggered a stronger response than sH1N1. • H1N1pdm induces greater response due to direct virus–cell interaction. • These results have potential to impact vaccine and therapeutic development

  2. Genetic variability available through cell fusion

    Energy Technology Data Exchange (ETDEWEB)

    Smith, H.H.; Mastrangelo-Hough, I.A.

    1977-01-01

    Results are reported for the following studies: plant hybridization through protoplast fusion using species of Nicotiana and Petunia; chromosome instability studies on culture-induced chromosome changes and chromosome elimination; chloroplast distribution in parasexual hybrids; chromosomal introgression following fusion; plant-animal fusion; and microcell-mediated chromosome transfer and chromosome-mediated gene transfer. (HLW)

  3. Cell fusion in the liver, revisited.

    Science.gov (United States)

    Lizier, Michela; Castelli, Alessandra; Montagna, Cristina; Lucchini, Franco; Vezzoni, Paolo; Faggioli, Francesca

    2018-02-27

    There is wide agreement that cell fusion is a physiological process in cells in mammalian bone, muscle and placenta. In other organs, such as the cerebellum, cell fusion is controversial. The liver contains a considerable number of polyploid cells: They are commonly believed to originate by genome endoreplication, although the contribution of cell fusion to polyploidization has not been excluded. Here, we address the topic of cell fusion in the liver from a historical point of view. We discuss experimental evidence clearly supporting the hypothesis that cell fusion occurs in the liver, specifically when bone marrow cells were injected into mice and shown to rescue genetic hepatic degenerative defects. Those experiments-carried out in the latter half of the last century-were initially interpreted to show "transdifferentiation", but are now believed to demonstrate fusion between donor macrophages and host hepatocytes, raising the possibility that physiologically polyploid cells, such as hepatocytes, could originate, at least partially, through homotypic cell fusion. In support of the homotypic cell fusion hypothesis, we present new data generated using a chimera-based model, a much simpler model than those previously used. Cell fusion as a road to polyploidization in the liver has not been extensively investigated, and its contribution to a variety of conditions, such as viral infections, carcinogenesis and aging, remains unclear.

  4. Localization of a region in the fusion protein of avian metapneumovirus that modulates cell-cell fusion.

    Science.gov (United States)

    Wei, Yongwei; Feng, Kurtis; Yao, Xiangjie; Cai, Hui; Li, Junan; Mirza, Anne M; Iorio, Ronald M; Li, Jianrong

    2012-11-01

    The genus Metapneumovirus within the subfamily Pneumovirinae of the family Paramyxoviridae includes two members, human metapneumovirus (hMPV) and avian metapneumovirus (aMPV), causing respiratory tract infections in humans and birds, respectively. Paramyxoviruses enter host cells by fusing the viral envelope with a host cell membrane. Membrane fusion of hMPV appears to be unique, in that fusion of some hMPV strains requires low pH. Here, we show that the fusion (F) proteins of aMPV promote fusion in the absence of the attachment protein and low pH is not required. Furthermore, there are notable differences in cell-cell fusion among aMPV subtypes. Trypsin was required for cell-cell fusion induced by subtype B but not subtypes A and C. The F protein of aMPV subtype A was highly fusogenic, whereas those from subtypes B and C were not. By construction and evaluation of chimeric F proteins composed of domains from the F proteins of subtypes A and B, we localized a region composed of amino acid residues 170 to 338 in the F protein that is responsible for the hyperfusogenic phenotype of the F from subtype A. Further mutagenesis analysis revealed that residues R295, G297, and K323 in this region collectively contributed to the hyperfusogenicity. Taken together, we have identified a region in the aMPV F protein that modulates the extent of membrane fusion. A model for fusion consistent with these data is presented.

  5. Cancer Cell Fusion: Mechanisms Slowly Unravel

    Directory of Open Access Journals (Sweden)

    Felicite K. Noubissi

    2016-09-01

    Full Text Available Although molecular mechanisms and signaling pathways driving invasion and metastasis have been studied for many years, the origin of the population of metastatic cells within the primary tumor is still not well understood. About a century ago, Aichel proposed that cancer cell fusion was a mechanism of cancer metastasis. This hypothesis gained some support over the years, and recently became the focus of many studies that revealed increasing evidence pointing to the possibility that cancer cell fusion probably gives rise to the metastatic phenotype by generating widespread genetic and epigenetic diversity, leading to the emergence of critical populations needed to evolve resistance to the treatment and development of metastasis. In this review, we will discuss the clinical relevance of cancer cell fusion, describe emerging mechanisms of cancer cell fusion, address why inhibiting cancer cell fusion could represent a critical line of attack to limit drug resistance and to prevent metastasis, and suggest one new modality for doing so.

  6. Distinct Requirements for HIV-Cell Fusion and HIV-mediated Cell-Cell Fusion*

    Science.gov (United States)

    Kondo, Naoyuki; Marin, Mariana; Kim, Jeong Hwa; Desai, Tanay M.; Melikyan, Gregory B.

    2015-01-01

    Whether HIV-1 enters cells by fusing with the plasma membrane or with endosomes is a subject of active debate. The ability of HIV-1 to mediate fusion between adjacent cells, a process referred to as “fusion-from-without” (FFWO), shows that this virus can fuse with the plasma membrane. To compare FFWO occurring at the cell surface with HIV-cell fusion through a conventional entry route, we designed an experimental approach that enabled the measurements of both processes in the same sample. The following key differences were observed. First, a very small fraction of viruses fusing with target cells participated in FFWO. Second, whereas HIV-1 fusion with adherent cells was insensitive to actin inhibitors, post-CD4/coreceptor binding steps during FFWO were abrogated. A partial dependence of HIV-cell fusion on actin remodeling was observed in CD4+ T cells, but this effect appeared to be due to the actin dependence of virus uptake. Third, deletion of the cytoplasmic tail of HIV-1 gp41 dramatically enhanced the ability of the virus to promote FFWO, while having a modest effect on virus-cell fusion. Distinct efficiencies and actin dependences of FFWO versus HIV-cell fusion are consistent with the notion that, except for a minor fraction of particles that mediate fusion between the plasma membranes of adjacent cells, HIV-1 enters through an endocytic pathway. We surmise, however, that cell-cell contacts enabling HIV-1 fusion with the plasma membrane could be favored at the sites of high density of target cells, such as lymph nodes. PMID:25589785

  7. Recombinant flagellin-PAL fusion protein of Legionella pneumophila induced cell-mediated and protective immunity against bacteremia in BALB/c mice.

    Science.gov (United States)

    Mohabati Mobarez, Ashraf; Ahmadrajabi, Roya; Khoramabadi, Nima; Salmanian, Ali Hatef

    2017-09-08

    We report a new recombinant fusion protein composed of full-length Legionella pneumophila flagellin A and peptidoglycan-associated lipoprotein (PAL), rFLA-PAL, capable of inducing protective immunity against L. pneumophila. The recombinant protein was over expressed in Escherichia coli strain BL21 (DE3) using pET-28a (+) expression vector (pET28a-flaA-pal) and purified by Ni 2+ exchange chromatography. Immunological properties of rFLA-PAL were assessed in a mouse model. Female BALB/c mice, immunized with rFLA-PAL, exhibited a rapid increase in serum antibody concentration against each of its protein portions. Furthermore, a strong activation of both innate and adaptive cell-mediated immunity was observed as indicated by antigen-specific splenocyte proliferation, IFN-γ and IL-12 production, and early production of TNF-α in the serum and in splenocyte cultures which were separately assessed against PAL and FLA. BALB/c mice were challenged with a lethal dose of L. pneumophila intravenously. In a 10-days follow-up after intravenous lethal challenge with L. pneumophila, a 100% survival rate was observed for mice immunized with rFLA-PAL, same as for those immunized with a sublethal dose of L. pneumophila. Based on the potent immune responses observed in mice immunized with rFLA-PAL, this recombinant fusion protein could be a potential vaccine candidate against the intracellular pathogen L. pneumophila.

  8. Dissipative Particle Dynamics of tension-induced membrane fusion

    DEFF Research Database (Denmark)

    Shillcock, Julian C.

    2009-01-01

    Recent studies of tension-induced membrane fusion using dissipative particle dynamics (DPD) simulations are briefly reviewed. The stochastic nature of the fusion process makes it necessary to simulate a large number of fusion attempts in order to obtain reliable fusion statistics and to extract...... meaningful values for the fusion probability and the average fusion times. All successful fusion events follow the same pathway. In this fusion pathway, configurations of individual lipids play an important role. Fusion starts with individual lipids assuming a splayed tail configuration with one tail......, three sub-processes have been identified in the fusion pathway. Their energy barriers are estimated to lie in the range 8-15kBT. The fusion probability is found to possess a maximum at intermediate tension values. As one decreases the tension, the fusion probability seems to vanish before...

  9. Cell Therapy to Obtain Spinal Fusion

    National Research Council Canada - National Science Library

    Olmsted-Davis, Elizabeth A; Davis, Alan R; Heggeness, Michael; Gannon, Francis; Dickinson, Mary; Hipp, John; West, Jennifer

    2008-01-01

    .... Current procedures are highly invasive with limited success. The goal of this study is to develop a safe efficacious system for inducing spine fusion which will eliminate the need for invasive surgery...

  10. Cell Therapy to Obtain Spinal Fusion

    National Research Council Canada - National Science Library

    Olmsted-Davis, Elizabeth A; Davis, Alan R; Moran, Kevin M; West, Jennifer

    2007-01-01

    .... Current procedures are highly invasive with limited success. The goal of this study is to develop a safe efficacious system for inducing spine fusion which will eliminate the need for invasive surgery...

  11. Induction of cell-cell fusion from without by human herpesvirus 6B

    DEFF Research Database (Denmark)

    Pedersen, Simon Metz; Øster, Bodil; Bundgaard, Bettina

    2006-01-01

    Human herpesvirus (HHV) 6A induce fusion from without (FFWO), whereas HHV-6B is believed to be ineffective in this process. Here, we demonstrate that HHV-6B induces rapid fusion in both epithelial cells and lymphocytes. The fusion was identified 1 h postinfection, could be inhibited by antibodies...... to HHV-6B gH and to the cellular receptor CD46, and was dependent on virus titer but independent of de novo protein synthesis and UV inactivation of the virus. Comparisons indicate that HHV-6A is only 10-fold more effective in inducing FFWO than HHV-6B. These data demonstrate that HHV-6B can induce FFWO...

  12. Rho GTPase activity modulates paramyxovirus fusion protein-mediated cell-cell fusion

    International Nuclear Information System (INIS)

    Schowalter, Rachel M.; Wurth, Mark A.; Aguilar, Hector C.; Lee, Benhur; Moncman, Carole L.; McCann, Richard O.; Dutch, Rebecca Ellis

    2006-01-01

    The paramyxovirus fusion protein (F) promotes fusion of the viral envelope with the plasma membrane of target cells as well as cell-cell fusion. The plasma membrane is closely associated with the actin cytoskeleton, but the role of actin dynamics in paramyxovirus F-mediated membrane fusion is unclear. We examined cell-cell fusion promoted by two different paramyxovirus F proteins in three cell types in the presence of constitutively active Rho family GTPases, major cellular coordinators of actin dynamics. Reporter gene and syncytia assays demonstrated that expression of either Rac1 V12 or Cdc42 V12 could increase cell-cell fusion promoted by the Hendra or SV5 glycoproteins, though the effect was dependent on the cell type expressing the viral glycoproteins. In contrast, RhoA L63 decreased cell-cell fusion promoted by Hendra glycoproteins but had little affect on SV5 F-mediated fusion. Also, data suggested that GTPase activation in the viral glycoprotein-containing cell was primarily responsible for changes in fusion. Additionally, we found that activated Cdc42 promoted nuclear rearrangement in syncytia

  13. Localization of a Region in the Fusion Protein of Avian Metapneumovirus That Modulates Cell-Cell Fusion

    Science.gov (United States)

    Wei, Yongwei; Feng, Kurtis; Yao, Xiangjie; Cai, Hui; Li, Junan; Mirza, Anne M.; Iorio, Ronald M.

    2012-01-01

    The genus Metapneumovirus within the subfamily Pneumovirinae of the family Paramyxoviridae includes two members, human metapneumovirus (hMPV) and avian metapneumovirus (aMPV), causing respiratory tract infections in humans and birds, respectively. Paramyxoviruses enter host cells by fusing the viral envelope with a host cell membrane. Membrane fusion of hMPV appears to be unique, in that fusion of some hMPV strains requires low pH. Here, we show that the fusion (F) proteins of aMPV promote fusion in the absence of the attachment protein and low pH is not required. Furthermore, there are notable differences in cell-cell fusion among aMPV subtypes. Trypsin was required for cell-cell fusion induced by subtype B but not subtypes A and C. The F protein of aMPV subtype A was highly fusogenic, whereas those from subtypes B and C were not. By construction and evaluation of chimeric F proteins composed of domains from the F proteins of subtypes A and B, we localized a region composed of amino acid residues 170 to 338 in the F protein that is responsible for the hyperfusogenic phenotype of the F from subtype A. Further mutagenesis analysis revealed that residues R295, G297, and K323 in this region collectively contributed to the hyperfusogenicity. Taken together, we have identified a region in the aMPV F protein that modulates the extent of membrane fusion. A model for fusion consistent with these data is presented. PMID:22915815

  14. Three Cell Fusions during Double Fertilization.

    Science.gov (United States)

    Sprunck, Stefanie; Dresselhaus, Thomas

    2015-05-07

    Fertilization of both egg and central cell is a major distinguishing feature of flowering plants. Now, Maruyama et al. report a third cell fusion event between the persistent synergid and the fertilized central cell shortly after double fertilization in Arabidopsis. This causes rapid dilution of pollen tube attractant(s), preventing polytubey. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Microfluidic device for high-yield pairing and fusion of stem cells with somatic cells

    Science.gov (United States)

    Gel, Murat; Hirano, Kunio; Oana, Hidehiro; Kotera, Hidetoshi; Tada, Takashi; Washizu, Masao

    2011-12-01

    Electro cell fusion has significant potential as a biotechnology tool with applications ranging from antibody production to cellular reprogramming. However due to low fusion efficiency of the conventional electro fusion methodology the true potential of the technique has not been reached. In this paper, we report a new method which takes cell fusion efficiency two orders magnitude higher than the conventional electro fusion method. The new method, based on one-toone pairing, fusion and selection of fused cells was developed using a microfabricated device. The device was composed of two microfluidic channels, a micro slit array and a petri dish integrated with electrodes. The electrodes positioned in each channel were used to generate electric field lines concentrating in the micro slits. Cells were introduced into channels and brought in to contact through the micro slit array using dielectrophoresis. The cells in contact were fused by applying a DC pulse to electrodes. As the electric field lines were concentrated at the micro slits the membrane potential was induced only at the vicinity of the micro slits, namely only at the cell-cell contact point. This mechanism assured the minimum damage to cells in the fusion as well as the ability to control the strength and location of induced membrane potential. We introduced mouse embryonic stem cells and mouse embryonic fibroblasts to the microfluidic channels and demonstrated high-yield fusion (> 80%). Post-fusion study showed the method can generate viable hybrids of stem cells and embryonic fibroblasts. Multinucleated hybrid cells adhering on the chip surface were routinely obtained by using this method and on-chip culturing.

  16. High magnetic field induced otolith fusion in the zebrafish larvae.

    Science.gov (United States)

    Pais-Roldán, Patricia; Singh, Ajeet Pratap; Schulz, Hildegard; Yu, Xin

    2016-04-11

    Magnetoreception in animals illustrates the interaction of biological systems with the geomagnetic field (geoMF). However, there are few studies that identified the impact of high magnetic field (MF) exposure from Magnetic Resonance Imaging (MRI) scanners (>100,000 times of geoMF) on specific biological targets. Here, we investigated the effects of a 14 Tesla MRI scanner on zebrafish larvae. All zebrafish larvae aligned parallel to the B0 field, i.e. the static MF, in the MRI scanner. The two otoliths (ear stones) in the otic vesicles of zebrafish larvae older than 24 hours post fertilization (hpf) fused together after the high MF exposure as short as 2 hours, yielding a single-otolith phenotype with aberrant swimming behavior. The otolith fusion was blocked in zebrafish larvae under anesthesia or embedded in agarose. Hair cells may play an important role on the MF-induced otolith fusion. This work provided direct evidence to show that high MF interacts with the otic vesicle of zebrafish larvae and causes otolith fusion in an "all-or-none" manner. The MF-induced otolith fusion may facilitate the searching for MF sensors using genetically amenable vertebrate animal models, such as zebrafish.

  17. Cell fusion through a microslit between adhered cells and observation of their nuclear behavior.

    Science.gov (United States)

    Wada, Ken-Ichi; Hosokawa, Kazuo; Kondo, Eitaro; Ito, Yoshihiro; Maeda, Mizuo

    2014-07-01

    This paper describes a novel cell fusion method which induces cell fusion between adhered cells through a microslit for preventing nuclear mixing. For this purpose, a microfluidic device which had ∼ 100 cell pairing structures (CPSs) making cell pairs through microslits with 2.1 ± 0.3 µm width was fabricated. After trapping NIH3T3 cells with hydrodynamic forces at the CPSs, the cells were fused through the microslit by the Sendai virus envelope method. With following timelapse observation, we discovered that the spread cells were much less susceptible to nuclear migration passing through the microslit compared with round cells, and that cytoplasmic fraction containing mitochondria was transferred through the microslit without nuclear mixing. These findings will provide an effective method for cell fusion without nuclear mixing, and will lead to an efficient method for reprograming and transdifferentiation of target cells toward regenerative medicine. © 2014 Wiley Periodicals, Inc.

  18. Mesenchymal stem cells generate distinct functional hybrids in vitro via cell fusion or entosis.

    Science.gov (United States)

    Sottile, Francesco; Aulicino, Francesco; Theka, Ilda; Cosma, Maria Pia

    2016-11-09

    Homotypic and heterotypic cell-to-cell fusion are key processes during development and tissue regeneration. Nevertheless, aberrant cell fusion can contribute to tumour initiation and metastasis. Additionally, a form of cell-in-cell structure called entosis has been observed in several human tumours. Here we investigate cell-to-cell interaction between mouse mesenchymal stem cells (MSCs) and embryonic stem cells (ESCs). MSCs represent an important source of adult stem cells since they have great potential for regenerative medicine, even though they are also involved in cancer progression. We report that MSCs can either fuse forming heterokaryons, or be invaded by ESCs through entosis. While entosis-derived hybrids never share their genomes and induce degradation of the target cell, fusion-derived hybrids can convert into synkaryons. Importantly we show that hetero-to-synkaryon transition occurs through cell division and not by nuclear membrane fusion. Additionally, we also observe that the ROCK-actin/myosin pathway is required for both fusion and entosis in ESCs but only for entosis in MSCs. Overall, we show that MSCs can undergo fusion or entosis in culture by generating distinct functional cellular entities. These two processes are profoundly different and their outcomes should be considered given the beneficial or possible detrimental effects of MSC-based therapeutic applications.

  19. Gene expression profiling of Drosophila tracheal fusion cells.

    Science.gov (United States)

    Chandran, Rachana R; Iordanou, Ekaterini; Ajja, Crystal; Wille, Michael; Jiang, Lan

    2014-07-01

    The Drosophila trachea is a premier genetic system to investigate the fundamental mechanisms of tubular organ formation. Tracheal fusion cells lead the branch fusion process to form an interconnected tubular network. Therefore, fusion cells in the Drosophila trachea will be an excellent model to study branch fusion in mammalian tubular organs, such as kidneys and blood vessels. The fusion process is a dynamic cellular process involving cell migration, adhesion, vesicle trafficking, cytoskeleton rearrangement, and membrane fusion. To understand how these cellular events are coordinated, we initiated the critical step to assemble a gene expression profile of fusion cells. For this study, we analyzed the expression of 234 potential tracheal-expressed genes in fusion cells during fusion cell development. 143 Tracheal genes were found to encode transcription factors, signal proteins, cytoskeleton and matrix proteins, transporters, and proteins with unknown function. These genes were divided into four subgroups based on their levels of expression in fusion cells compared to neighboring non-fusion cells revealed by in situ hybridization: (1) genes that have relative high abundance in fusion cells, (2) genes that are dynamically expressed in fusion cells, (3) genes that have relative low abundance in fusion cells, and (4) genes that are expressed at similar levels in fusion cells and non-fusion tracheal cells. This study identifies the expression profile of fusion cells and hypothetically suggests genes which are necessary for the fusion process and which play roles in distinct stages of fusion, as indicated by the location and timing of expression. These data will provide the basis for a comprehensive understanding of the molecular and cellular mechanisms of branch fusion. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. A pharmacological study of Arabidopsis cell fusion between the persistent synergid and endosperm.

    Science.gov (United States)

    Motomura, Kazuki; Kawashima, Tomokazu; Berger, Frédéric; Kinoshita, Tetsu; Higashiyama, Tetsuya; Maruyama, Daisuke

    2018-01-29

    Cell fusion is a pivotal process in fertilization and multinucleate cell formation. A plant cell is ubiquitously surrounded by a hard cell wall, and very few cell fusions have been observed except for gamete fusions. We recently reported that the fertilized central cell (the endosperm) absorbs the persistent synergid, a highly differentiated cell necessary for pollen tube attraction. The synergid-endosperm fusion (SE fusion) appears to eliminate the persistent synergid from fertilized ovule in Arabidopsis thaliana Here, we analyzed the effects of various inhibitors on SE fusion in an in vitro culture system. Different from other cell fusions, neither disruption of actin polymerization nor protein secretion impaired SE fusion. However, transcriptional and translational inhibitors decreased the SE fusion success rate and also inhibited endosperm division. Failures of SE fusion and endosperm nuclear proliferation were also induced by roscovitine, an inhibitor of cyclin-dependent kinases (CDK). These data indicate unique aspects of SE fusion such as independence of filamentous actin support and the importance of CDK-mediated mitotic control. © 2018. Published by The Company of Biologists Ltd.

  1. Cell fusion assay by expression of respiratory syncytial virus (RSV) fusion protein to analyze the mutation of palivizumab-resistant strains.

    Science.gov (United States)

    Yasui, Yosuke; Yamaji, Yoshiaki; Sawada, Akihito; Ito, Takashi; Nakayama, Tetsuo

    2016-05-01

    Respiratory syncytial virus (RSV) consists of fusion (F), glyco (G), and small hydrophobic (SH) proteins as envelope proteins, and infects through cell fusion. F protein is expressed on the surface of infected cells, and induces cell fusion. In the present report, expression plasmids of the F, G and SH proteins were constructed and cell fusion activity was investigated under T7 RNA polymerase. F protein alone induced cell fusion at a lower concentration than previously reported, and co-expression of F and SH proteins induced cell fusion more efficiently than F protein alone. Palivizumab is the only prophylactic agent against RSV infection. Palivizumab-resistant strains having mutations of the F protein of K272E and S275F were reported. These mutations were introduced into an F-expression plasmid, and exhibited no inhibition of cell fusion with palivizumab. Among the RSV F protein mutants, N276S has been reported to have partial resistance against palivizumab, but the F expression plasmid with the N276S mutation showed a reduction in cell fusion in the presence of palivizumab, showing no resistance to palivizumab. The present expression system was useful for investigating the mechanisms of RSV cell fusion. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Cell fusion-independent differentiation of neural stem cells to the endothelial lineage.

    Science.gov (United States)

    Wurmser, Andrew E; Nakashima, Kinichi; Summers, Robert G; Toni, Nicolas; D'Amour, Kevin A; Lie, Dieter C; Gage, Fred H

    2004-07-15

    Somatic stem cells have been claimed to possess an unexpectedly broad differentiation potential (referred to here as plasticity) that could be induced by exposing stem cells to the extracellular developmental signals of other lineages in mixed-cell cultures. Recently, this and other experimental evidence supporting the existence of stem-cell plasticity have been refuted because stem cells have been shown to adopt the functional features of other lineages by means of cell-fusion-mediated acquisition of lineage-specific determinants (chromosomal DNA) rather than by signal-mediated differentiation. In this study we co-cultured mouse neural stem cells (NSCs), which are committed to become neurons and glial cells, with human endothelial cells, which form the lining of blood vessels. We show that in the presence of endothelial cells six per cent of the NSC population converted to cells that did not express neuronal or glial markers, but instead showed the stable expression of multiple endothelial markers and the capacity to form capillary networks. This was surprising because NSCs and endothelial cells are believed to develop from the ectoderm and mesoderm, respectively. Experiments in which endothelial cells were killed by fixation before co-culture with live NSCs (to prevent cell fusion) and karyotyping analyses, revealed that NSCs had differentiated into endothelial-like cells independently of cell fusion. We conclude that stem-cell plasticity is a true characteristic of NSCs and that the conversion of NSCs to unanticipated cell types can be accomplished without cell fusion.

  3. Paclitaxel stimulates chromosomal fusion and instability in cells with dysfunctional telomeres: Implication in multinucleation and chemosensitization

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jeong-Eun [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Woo, Seon Rang [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Department of Biochemistry, College of Medicine, Korea University, Seoul 136-705 (Korea, Republic of); Kang, Chang-Mo [Laboratory of Cytogenetics and Tissue Regeneration, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Juhn, Kyoung-Mi; Ju, Yeun-Jin; Shin, Hyun-Jin; Joo, Hyun-Yoo; Park, Eun Ran; Park, In-chul; Hong, Sung Hee; Hwang, Sang-Gu [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Lee, Jung-Kee [Department of Life Science and Genetic Engineering, Paichai University, Daejeon 302-735 (Korea, Republic of); Kim, Hae Kwon [Department of Biotechnology, Seoul Woman' s University, Seoul 139-774 (Korea, Republic of); Cho, Myung-Haing [Laboratory of Toxicology, College of Veterinary Medicine, Seoul National University, Seoul 151-74-2 (Korea, Republic of); Park, Gil Hong [Department of Biochemistry, College of Medicine, Korea University, Seoul 136-705 (Korea, Republic of); Lee, Kee-Ho, E-mail: khlee@kirams.re.kr [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of)

    2011-01-14

    Research highlights: {yields} Paclitaxel serves as a stimulator of chromosomal fusion in cells in which telomeres are dysfunctional. {yields} Typical fusions involve p-arms, but paclitaxel-induced fusions occur between both q- and p-arms. {yields} Paclitaxel-stimulated fusions in cells in which telomeres are dysfunctional evoke prolonged G2/M cell cycle arrest and delay multinucleation. {yields} Upon telomere erosion, paclitaxel promotes chromosomal instability and subsequent apoptosis. {yields} Chromosomal fusion enhances paclitaxel chemosensitivity under telomere dysfunction. -- Abstract: The anticancer effect of paclitaxel is attributable principally to irreversible promotion of microtubule stabilization and is hampered upon development of chemoresistance by tumor cells. Telomere shortening, and eventual telomere erosion, evoke chromosomal instability, resulting in particular cellular responses. Using telomerase-deficient cells derived from mTREC-/-p53-/- mice, here we show that, upon telomere erosion, paclitaxel propagates chromosomal instability by stimulating chromosomal end-to-end fusions and delaying the development of multinucleation. The end-to-end fusions involve both the p- and q-arms in cells in which telomeres are dysfunctional. Paclitaxel-induced chromosomal fusions were accompanied by prolonged G2/M cell cycle arrest, delayed multinucleation, and apoptosis. Telomere dysfunctional cells with mutlinucleation eventually underwent apoptosis. Thus, as telomere erosion proceeds, paclitaxel stimulates chromosomal fusion and instability, and both apoptosis and chemosensitization eventually develop.

  4. Tension-induced vesicle fusion: pathways and pore dynamics

    DEFF Research Database (Denmark)

    Shillcock, Julian C.

    2008-01-01

    The dynamics of tension-induced fusion of two vesicles is studied using dissipative particle dynamics (DPD) simulations. The vesicle membranes use an improved DPD parameter set that results in their sustaining only a 10–30% relative area stretch before rupturing on the microsecond timescale...... fusion time on membrane tension implies that the fusion process is completed by overcoming two energy barriers with scales of 13kBT and 11kBT. The fusion pore radius as a function of time has also been extracted from the simulations, and provides a quantitative measure of the fusion dynamics which...

  5. Induction of Cell-Cell Fusion by Ebola Virus Glycoprotein: Low pH Is Not a Trigger.

    Directory of Open Access Journals (Sweden)

    Ruben M Markosyan

    2016-01-01

    Full Text Available Ebola virus (EBOV is a highly pathogenic filovirus that causes hemorrhagic fever in humans and animals. Currently, how EBOV fuses its envelope membrane within an endosomal membrane to cause infection is poorly understood. We successfully measure cell-cell fusion mediated by the EBOV fusion protein, GP, assayed by the transfer of both cytoplasmic and membrane dyes. A small molecule fusion inhibitor, a neutralizing antibody, as well as mutations in EBOV GP known to reduce viral infection, all greatly reduce fusion. By monitoring redistribution of small aqueous dyes between cells and by electrical capacitance measurements, we discovered that EBOV GP-mediated fusion pores do not readily enlarge-a marked difference from the behavior of other viral fusion proteins. EBOV GP must be cleaved by late endosome-resident cathepsins B or L in order to become fusion-competent. Cleavage of cell surface-expressed GP appears to occur in endosomes, as evidenced by the fusion block imposed by cathepsin inhibitors, agents that raise endosomal pH, or an inhibitor of anterograde trafficking. Treating effector cells with a recombinant soluble cathepsin B or thermolysin, which cleaves GP into an active form, increases the extent of fusion, suggesting that a fraction of surface-expressed GP is not cleaved. Whereas the rate of fusion is increased by a brief exposure to acidic pH, fusion does occur at neutral pH. Importantly, the extent of fusion is independent of external pH in experiments in which cathepsin activity is blocked and EBOV GP is cleaved by thermolysin. These results imply that low pH promotes fusion through the well-known pH-dependent activity of cathepsins; fusion induced by cleaved EBOV GP is a process that is fundamentally independent of pH. The cell-cell fusion system has revealed some previously unappreciated features of EBOV entry, which could not be readily elucidated in the context of endosomal entry.

  6. The actin cytoskeleton inhibits pore expansion during PIV5 fusion protein-promoted cell-cell fusion

    OpenAIRE

    Wurth, Mark A.; Schowalter, Rachel M.; Smith, Everett Clinton; Moncman, Carole L.; Dutch, Rebecca Ellis; McCann, Richard O.

    2010-01-01

    Paramyxovirus fusion (F) proteins promote both virus-cell fusion, required for viral entry, and cell-cell fusion, resulting in syncytia formation. We used the F-actin stabilizing drug, jasplakinolide, and the G-actin sequestrant, latrunculin A, to examine the role of actin dynamics in cell-cell fusion mediated by the parainfluenza virus 5 (PIV5) F protein. Jasplakinolide treatment caused a dose-dependent increase in cell-cell fusion as measured by both syncytia and reporter gene assays, and l...

  7. Fusion of small unilamellar vesicles with viable EDTA-treated Escherichia coli cells.

    OpenAIRE

    Marvin, H J; ter Beest, M B; Hoekstra, D; Witholt, B

    1989-01-01

    Fusion characteristics of EDTA-treated Escherichia coli cells with small unilamellar vesicles were investigated, using a membrane fusion assay based on resonance energy transfer. Ca2+-EDTA treatments of Escherichia coli O111:B4 (wild type), E. coli C600 (rough), and E. coli D21f2 (deep rough) which permeabilize the outer membrane by inducing the release of lipopolysaccharide and outer membrane proteins resulted in fusion activity of the intact and viable bacteria with small unilamellar vesicl...

  8. Cell-surface phosphatidylserine regulates osteoclast precursor fusion.

    Science.gov (United States)

    Verma, Santosh K; Leikina, Evgenia; Melikov, Kamran; Gebert, Claudia; Kram, Vardit; Young, Marian F; Uygur, Berna; Chernomordik, Leonid V

    2018-01-05

    Bone-resorbing multinucleated osteoclasts that play a central role in the maintenance and repair of our bones are formed from bone marrow myeloid progenitor cells by a complex differentiation process that culminates in fusion of mononuclear osteoclast precursors. In this study, we uncoupled the cell fusion step from both pre-fusion stages of osteoclastogenic differentiation and the post-fusion expansion of the nascent fusion connections. We accumulated ready-to-fuse cells in the presence of the fusion inhibitor lysophosphatidylcholine and then removed the inhibitor to study synchronized cell fusion. We found that osteoclast fusion required the dendrocyte-expressed seven transmembrane protein (DC-STAMP)-dependent non-apoptotic exposure of phosphatidylserine at the surface of fusion-committed cells. Fusion also depended on extracellular annexins, phosphatidylserine-binding proteins, which, along with annexin-binding protein S100A4, regulated fusogenic activity of syncytin 1. Thus, in contrast to fusion processes mediated by a single protein, such as epithelial cell fusion in Caenorhabditis elegans , the cell fusion step in osteoclastogenesis is controlled by phosphatidylserine-regulated activity of several proteins.

  9. A sensitive HIV-1 envelope induced fusion assay identifies fusion enhancement of thrombin

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, De-Chun; Zhong, Guo-Cai; Su, Ju-Xiang [Department of Microbiology, Harbin Medical University, 194 Xuefu Road, Harbin, Heilongjiang 150081 (China); Liu, Yan-Hong [Second Affiliated Hospital of Harbin Medical University, 246 Xuefu Road, Harbin, Heilongjiang 150081 (China); Li, Yan; Wang, Jia-Ye [Department of Microbiology, Harbin Medical University, 194 Xuefu Road, Harbin, Heilongjiang 150081 (China); Hattori, Toshio [Department of Emerging Infectious Diseases, Division of Internal Medicine, Graduate School of Medicine, Tohoku University, Sendai 9808574 (Japan); Ling, Hong, E-mail: lingh@ems.hrbmu.edu.cn [Department of Microbiology, Harbin Medical University, 194 Xuefu Road, Harbin, Heilongjiang 150081 (China); Department of Parasitology, Harbin Medical University, 194 Xuefu Road, Harbin, Heilongjiang 150081 (China); Key Lab of Heilongjiang Province for Infection and Immunity, Key Lab of Heilongjiang Province Education Bureau for Etiology, Harbin, Heilongjiang 150081 (China); Zhang, Feng-Min, E-mail: fengminzhang@yahoo.com.cn [Department of Microbiology, Harbin Medical University, 194 Xuefu Road, Harbin, Heilongjiang 150081 (China); Key Lab of Heilongjiang Province for Infection and Immunity, Key Lab of Heilongjiang Province Education Bureau for Etiology, Harbin, Heilongjiang 150081 (China)

    2010-01-22

    To evaluate the interaction between HIV-1 envelope glycoprotein (Env) and target cell receptors, various cell-cell-fusion assays have been developed. In the present study, we established a novel fusion system. In this system, the expression of the sensitive reporter gene, firefly luciferase (FL) gene, in the target cells was used to evaluate cell fusion event. Simultaneously, constitutively expressed Renilla luciferase (RL) gene was used to monitor effector cell number and viability. FL gave a wider dynamic range than other known reporters and the introduction of RL made the assay accurate and reproducible. This system is especially beneficial for investigation of potential entry-influencing agents, for its power of ruling out the false inhibition or enhancement caused by the artificial cell-number variation. As a case study, we applied this fusion system to observe the effect of a serine protease, thrombin, on HIV Env-mediated cell-cell fusion and have found the fusion enhancement activity of thrombin over two R5-tropic HIV strains.

  10. Syncytin is involved in breast cancer-endothelial cell fusions

    DEFF Research Database (Denmark)

    Bjerregaard, Bolette; Holck, S.; Christensen, I.J.

    2006-01-01

    Cancer cells can fuse spontaneously with normal host cells, including endothelial cells, and such fusions may strongly modulate the biological behaviour of tumors. However, the underlying mechanisms are unknown. We now show that human breast cancer cell lines and 63 out of 165 (38%) breast cancer...... specimens express syncytin, an endogenous retroviral envelope protein, previously implicated in fusions between placental trophoblast cells. Additionally, endothelial and cancer cells are shown to express ASCT-2, a receptor for syncytin. Syncytin antisense treatment decreases syncytin expression...... and inhibits fusions between breast cancer cells and endothelial cells. Moreover, a syncytin inhibitory peptide also inhibits fusions between cancer and endothelial cells. These results are the first to show that syncytin is expressed by human cancer cells and is involved in cancer-endothelial cell fusions....

  11. Electrochemically induced nuclear fusion of deuterium

    International Nuclear Information System (INIS)

    Jorne, J.

    1990-01-01

    In this paper cold fusion of deuterium by electrolysis of heavy water onto a palladium (or titanium) cathode is reported. Contrary to the assumption of Fleishmann and Pons that electrochemically compressed D + exists inside the palladium cathode, the observations of Jones et al. can be partially explained by the simultaneous presence of deuteride D - and the highly mobile positive deuterium ion D + . The opposite charges reduce the intranuclear distance and enhance the tunneling fusion rate. Furthermore, alloying of lithium with palladium can stabilize a negatively charged deuteride ion due to the salinelike character of lithium deuteride. The enormous pressure (or fugacity), achieved by the applied electrochemical potential (10 30 atm), is a virtual pressure that would have existed in equilibrium with palladium deuteride (PdD x ). It is speculated that nuclear fusion occurs at the surface, and the PdD x serves as a reservoir for the supply of deuteride ions

  12. Oral cancer/endothelial cell fusion experiences nuclear fusion and acquisition of enhanced survival potential

    Energy Technology Data Exchange (ETDEWEB)

    Song, Kai [Department of Oral and Maxillofacial Surgery, The Affiliated Hospital of Qingdao University, Shandong Province (China); The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China); Song, Yong [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China); Department of Stomatology, Liu Zhou People' s Hospital, Guangxi (China); Zhao, Xiao-Ping; Shen, Hui; Wang, Meng; Yan, Ting-lin [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China); Liu, Ke, E-mail: liuke.1999@aliyun.com [Department of Oral and Maxillofacial-Head and Neck oncology, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China); The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China); Shang, Zheng-jun, E-mail: shangzhengjun@hotmail.com [Department of Oral and Maxillofacial-Head and Neck oncology, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China); The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China)

    2014-10-15

    Most previous studies have linked cancer–macrophage fusion with tumor progression and metastasis. However, the characteristics of hybrid cells derived from oral cancer and endothelial cells and their involvement in cancer remained unknown. Double-immunofluorescent staining and fluorescent in situ hybridization (FISH) were performed to confirm spontaneous cell fusion between eGFP-labeled human umbilical vein endothelial cells (HUVECs) and RFP-labeled SCC9, and to detect the expression of vementin and cytokeratin 18 in the hybrids. The property of chemo-resistance of such hybrids was examined by TUNEL assay. The hybrid cells in xenografted tumor were identified by FISH and GFP/RFP dual-immunofluoresence staining. We showed that SCC9 cells spontaneously fused with cocultured endothelial cells, and the resultant hybrid cells maintained the division and proliferation activity after re-plating and thawing. Such hybrids expressed markers of both parental cells and became more resistant to chemotherapeutic drug cisplatin as compared to the parental SCC9 cells. Our in vivo data indicated that the hybrid cells contributed to tumor composition by using of immunostaining and FISH analysis, even though the hybrid cells and SCC9 cells were mixed with 1:10,000, according to the FACS data. Our study suggested that the fusion events between oral cancer and endothelial cells undergo nuclear fusion and acquire a new property of drug resistance and consequently enhanced survival potential. These experimental findings provide further supportive evidence for the theory that cell fusion is involved in cancer progression. - Highlights: • The fusion events between oral cancer and endothelial cells undergo nuclear fusion. • The resulting hybrid cells acquire a new property of drug resistance. • The resulting hybrid cells express the markers of both parental cells (i.e. vimentin and cytokeratin 18). • The hybrid cells contribute to tumor repopulation in vivo.

  13. Laser induced photonuclear and fusion-reactions

    International Nuclear Information System (INIS)

    LoDato, V.A.

    1977-01-01

    The energy release from the fusion-fission pellets is demonstrated. It is shown that the coupling of the fusion-fission process is extremely efficient provided one can obtain the proper compression heating. The pellet of an outer core of (Li6D-Li6T) with an inner core of U238 is shown to be an efficient and practical fuel and can be ignited by the present generation of lasers to produce thermonuclear burn. The demonstration of the efficiency for photonuclear and photofission pellets is shown. However no suitable gamma ray source exists at present to initiate these processes. (orig.) [de

  14. The actin cytoskeleton inhibits pore expansion during PIV5 fusion protein-promoted cell-cell fusion

    International Nuclear Information System (INIS)

    Wurth, Mark A.; Schowalter, Rachel M.; Smith, Everett Clinton; Moncman, Carole L.; Ellis Dutch, Rebecca; McCann, Richard O.

    2010-01-01

    Paramyxovirus fusion (F) proteins promote both virus-cell fusion, required for viral entry, and cell-cell fusion, resulting in syncytia formation. We used the F-actin stabilizing drug, jasplakinolide, and the G-actin sequestrant, latrunculin A, to examine the role of actin dynamics in cell-cell fusion mediated by the parainfluenza virus 5 (PIV5) F protein. Jasplakinolide treatment caused a dose-dependent increase in cell-cell fusion as measured by both syncytia and reporter gene assays, and latrunculin A treatment also resulted in fusion stimulation. Treatment with jasplakinolide or latrunculin A partially rescued a fusion pore opening defect caused by deletion of the PIV5 F protein cytoplasmic tail, but these drugs had no effect on fusion inhibited at earlier stages by either temperature arrest or by a PIV5 heptad repeat peptide. These data suggest that the cortical actin cytoskeleton is an important regulator of fusion pore enlargement, an energetically costly stage of viral fusion protein-mediated membrane merger.

  15. Influenza-virus membrane fusion by cooperative fold-back of stochastically induced hemagglutinin intermediates.

    Science.gov (United States)

    Ivanovic, Tijana; Choi, Jason L; Whelan, Sean P; van Oijen, Antoine M; Harrison, Stephen C

    2013-02-19

    Influenza virus penetrates cells by fusion of viral and endosomal membranes catalyzed by the viral hemagglutinin (HA). Structures of the initial and final states of the HA trimer define the fusion endpoints, but do not specify intermediates. We have characterized these transitions by analyzing low-pH-induced fusion kinetics of individual virions and validated the analysis by computer simulation. We detect initial engagement with the target membrane of fusion peptides from independently triggered HAs within the larger virus-target contact patch; fusion then requires engagement of three or four neighboring HA trimers. Effects of mutations in HA indicate that withdrawal of the fusion peptide from a pocket in the pre-fusion trimer is rate-limiting for both events, but the requirement for cooperative action of several HAs to bring the fusing membranes together leads to a long-lived intermediate state for single, extended HA trimers. This intermediate is thus a fundamental aspect of the fusion mechanism. DOI:http://dx.doi.org/10.7554/eLife.00333.001.

  16. Renal epithelial cells can release ATP by vesicular fusion

    Directory of Open Access Journals (Sweden)

    Randi G Bjaelde

    2013-09-01

    Full Text Available Renal epithelial cells have the ability to release nucleotides as paracrine factors. In the intercalated cells of the collecting duct, ATP is released by connexin30 (cx30, which is selectively expressed in this cell type. However, ATP is released by virtually all renal epithelia and the aim of the present study was to identify possible alternative nucleotide release pathways in a renal epithelial cell model. We used MDCK (type1 cells to screen for various potential ATP release pathways. In these cells, inhibition of the vesicular H+-ATPases (bafilomycin reduced both the spontaneous and hypotonically (80%-induced nucleotide release. Interference with vesicular fusion using N-ethylamide markedly reduced the spontaneous nucleotide release, as did interference with trafficking from the endoplasmic reticulum to the Golgi apparatus (brefeldin A1 and vesicular transport (nocodazole. These findings were substantiated using a siRNA directed against SNAP-23, which significantly reduced spontaneous ATP release. Inhibition of pannexin and connexins did not affect the spontaneous ATP release in this cell type, which consists of ∼90% principal cells. TIRF-microscopy of either fluorescently-labeled ATP (MANT-ATP or quinacrine-loaded vesicles, revealed that spontaneous release of single vesicles could be promoted by either hypoosmolality (50% or ionomycin. This vesicular release decreased the overall cellular fluorescence by 5.8% and 7.6% respectively. In summary, this study supports the notion that spontaneous and induced ATP release can occur via exocytosis in renal epithelial cells.

  17. Localized cyclic AMP-dependent protein kinase activity is required for myogenic cell fusion

    International Nuclear Information System (INIS)

    Mukai, Atsushi; Hashimoto, Naohiro

    2008-01-01

    Multinucleated myotubes are formed by fusion of mononucleated myogenic progenitor cells (myoblasts) during terminal skeletal muscle differentiation. In addition, myoblasts fuse with myotubes, but terminally differentiated myotubes have not been shown to fuse with each other. We show here that an adenylate cyclase activator, forskolin, and other reagents that elevate intracellular cyclic AMP (cAMP) levels induced cell fusion between small bipolar myotubes in vitro. Then an extra-large myotube, designated a 'myosheet,' was produced by both primary and established mouse myogenic cells. Myotube-to-myotube fusion always occurred between the leading edge of lamellipodia at the polar end of one myotube and the lateral plasma membrane of the other. Forskolin enhanced the formation of lamellipodia where cAMP-dependent protein kinase (PKA) was accumulated. Blocking enzymatic activity or anchoring of PKA suppressed forskolin-enhanced lamellipodium formation and prevented fusion of multinucleated myotubes. Localized PKA activity was also required for fusion of mononucleated myoblasts. The present results suggest that localized PKA plays a pivotal role in the early steps of myogenic cell fusion, such as cell-to-cell contact/recognition through lamellipodium formation. Furthermore, the localized cAMP-PKA pathway might be involved in the specification of the fusion-competent areas of the plasma membrane in lamellipodia of myogenic cells

  18. BCR-ABL fusion genes are inducible by X-irradiation in vitro

    International Nuclear Information System (INIS)

    Ito, Takashi; Seyama, Toshio; Mizuno, Terumi; Hayashi, Tomonori; Nakamura, Nori; Akiyama, Mitoshi; Dohi, Kiyohiko.

    1992-01-01

    The Philadelphia chromosome consists of a reciprocal translocation between the ABL oncogene at chromosome 9q34 and the BCR gene at chromosome 22q resulting in the expression of chimeric BCR-ABL mRNAs specific to chronic myelogenous leukemia (CML). The presence of the fusion genes can be detected with high specificity and sensitivity by means of reverse transcription and polymerase chain reaction. Using this assay, it was possible to detect BCR-ABL fusion genes induced among HL60 cells after 100 Gy of X-irradiation in vitro. A total of five fusion gene transcripts were obtained. These fusion genes contained not only CML-specific BCR-ABL rearrangements, but also other forms of BCR-ABL fusions. These latter genes had junctions of BCR exon 4/ABL exon 2 intervened by a segment of DNA of unknown origin, BCR exon 5/ABL exon 2, and BCR exon 4/ABL exon 2. The results appear to be the first evidence for the induction of the BCR-ABL fusion gene by X-irradiation. In terms of leukemogenesis, it is suggested that only those cells bearing certain CML-related BCR-ABL fusion genes are positively selected by virtue of a growth advantage in vivo. (author)

  19. Quasi-elastic scattering an alternative tool for mapping the fusion barriers for heavy-ion induced fusion reaction

    International Nuclear Information System (INIS)

    Behera, B.R.

    2016-01-01

    Heavy element synthesis through heavy-ion induced fusion reaction is an active field in contemporary nuclear physics. Exact knowledge of fusion barrier is one of the essential parameters for planning any experiments for heavy element production. Theoretically there are many models available to predict the exact barrier. Though these models are successful for predicting the fusion of medium mass nuclei, it somehow fails for predicting the exact location of barrier for fusion of heavy nuclei. Experimental determination of barrier for such reactions is required for future experiments for the synthesis of heavy elements. Traditionally fusion barrier is determined taking a double derivative of fusion excitation function. However, such method is difficult in case of fusion of heavy nuclei due to its very low fusion/capture cross section and its experimental complications. Alternatively fusion barrier can be determined by measuring the quasi-elastic cross section at backward angles. This method can be applied for determining the fusion barrier for the fusion of heavy nuclei. Experimental determination of fusion barrier by different methods and comparison of the fusion excitation function and quasi-elastic scattering methods for the determination of fusion barrier are reviewed. At IUAC, New Delhi recently a program has been started for the measurement of fusion barrier through quasi-elastic scattering methods. The experimental facility and the first results of the experiments carried out with this facility are presented. (author)

  20. Monocyte-lymphocyte fusion induced by the HIV-1 envelope generates functional heterokaryons with an activated monocyte-like phenotype

    International Nuclear Information System (INIS)

    Martínez-Méndez, David; Rivera-Toledo, Evelyn; Ortega, Enrique; Licona-Limón, Ileana; Huerta, Leonor

    2017-01-01

    Enveloped viruses induce cell-cell fusion when infected cells expressing viral envelope proteins interact with target cells, or through the contact of cell-free viral particles with adjoining target cells. CD4 + T lymphocytes and cells from the monocyte-macrophage lineage express receptors for HIV envelope protein. We have previously reported that lymphoid Jurkat T cells expressing the HIV-1 envelope protein (Env) can fuse with THP-1 monocytic cells, forming heterokaryons with a predominantly myeloid phenotype. This study shows that the expression of monocytic markers in heterokaryons is stable, whereas the expression of lymphoid markers is mostly lost. Like THP-1 cells, heterokaryons exhibited FcγR-dependent phagocytic activity and showed an enhanced expression of the activation marker ICAM-1 upon stimulation with PMA. In addition, heterokaryons showed morphological changes compatible with maturation, and high expression of the differentiation marker CD11b in the absence of differentiation-inducing agents. No morphological change nor increase in CD11b expression were observed when an HIV-fusion inhibitor blocked fusion, or when THP-1 cells were cocultured with Jurkat cells expressing a non-fusogenic Env protein, showing that differentiation was not induced merely by cell-cell interaction but required cell-cell fusion. Inhibition of TLR2/TLR4 signaling by a TIRAP inhibitor greatly reduced the expression of CD11b in heterokaryons. Thus, lymphocyte-monocyte heterokaryons induced by HIV-1 Env are stable and functional, and fusion prompts a phenotype characteristic of activated monocytes via intracellular TLR2/TLR4 signaling. - Highlights: • Jurkat T cells expressing the HIV-1 envelope fuse with THP-1 monocytes. • Heterokaryons display a dominant myeloid phenotype and monocyte function. • Heterokaryons exhibit activation features in the absence of activation agents. • Activation is not due to cell-cell interaction but requires cell-cell fusion. • The

  1. Monocyte-lymphocyte fusion induced by the HIV-1 envelope generates functional heterokaryons with an activated monocyte-like phenotype

    Energy Technology Data Exchange (ETDEWEB)

    Martínez-Méndez, David; Rivera-Toledo, Evelyn; Ortega, Enrique; Licona-Limón, Ileana; Huerta, Leonor, E-mail: leonorhh@biomedicas.unam.mx

    2017-03-01

    Enveloped viruses induce cell-cell fusion when infected cells expressing viral envelope proteins interact with target cells, or through the contact of cell-free viral particles with adjoining target cells. CD4{sup +} T lymphocytes and cells from the monocyte-macrophage lineage express receptors for HIV envelope protein. We have previously reported that lymphoid Jurkat T cells expressing the HIV-1 envelope protein (Env) can fuse with THP-1 monocytic cells, forming heterokaryons with a predominantly myeloid phenotype. This study shows that the expression of monocytic markers in heterokaryons is stable, whereas the expression of lymphoid markers is mostly lost. Like THP-1 cells, heterokaryons exhibited FcγR-dependent phagocytic activity and showed an enhanced expression of the activation marker ICAM-1 upon stimulation with PMA. In addition, heterokaryons showed morphological changes compatible with maturation, and high expression of the differentiation marker CD11b in the absence of differentiation-inducing agents. No morphological change nor increase in CD11b expression were observed when an HIV-fusion inhibitor blocked fusion, or when THP-1 cells were cocultured with Jurkat cells expressing a non-fusogenic Env protein, showing that differentiation was not induced merely by cell-cell interaction but required cell-cell fusion. Inhibition of TLR2/TLR4 signaling by a TIRAP inhibitor greatly reduced the expression of CD11b in heterokaryons. Thus, lymphocyte-monocyte heterokaryons induced by HIV-1 Env are stable and functional, and fusion prompts a phenotype characteristic of activated monocytes via intracellular TLR2/TLR4 signaling. - Highlights: • Jurkat T cells expressing the HIV-1 envelope fuse with THP-1 monocytes. • Heterokaryons display a dominant myeloid phenotype and monocyte function. • Heterokaryons exhibit activation features in the absence of activation agents. • Activation is not due to cell-cell interaction but requires cell-cell fusion. • The

  2. High-Frequency Gravitational Wave Induced Nuclear Fusion

    International Nuclear Information System (INIS)

    Fontana, Giorgio; Baker, Robert M. L. Jr.

    2007-01-01

    Nuclear fusion is a process in which nuclei, having a total initial mass, combine to produce a single nucleus, having a final mass less than the total initial mass. Below a given atomic number the process is exothermic; that is, since the final mass is less than the combined initial mass and the mass deficit is converted into energy by the nuclear fusion. On Earth nuclear fusion does not happen spontaneously because electrostatic barriers prevent the phenomenon. To induce controlled, industrial scale, nuclear fusion, only a few methods have been discovered that look promising, but net positive energy production is not yet possible because of low overall efficiency of the systems. In this paper we propose that an intense burst of High Frequency Gravitational Waves (HFGWs) could be focused or beamed to a target mass composed of appropriate fuel or target material to efficiently rearrange the atomic or nuclear structure of the target material with consequent nuclear fusion. Provided that efficient generation of HFGW can be technically achieved, the proposed fusion reactor could become a viable solution for the energy needs of mankind and alternatively a process for beaming energy to produce a source of fusion energy remotely - even inside solid materials

  3. Tension-induced fusion of bilayer membranes and vesicles

    Science.gov (United States)

    Shillcock, Julian C.; Lipowsky, Reinhard

    2005-03-01

    Maintaining the integrity of their protective plasma membrane is a primary requirement of cells. Accordingly, cellular events that breach the membrane are tightly regulated. Artificial vesicles used in drug delivery must also stay intact until they have reached the desired target. In both cases, the intrinsic resistance of the membrane to rupture must be overcome to allow the efflux of the vesicle's contents. Here, we use mesoscopic simulations to study the fusion of 28-nm-diameter vesicles to 50 × 50 nm2 planar membrane patches over 2 μs. We monitor the time evolution of 93 different fusion attempts. This allows us to construct a global morphology diagram, using the initial tensions of the vesicle and the planar membrane patch as control parameters, and to determine the corresponding fusion statistics. All successful fusion events are observed to occur within 350 ns, which reflects the presence of alternative pathways for the tension relaxation.

  4. Trophoblast cell fusion and differentiation are mediated by both the protein kinase C and a pathways.

    Directory of Open Access Journals (Sweden)

    Waka Omata

    Full Text Available The syncytiotrophoblast of the human placenta is an epithelial barrier that interacts with maternal blood and is a key for the transfer of nutrients and other solutes to the developing fetus. The syncytiotrophoblast is a true syncytium and fusion of progenitor cytotrophoblasts is the cardinal event leading to the formation of this layer. BeWo cells are often used as a surrogate for cytotrophoblasts, since they can be induced to fuse, and then express certain differentiation markers associated with trophoblast syncytialization. Dysferlin, a syncytiotrophoblast membrane repair protein, is up-regulated in BeWo cells induced to fuse by treatment with forskolin; this fusion is thought to occur through cAMP/protein kinase A-dependent mechanisms. We hypothesized that dysferlin may also be up-regulated in response to fusion through other pathways. Here, we show that BeWo cells can also be induced to fuse by treatment with an activator of protein kinase C, and that this fusion is accompanied by increased expression of dysferlin. Moreover, a dramatic synergistic increase in dysferlin expression is observed when both the protein kinase A and protein kinase C pathways are activated in BeWo cells. This synergy in fusion is also accompanied by dramatic increases in mRNA for the placental fusion proteins syncytin 1, syncytin 2, as well as dysferlin. Dysferlin, however, was shown to be dispensable for stimulus-induced BeWo cell syncytialization, since dysferlin knockdown lines fused to the same extent as control cells. The classical trophoblast differentiation marker human chorionic gonadotropin was also monitored and changes in the expression closely parallel that of dysferlin in all of the experimental conditions employed. Thus different biochemical markers of trophoblast fusion behave in concert supporting the hypothesis that activation of both protein kinase C and A pathways lead to trophoblastic differentiation.

  5. Protective antitumor activity induced by a fusion vaccine with murine ...

    African Journals Online (AJOL)

    Targeting angiogenesis is an effective strategy for anticancer therapy. The vascular endothelialcadherin (VE-cad) regulated angiogenesis is a potential target for anti-angiogenesis. Here, we develop a fusion vaccine plasmid DNA pSec-MBD2-VE-cad from VE-cad and murine beta defensin2 (MBD2) to induce immunity for ...

  6. Shear-Induced Membrane Fusion in Viscous Solutions

    KAUST Repository

    Kogan, Maxim

    2014-05-06

    Large unilamellar lipid vesicles do not normally fuse under fluid shear stress. They might deform and open pores to relax the tension to which they are exposed, but membrane fusion occurring solely due to shear stress has not yet been reported. We present evidence that shear forces in a viscous solution can induce lipid bilayer fusion. The fusion of 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC) liposomes is observed in Couette flow with shear rates above 3000 s-1 provided that the medium is viscous enough. Liposome samples, prepared at different viscosities using a 0-50 wt % range of sucrose concentration, were studied by dynamic light scattering, lipid fusion assays using Förster resonance energy transfer (FRET), and linear dichroism (LD) spectroscopy. Liposomes in solutions with 40 wt % (or more) sucrose showed lipid fusion under shear forces. These results support the hypothesis that under suitable conditions lipid membranes may fuse in response to mechanical-force- induced stress. © 2014 American Chemical Society.

  7. Engineered three-dimensional multicellular culture model to recapitulate morphogenetic fusion using human cells

    Science.gov (United States)

    Tissue fusion during early mammalian development requires crosstalk between multiple cell types. For example, paracrine signaling between palatal epithelial cells and palatal mesenchyme mediates the fusion of opposing palatal shelves during embryonic development. Fusion events in...

  8. Recombinant TAT-BMI-1 fusion protein induces ex vivo expansion of human umbilical cord blood-derived hematopoietic stem cells.

    Science.gov (United States)

    Codispoti, Bruna; Rinaldo, Nicola; Chiarella, Emanuela; Lupia, Michela; Spoleti, Cristina Barbara; Marafioti, Maria Grazia; Aloisio, Annamaria; Scicchitano, Stefania; Giordano, Marco; Nappo, Giovanna; Lucchino, Valeria; Moore, Malcolm A S; Zhou, Pengbo; Mesuraca, Maria; Bond, Heather Mandy; Morrone, Giovanni

    2017-07-04

    Transplantation of hematopoietic stem cells (HSCs) is a well-established therapeutic approach for numerous disorders. HSCs are typically derived from bone marrow or peripheral blood after cytokine-induced mobilization. Umbilical cord blood (CB) represents an appealing alternative HSC source, but the small amounts of the individual CB units have limited its applications. The availability of strategies for safe ex vivo expansion of CB-derived HSCs (CB-HSCs) may allow to extend the use of these cells in adult patients and to avoid the risk of insufficient engraftment or delayed hematopoietic recovery.Here we describe a system for the ex vivo expansion of CB-HSCs based on their transient exposure to a recombinant TAT-BMI-1 chimeric protein. BMI-1 belongs to the Polycomb family of epigenetic modifiers and is recognized as a central regulator of HSC self-renewal. Recombinant TAT-BMI-1 produced in bacteria was able to enter the target cells via the HIV TAT-derived protein transduction peptide covalently attached to BMI-1, and conserved its biological activity. Treatment of CB-CD34+ cells for 3 days with repeated addition of 10 nM purified TAT-BMI-1 significantly enhanced total cell expansion as well as that of primitive hematopoietic progenitors in culture. Importantly, TAT-BMI-1-treated CB-CD34+ cells displayed a consistently higher rate of multi-lineage long-term repopulating activity in primary and secondary xenotransplants in immunocompromised mice. Thus, recombinant TAT-BMI-1 may represent a novel, effective reagent for ex vivo expansion of CB-HSC for therapeutic purposes.

  9. Cell fusion of bone marrow cells and somatic cell reprogramming by embryonic stem cells

    OpenAIRE

    Bonde, Sabrina; Pedram, Mehrdad; Stultz, Ryan; Zavazava, Nicholas

    2010-01-01

    Bone marrow transplantation is a curative treatment for many diseases, including leukemia, autoimmune diseases, and a number of immunodeficiencies. Recently, it was claimed that bone marrow cells transdifferentiate, a much desired property as bone marrow cells are abundant and therefore could be used in regenerative medicine to treat incurable chronic diseases. Using a Cre/loxP system, we studied cell fusion after bone marrow transplantation. Fused cells were chiefly Gr-1+, a myeloid cell mar...

  10. Oral cancer/endothelial cell fusion experiences nuclear fusion and acquisition of enhanced survival potential.

    Science.gov (United States)

    Song, Kai; Song, Yong; Zhao, Xiao-Ping; Shen, Hui; Wang, Meng; Yan, Ting-Lin; Liu, Ke; Shang, Zheng-Jun

    2014-10-15

    Most previous studies have linked cancer-macrophage fusion with tumor progression and metastasis. However, the characteristics of hybrid cells derived from oral cancer and endothelial cells and their involvement in cancer remained unknown. Double-immunofluorescent staining and fluorescent in situ hybridization (FISH) were performed to confirm spontaneous cell fusion between eGFP-labeled human umbilical vein endothelial cells (HUVECs) and RFP-labeled SCC9, and to detect the expression of vementin and cytokeratin 18 in the hybrids. The property of chemo-resistance of such hybrids was examined by TUNEL assay. The hybrid cells in xenografted tumor were identified by FISH and GFP/RFP dual-immunofluoresence staining. We showed that SCC9 cells spontaneously fused with cocultured endothelial cells, and the resultant hybrid cells maintained the division and proliferation activity after re-plating and thawing. Such hybrids expressed markers of both parental cells and became more resistant to chemotherapeutic drug cisplatin as compared to the parental SCC9 cells. Our in vivo data indicated that the hybrid cells contributed to tumor composition by using of immunostaining and FISH analysis, even though the hybrid cells and SCC9 cells were mixed with 1:10,000, according to the FACS data. Our study suggested that the fusion events between oral cancer and endothelial cells undergo nuclear fusion and acquire a new property of drug resistance and consequently enhanced survival potential. These experimental findings provide further supportive evidence for the theory that cell fusion is involved in cancer progression. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Investigation of fusion gene expression in HCT116 cells.

    Science.gov (United States)

    Zhang, Yanmei; Ren, Juan; Fang, Mengdie; Wang, Xiaoju

    2017-12-01

    Colon cancer is the most common type of gastrointestinal cancer. A number of specific and sensitive biomarkers facilitate the diagnosis and monitoring of patients with colon cancer. Fusion genes are typically identified in cancer and a majority of the newly identified fusion genes are oncogenic in nature. Therefore, fusion genes are potential biomarkers and/or therapy targets in cancer. In the present study, the regulation of specific candidate fusion genes were investigated using Brother of the Regulator of Imprinted Sites (BORIS) in the HCT116 colon cancer cell line, which is a paralog of the fusion gene regulator CCCTC-binding factor (CTCF). The copy number of BORIS increased correspondingly to the progression of colorectal carcinoma from the M0 to the M1a stage. It was identified that EIF3E(e1)-RSPO2(e2) , EIF3E(e1)-RSPO2(e3) , PTPRK(e1)-RSPO3(e2) , PTPRK(e7)-RSPO3(e2), TADA2A-MEF2B and MED13L-CD4 are fusion transcripts present in the transcriptome of the HCT116 colon cancer cell line. CDC42SE2-KIAAO146 is a genomic fusion transcript, which originates from DNA arrangement in HCT116 cells. BORIS suppresses the expression of EIF3E , RSPO2 , PTPRK , RSPO3 , TADA2A and CD4 to inhibit the expression of fusion transcripts in HCT116 cells. It was hypothesized that the fusion transcripts investigated in the present study may not be oncogenic in HCT116 cells. As BORIS is not colorectal carcinoma-specific, the fusion genes investigated may be a biomarker assemblage for monitoring the progression of colorectal carcinoma.

  12. Effect of irradiation-induced defects on fusion reactor ceramics

    International Nuclear Information System (INIS)

    Clinard, F.W. Jr.

    1986-01-01

    Structural, thermal, and electrical properties critical to performance of ceramics in a fusion environment can be profoundly altered by irradiation effects. Neutron damage may cause swelling, reduction of thermal conductivity, increase in dielectric loss, and either reduction or enhancement of strength depending on the crystal structure and defect content of the material. Absorption of ionizing energy inevitably leads to degradation of insulating properties, but these changes can be reduced by alterations in structural or compositional makeup. Assessment of the irradiation response of candidate ceramics Al 2 O 3 , MgAl 2 O 4 , SiC and Si 3 N 4 shows that each may find use in advanced fusion devices. The present understanding of irradiation-induced defects in ceramics, while far from complete, nevertheless points the way to methods for developing improved materials for fusion applications

  13. Enhanced human somatic cell reprogramming efficiency by fusion of the MYC transactivation domain and OCT4

    Directory of Open Access Journals (Sweden)

    Ling Wang

    2017-12-01

    Full Text Available The development of human induced pluripotent stem cells (iPSCs holds great promise for regenerative medicine. However the iPSC induction efficiency is still very low and with lengthy reprogramming process. We utilized the highly potent transactivation domain (TAD of MYC protein to engineer the human OCT4 fusion proteins. Applying the MYC-TAD-OCT4 fusion proteins in mouse iPSC generation leads to shorter reprogramming dynamics, with earlier activation of pluripotent markers in reprogrammed cells than wild type OCT4 (wt-OCT4. Dramatic enhancement of iPSC colony induction efficiency and shortened reprogramming dynamics were observed when these MYC-TAD-OCT4 fusion proteins were used to reprogram primary human cells. The OCT4 fusion proteins induced human iPSCs are pluripotent. We further show that the MYC Box I (MBI is dispensable while both MBII and the linking region between MBI/II are essential for the enhanced reprogramming activity of MYC-TAD-OCT4 fusion protein. Consistent with an enhanced transcription activity, the engineered OCT4 significantly stimulated the expression of genes specifically targeted by OCT4-alone, OCT4/SOX2, and OCT4/SOX2/KLF4 during human iPSC induction, compared with the wt-OCT4. The MYC-TAD-OCT4 fusion proteins we generated will be valuable tools for studying the reprogramming mechanisms and for efficient iPSC generation for humans as well as for other species.

  14. Expression of RecA and cell-penetrating peptide (CPP) fusion protein in bacteria and in mammalian cells.

    Science.gov (United States)

    Chang, Xiubao; Hou, Yuexian

    2018-01-01

    Genome editing is a powerful tool to modify a specific gene and to correct a disease-causing mutation. Recently developed new techniques, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9), significantly facilitate the progression in this field. However, mutations associated with the double strand DNA breaks (DSBs) introduced by these systems hampered their direct usage in clinic. In order to prevent the mutations caused by DSBs, we have designed a novel mean to induce homology-directed recombination (HDR) without DSBs, i.e., the fusion protein of RecA with cell-penetrating peptide (CPP). The involvement of RecA in these fusion proteins will play important roles in formation of the nucleoprotein filament with single strand DNA (ssDNA) in vitro and promoting HDR in vivo ; whereas the involvement of CPP in these fusion proteins will mainly play a role in facilitating cellular intake/uptake of the nucleoprotein filaments. Our results indicated that certain amount of the fusion proteins expressed in bacteria is in soluble fraction, whereas majority of the fusion proteins expressed in baby hamster kidney (BHK) cells is in soluble fraction. Interestingly, expression of these fusion proteins in bacteria completely blocked cell growth, whereas expression of them in BHK cells significantly inhibited cell growth, implying that these fusion proteins may bind to ssDNA regions, such as ssDNA regions in DNA replication forks, and inhibit cell growth. These results suggest that we have functional RecA.CPP fusion proteins ready to test our novel idea of inducing HDR without DSB.

  15. Targeted cell fusion facilitates stable heterokaryon generation in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Michael A Long

    Full Text Available Induced cell fusion has enabled several important discoveries, including the phenomenon of nuclear reprogramming and may yet be applied as a novel therapy for degenerative diseases. However, existing fusogens lack the efficiency required to enable investigation of the epigenetic modifications underlying nuclear reprogramming and the specificity required for clinical application. Here we present a chimeric measles hemagglutinin, Hα7, which specifically and efficiently mediates the fusion of diverse cell types with skeletal muscle both in vitro and in vivo. When compared directly to polyethylene glycol, Hα7 consistently generated a substantial increase in heterokaryon yield and exhibited insignificant levels of toxicity. Moreover, this increased fusion efficiency enabled detection of chromatin modifications associated with nuclear reprogramming following Hα7-mediated fusion of human fibroblasts and mouse myotubes. Finally, Hα7 was also capable of increasing the contribution of transplanted fibroblasts to skeletal muscle repair in vivo, suggesting that this strategy could be used for therapeutic gene delivery.

  16. Cell Fusion in the War on Cancer: A Perspective on the Inception of Malignancy.

    Science.gov (United States)

    Platt, Jeffrey L; Zhou, Xiaofeng; Lefferts, Adam R; Cascalho, Marilia

    2016-07-13

    Cell fusion occurs in development and in physiology and rarely in those settings is it associated with malignancy. However, deliberate fusion of cells and possibly untoward fusion of cells not suitably poised can eventuate in aneuploidy, DNA damage and malignant transformation. How often cell fusion may initiate malignancy is unknown. However, cell fusion could explain the high frequency of cancers in tissues with low underlying rates of cell proliferation and mutation. On the other hand, cell fusion might also engage innate and adaptive immune surveillance, thus helping to eliminate or retard malignancies. Here we consider whether and how cell fusion might weigh on the overall burden of cancer in modern societies.

  17. pH regulation in early endosomes and interferon-inducible transmembrane proteins control avian retrovirus fusion.

    Science.gov (United States)

    Desai, Tanay M; Marin, Mariana; Mason, Caleb; Melikyan, Gregory B

    2017-05-12

    Enveloped viruses infect host cells by fusing their membranes with those of the host cell, a process mediated by viral glycoproteins upon binding to cognate host receptors or entering into acidic intracellular compartments. Whereas the effect of receptor density on viral infection has been well studied, the role of cell type-specific factors/processes, such as pH regulation, has not been characterized in sufficient detail. Here, we examined the effects of cell-extrinsic factors (buffer environment) and cell-intrinsic factors (interferon-inducible transmembrane proteins, IFITMs), on the pH regulation in early endosomes and on the efficiency of acid-dependent fusion of the avian sarcoma and leukosis virus (ASLV), with endosomes. First, we found that a modest elevation of external pH can raise the pH in early endosomes in a cell type-dependent manner and thereby delay the acid-induced fusion of endocytosed ASLV. Second, we observed a cell type-dependent delay between the low pH-dependent and temperature-dependent steps of viral fusion, consistent with the delayed enlargement of the fusion pore. Third, ectopic expression of IFITMs, known to potently block influenza virus fusion with late compartments, was found to only partially inhibit ASLV fusion with early endosomes. Interestingly, IFITM expression promoted virus uptake and the acidification of endosomal compartments, resulting in an accelerated fusion rate when driven by the glycosylphosphatidylinositol-anchored, but not by the transmembrane isoform of the ASLV receptor. Collectively, these results highlight the role of cell-extrinsic and cell-intrinsic factors in regulating the efficiency and kinetics of virus entry and fusion with target cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Cancer Vaccine by Fusions of Dendritic and Cancer Cells

    Directory of Open Access Journals (Sweden)

    Shigeo Koido

    2009-01-01

    Full Text Available Dendritic cells (DCs are potent antigen-presenting cells and play a central role in the initiation and regulation of primary immune responses. Therefore, their use for the active immunotherapy against cancers has been studied with considerable interest. The fusion of DCs with whole tumor cells represents in many ways an ideal approach to deliver, process, and subsequently present a broad array of tumor-associated antigens, including those yet to be unidentified, in the context of DCs-derived costimulatory molecules. DCs/tumor fusion vaccine stimulates potent antitumor immunity in the animal tumor models. In the human studies, T cells stimulated by DC/tumor fusion cells are effective in lysis of tumor cells that are used as the fusion partner. In the clinical trials, clinical and immunological responses were observed in patients with advanced stage of malignant tumors after being vaccinated with DC/tumor fusion cells, although the antitumor effect is not as vigorous as in the animal tumor models. This review summarizes recent advances in concepts and techniques that are providing new impulses to DCs/tumor fusions-based cancer vaccination.

  19. Measles Virus Mutants Possessing the Fusion Protein with Enhanced Fusion Activity Spread Effectively in Neuronal Cells, but Not in Other Cells, without Causing Strong Cytopathology

    Science.gov (United States)

    Ohno, Shinji; Shirogane, Yuta; Suzuki, Satoshi O.; Koga, Ritsuko

    2014-01-01

    ABSTRACT Subacute sclerosing panencephalitis (SSPE) is caused by persistent measles virus (MV) infection in the central nervous system (CNS). Since human neurons, its main target cells, do not express known MV receptors (signaling lymphocyte activation molecule [SLAM] and nectin 4), it remains to be understood how MV infects and spreads in them. We have recently reported that fusion-enhancing substitutions in the extracellular domain of the MV fusion (F) protein (T461I and S103I/N462S/N465S), which are found in multiple SSPE virus isolates, promote MV spread in human neuroblastoma cell lines and brains of suckling hamsters. In this study, we show that hyperfusogenic viruses with these substitutions also spread efficiently in human primary neuron cultures without inducing syncytia. These substitutions were found to destabilize the prefusion conformation of the F protein trimer, thereby enhancing fusion activity. However, these hyperfusogenic viruses exhibited stronger cytopathology and produced lower titers at later time points in SLAM- or nectin 4-expressing cells compared to the wild-type MV. Although these viruses spread efficiently in the brains of SLAM knock-in mice, they did not in the spleens. Taken together, the results suggest that enhanced fusion activity is beneficial for MV to spread in neuronal cells where no cytopathology occurs, but detrimental to other types of cells due to strong cytopathology. Acquisition of enhanced fusion activity through substitutions in the extracellular domain of the F protein may be crucial for MV's extensive spread in the CNS and development of SSPE. IMPORTANCE Subacute sclerosing panencephalitis (SSPE) is a fatal disease caused by persistent measles virus (MV) infection in the central nervous system (CNS). Its cause is not well understood, and no effective therapy is currently available. Recently, we have reported that enhanced fusion activity of MV through the mutations in its fusion protein is a major determinant of

  20. DNA-protein immunization using Leishmania peroxidoxin-1 induces a strong CD4+ T cell response and partially protects mice from cutaneous leishmaniasis: role of fusion murine granulocyte-macrophage colony-stimulating factor DNA adjuvant.

    Directory of Open Access Journals (Sweden)

    Abebe Genetu Bayih

    2014-12-01

    Full Text Available To date, no universally effective and safe vaccine has been developed for general human use. Leishmania donovani Peroxidoxin-1 (LdPxn-1 is a member of the antioxidant family of proteins and is predominantly expressed in the amastigote stage of the parasite. The aim of this study was to evaluate the immunogenicity and protective efficacy of LdPxn-1 in BALB/c mice in heterologous DNA-Protein immunization regimen in the presence of fusion murine granulocyte-macrophage colony-stimulating factor (mGMCSF DNA adjuvant.A fusion DNA of LdPxn1 and mGMCSF was cloned into a modified pcDNA vector. To confirm the expression in mammalian system, Chinese hamster ovary cells were transfected with the plasmid vector containing LdPxn1 gene. BALB/c mice were immunized twice with pcDNA-mGMCSF-LdPxn-1 or pcDNA-LdPxn1 DNA and boosted once with recombinant LdPxn-1 protein. Three weeks after the last immunization, mice were infected with Leishmania major promastigotes. The result showed that immunization with pcDNA-mGMCSF-LdPxn1 elicited a mixed Th-1/Th-2 immune response with significantly higher production of IFN-γ than controls. Intracellular cytokine staining of antigen-stimulated spleen cells showed that immunization with this antigen elicited significantly higher proportion of CD4+ T cells that express IFN-γ, TNF-α, or IL-2. The antigen also induced significantly higher proportion of multipotent CD4+ cells that simultaneously express the three Th-1 cytokines. Moreover, a significant reduction in the footpad swelling was seen in mice immunized with pcDNA-mGMCSF-LdPxn1 antigen. Expression study in CHO cells demonstrated that pcDNA-mGMCSF-LdPxn-1 was expressed in mammalian system.The result demonstrates that immunization of BALB/c mice with a plasmid expressing LdPxn1 in the presence of mGMCSF adjuvant elicits a strong specific immune response with high level induction of multipotent CD4+ cells that mediate protection of the mice from Leishmania major infection. To

  1. Nuclear Fusion Effects Induced in Intense Laser-Generated Plasmas

    Directory of Open Access Journals (Sweden)

    Lorenzo Torrisi

    2013-01-01

    Full Text Available Deutered polyethylene (CD2n thin and thick targets were irradiated in high vacuum by infrared laser pulses at 1015W/cm2 intensity. The high laser energy transferred to the polymer generates plasma, expanding in vacuum at supersonic velocity, accelerating hydrogen and carbon ions. Deuterium ions at kinetic energies above 4 MeV have been measured by using ion collectors and SiC detectors in time-of-flight configuration. At these energies the deuterium–deuterium collisions may induce over threshold fusion effects, in agreement with the high D-D cross-section valuesaround 3 MeV energy. At the first instants of the plasma generation, during which high temperature, density and ionacceleration occur, the D-D fusions occur as confirmed by the detection of mono-energetic protonsand neutrons with a kinetic energy of 3.0 MeV and 2.5 MeV, respectively, produced by the nuclear reaction. The number of fusion events depends strongly on the experimental set-up, i.e. on the laser parameters (intensity, wavelength, focal spot dimension, target conditions (thickness, chemical composition, absorption coefficient, presence of secondary targets and used geometry (incidence angle, laser spot, secondary target positions.A number of D-D fusion events of the order of 106÷7 per laser shot has been measured.

  2. Rapid Elimination of the Persistent Synergid through a Cell Fusion Mechanism

    KAUST Repository

    Maruyama, Daisuke

    2015-05-01

    In flowering plants, fertilization-dependent degeneration of the persistent synergid cell ensures one-on-one pairings of male and female gametes. Here, we report that the fusion of the persistent synergid cell and the endosperm selectively inactivates the persistent synergid cell in Arabidopsis thaliana. The synergid-endosperm fusion causes rapid dilution of pre-secreted pollen tube attractant in the persistent synergid cell and selective disorganization of the synergid nucleus during the endosperm proliferation, preventing attractions of excess number of pollen tubes (polytubey). The synergid-endosperm fusion is induced by fertilization of the central cell, while the egg cell fertilization predominantly activates ethylene signaling, an inducer of the synergid nuclear disorganization. Therefore, two female gametes (the egg and the central cell) control independent pathways yet coordinately accomplish the elimination of the persistent synergid cell by double fertilization. Two female gametes (the egg cell and the central cell) in flowering plants coordinately prevent attractions of excess number of pollen tubes via two mechanisms to inactivate persistent synergid cell. © 2015 Elsevier Inc.

  3. Organotypic three-dimensional culture model of mesenchymal and epithelial cells to examine tissue fusion events.

    Science.gov (United States)

    Tissue fusion during early mammalian development requires coordination of multiple cell types, the extracellular matrix, and complex signaling pathways. Fusion events during processes including heart development, neural tube closure, and palatal fusion are dependent on signaling ...

  4. IGF1 is a common target gene of Ewing's sarcoma fusion proteins in mesenchymal progenitor cells.

    Directory of Open Access Journals (Sweden)

    Luisa Cironi

    Full Text Available BACKGROUND: The EWS-FLI-1 fusion protein is associated with 85-90% of Ewing's sarcoma family tumors (ESFT, the remaining 10-15% of cases expressing chimeric genes encoding EWS or FUS fused to one of several ets transcription factor family members, including ERG-1, FEV, ETV1 and ETV6. ESFT are dependent on insulin-like growth factor-1 (IGF-1 for growth and survival and recent evidence suggests that mesenchymal progenitor/stem cells constitute a candidate ESFT origin. METHODOLOGY/PRINCIPAL FINDINGS: To address the functional relatedness between ESFT-associated fusion proteins, we compared mouse progenitor cell (MPC permissiveness for EWS-FLI-1, EWS-ERG and FUS-ERG expression and assessed the corresponding expression profile changes. Whereas all MPC isolates tested could stably express EWS-FLI-1, only some sustained stable EWS-ERG expression and none could express FUS-ERG for more than 3-5 days. Only 14% and 4% of the total number of genes that were respectively induced and repressed in MPCs by the three fusion proteins were shared. However, all three fusion proteins, but neither FLI-1 nor ERG-1 alone, activated the IGF1 promoter and induced IGF1 expression. CONCLUSION/SIGNIFICANCE: Whereas expression of different ESFT-associated fusion proteins may require distinct cellular microenvironments and induce transcriptome changes of limited similarity, IGF1 induction may provide one common mechanism for their implication in ESFT pathogenesis.

  5. Outer nuclear membrane fusion of adjacent nuclei in varicella-zoster virus-induced syncytia.

    Science.gov (United States)

    Wang, Wei; Yang, Lianwei; Huang, Xiumin; Fu, Wenkun; Pan, Dequan; Cai, Linli; Ye, Jianghui; Liu, Jian; Xia, Ningshao; Cheng, Tong; Zhu, Hua

    2017-12-01

    Syncytia formation has been considered important for cell-to-cell spread and pathogenesis of many viruses. As a syncytium forms, individual nuclei often congregate together, allowing close contact of nuclear membranes and possibly fusion to occur. However, there is currently no reported evidence of nuclear membrane fusion between adjacent nuclei in wild-type virus-induced syncytia. Varicella-zoster virus (VZV) is one typical syncytia-inducing virus that causes chickenpox and shingles in humans. Here, we report, for the first time, an interesting observation of apparent fusion of the outer nuclear membranes from juxtaposed nuclei that comprise VZV syncytia both in ARPE-19 human epithelial cells in vitro and in human skin xenografts in the SCID-hu mouse model in vivo. This work reveals a novel aspect of VZV-related cytopathic effect in the context of multinucleated syncytia. Additionally, the information provided by this study could be helpful for future studies on interactions of viruses with host cell nuclei. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Global epigenomic analysis indicates protocadherin-7 activates osteoclastogenesis by promoting cell–cell fusion

    Energy Technology Data Exchange (ETDEWEB)

    Nakamura, Haruhiko [Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033 (Japan); Department of Cell Signaling, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8549 (Japan); Nakashima, Tomoki [Department of Cell Signaling, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8549 (Japan); Japan Science and Technology Agency, PRESTO, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8549 (Japan); Hayashi, Mikihito [Department of Cell Signaling, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8549 (Japan); Japan Science and Technology Agency, ERATO, Takayanagi Osteonetwork Project, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Izawa, Naohiro; Yasui, Tetsuro [Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033 (Japan); Aburatani, Hiroyuki [Genome Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1, Komaba, Meguro-ku, Tokyo 153-8904 (Japan); Tanaka, Sakae [Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033 (Japan); Takayanagi, Hiroshi, E-mail: takayana@m.u-tokyo.ac.jp [Japan Science and Technology Agency, ERATO, Takayanagi Osteonetwork Project, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)

    2014-12-12

    Highlights: • Identification of epigenetically regulated genes during osteoclastogenesis. • Pcdh7 is regulated by H3K4me3 and H3K27me3 during osteoclastogenesis. • Pcdh7 expression is increased by RANKL during osteoclastogenesis. • Establishment of novel cell fusion analysis for osteoclasts by imaging cytometer. • Pcdh7 regulates osteoclastogenesis by promoting cell fusion related gene expressions. - Abstract: Gene expression is dependent not only on genomic sequences, but also epigenetic control, in which the regulation of chromatin by histone modification plays a crucial role. Histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3) are related to transcriptionally activated and silenced sequences, respectively. Osteoclasts, the multinucleated cells that resorb bone, are generated by the fusion of precursor cells of monocyte/macrophage lineage. To elucidate the molecular and epigenetic regulation of osteoclast differentiation, we performed a chromatin immunoprecipitation sequencing (ChIP-seq) analysis for H3K4me3 and H3K27me3 in combination with RNA sequencing. We focused on the histone modification change from H3K4me3(+)H3K27me3(+) to H3K4me3(+)H3K27me3(–) and identified the protocadherin-7 gene (Pcdh7) to be among the genes epigenetically regulated during osteoclastogenesis. Pcdh7 was induced by RANKL stimulation in an NFAT-dependent manner. The knockdown of Pcdh7 inhibited RANKL-induced osteoclast differentiation due to the impairment of cell–cell fusion, accompanied by a decreased expression of the fusion-related genes Dcstamp, Ocstamp and Atp6v0d2. This study demonstrates that Pcdh7 plays a key role in osteoclastogenesis by promoting cell–cell fusion.

  7. Global epigenomic analysis indicates protocadherin-7 activates osteoclastogenesis by promoting cell–cell fusion

    International Nuclear Information System (INIS)

    Nakamura, Haruhiko; Nakashima, Tomoki; Hayashi, Mikihito; Izawa, Naohiro; Yasui, Tetsuro; Aburatani, Hiroyuki; Tanaka, Sakae; Takayanagi, Hiroshi

    2014-01-01

    Highlights: • Identification of epigenetically regulated genes during osteoclastogenesis. • Pcdh7 is regulated by H3K4me3 and H3K27me3 during osteoclastogenesis. • Pcdh7 expression is increased by RANKL during osteoclastogenesis. • Establishment of novel cell fusion analysis for osteoclasts by imaging cytometer. • Pcdh7 regulates osteoclastogenesis by promoting cell fusion related gene expressions. - Abstract: Gene expression is dependent not only on genomic sequences, but also epigenetic control, in which the regulation of chromatin by histone modification plays a crucial role. Histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3) are related to transcriptionally activated and silenced sequences, respectively. Osteoclasts, the multinucleated cells that resorb bone, are generated by the fusion of precursor cells of monocyte/macrophage lineage. To elucidate the molecular and epigenetic regulation of osteoclast differentiation, we performed a chromatin immunoprecipitation sequencing (ChIP-seq) analysis for H3K4me3 and H3K27me3 in combination with RNA sequencing. We focused on the histone modification change from H3K4me3(+)H3K27me3(+) to H3K4me3(+)H3K27me3(–) and identified the protocadherin-7 gene (Pcdh7) to be among the genes epigenetically regulated during osteoclastogenesis. Pcdh7 was induced by RANKL stimulation in an NFAT-dependent manner. The knockdown of Pcdh7 inhibited RANKL-induced osteoclast differentiation due to the impairment of cell–cell fusion, accompanied by a decreased expression of the fusion-related genes Dcstamp, Ocstamp and Atp6v0d2. This study demonstrates that Pcdh7 plays a key role in osteoclastogenesis by promoting cell–cell fusion

  8. Fusion

    International Nuclear Information System (INIS)

    Naraghi, M.

    1976-01-01

    It is proposed that Iran as a world's potential supplier of fossile fuel should participate in fusion research and gain experience in this new field. Fusion, as an ultimate source of energy in future, and the problems concerned with the fusion reactors are reviewed. Furthermore; plasma heating, magnetic and inertial confinement in a fusion reactor are discussed. A brief description of tokamak, theta pinch and magnetic mirror reactors is also included

  9. Dynamics of cell aggregates fusion: Experiments and simulations

    Science.gov (United States)

    Thomas, Gilberto L.; Mironov, Vladimir; Nagy-Mehez, Agnes; Mombach, José C. M.

    2014-02-01

    Fusion of cell tissues is an ubiquitous phenomenon and has important technological applications including tissue biofabrication. In this work we present experimental results of aggregates fusion using adipose derived stem cells (ADSC) and a three dimensional computer simulation of the process using the cellular Potts model with aggregates reaching 10,000 cells. We consider fusion of round aggregates and monitor the dimensionless neck area of contact between the two aggregates to characterize the process, as done for the coalescence of liquid droplets and polymers. Both experiments and simulations show that the evolution of this quantity obeys a power law in time. We also study quantitatively individual cell motion with the simulation and it corresponds to an anomalous diffusion.

  10. Protein-induced fusion can be modulated by target membrane lipids through a structural switch at the level of the fusion peptide

    NARCIS (Netherlands)

    Pecheur, EI; Martin, [No Value; Bienvenue, A; Ruysschaert, JM; Hoekstra, D

    2000-01-01

    Regulatory features of protein-induced membrane fusion are largely unclear, particularly at the level of the fusion peptide. Fusion peptides being part of larger protein complexes, such investigations are met with technical limitations. Here, we show that the fusion activity of influenza virus or

  11. Fusion

    CERN Document Server

    Mahaffey, James A

    2012-01-01

    As energy problems of the world grow, work toward fusion power continues at a greater pace than ever before. The topic of fusion is one that is often met with the most recognition and interest in the nuclear power arena. Written in clear and jargon-free prose, Fusion explores the big bang of creation to the blackout death of worn-out stars. A brief history of fusion research, beginning with the first tentative theories in the early 20th century, is also discussed, as well as the race for fusion power. This brand-new, full-color resource examines the various programs currently being funded or p

  12. Requirement of myomaker-mediated stem cell fusion for skeletal muscle hypertrophy.

    Science.gov (United States)

    Goh, Qingnian; Millay, Douglas P

    2017-02-10

    Fusion of skeletal muscle stem/progenitor cells is required for proper development and regeneration, however the significance of this process during adult muscle hypertrophy has not been explored. In response to muscle overload after synergist ablation in mice, we show that myomaker, a muscle specific membrane protein essential for myoblast fusion, is activated mainly in muscle progenitors and not myofibers. We rendered muscle progenitors fusion-incompetent through genetic deletion of myomaker in muscle stem cells and observed a complete reduction of overload-induced hypertrophy. This blunted hypertrophic response was associated with a reduction in Akt and p70s6k signaling and protein synthesis, suggesting a link between myonuclear accretion and activation of pro-hypertrophic pathways. Furthermore, fusion-incompetent muscle exhibited increased fibrosis after muscle overload, indicating a protective role for normal stem cell activity in reducing myofiber strain associated with hypertrophy. These findings reveal an essential contribution of myomaker-mediated stem cell fusion during physiological adult muscle hypertrophy.

  13. Extended Synaptotagmin 1 Interacts with Herpes Simplex Virus 1 Glycoprotein M and Negatively Modulates Virus-Induced Membrane Fusion.

    Science.gov (United States)

    El Kasmi, Imane; Khadivjam, Bita; Lackman, Miki; Duron, Johanne; Bonneil, Eric; Thibault, Pierre; Lippé, Roger

    2018-01-01

    Enveloped viruses typically encode their own fusion machinery to enter cells. Herpesviruses are unusual, as they fuse with a number of cellular compartments throughout their life cycles. As uncontrolled fusion of the host membranes should be avoided in these events, tight regulation of the viral fusion machinery is critical. While studying herpes simplex virus 1 (HSV-1) glycoprotein gM, we identified the cellular protein E-Syt1 (extended synaptotagmin 1) as an interaction partner. The interaction took place in both infected and transfected cells, suggesting other viral proteins were not required for the interaction. Most interestingly, E-Syt1 is a member of the synaptotagmin family of membrane fusion regulators. However, the protein is known to promote the tethering of the endoplasmic reticulum (ER) to the plasma membrane. We now show that E-Syt1, along with the related E-Syt3, negatively modulates viral release into the extracellular milieu, cell-to-cell viral spread, and viral entry, all processes that implicate membrane fusion events. Similarly, these E-Syt proteins impacted the formation of virus-induced syncytia. Altogether, these findings hint at the modulation of the viral fusion machinery by the E-Syt family of proteins. IMPORTANCE Viruses typically encode their own fusion apparatus to enable them to enter cells. For many viruses, this means a single fusogenic protein. However, herpesviruses are large entities that express several accessory viral proteins to regulate their fusogenic activity. The present study hints at the additional participation of cellular proteins in this process, suggesting the host can also modulate viral fusion to some extent. Hence E-Syt proteins 1 and 3 seem to negatively modulate the different viral fusion events that take place during the HSV-1 life cycle. This could represent yet another innate immunity response to the virus. Copyright © 2017 American Society for Microbiology.

  14. Cell Therapy To Obtain Spinal Fusion

    Science.gov (United States)

    2010-07-01

    and allografts have been commonly used in spinal fusion, revision total hip arthroplasty , maxillofacial reconstruction, and repair of segmental...Goldberg VM. Selection of bone grafts for revision total hip arthroplasty . Clin Orthop Relat Res 2000 Dec(381):68-76. 3. Nishida J, Shimamura T. Methods...require bone grafting(4), as do millions of total joint arthroplasties , spinal arthrodeses, maxillofacial surgeries and implant fixations(5). In

  15. Sulforaphane Induced Apoptosis via Promotion of Mitochondrial Fusion and ERK1/2-Mediated 26S Proteasome Degradation of Novel Pro-survival Bim and Upregulation of Bax in Human Non-Small Cell Lung Cancer Cells.

    Science.gov (United States)

    Geng, Yang; Zhou, Yan; Wu, Sai; Hu, Yabin; Lin, Kai; Wang, Yalin; Zheng, Zhongnan; Wu, Wei

    2017-01-01

    Previous studies in our laboratory showed that sulforaphane (SFN) induced apoptosis by sustained activation of extracellular regulated protein kinases 1/2 (ERK1/2). However, the underlying mechanisms associated with SFN-induced apoptosis and downstream cascades which are modulated by ERK1/2 were not elucidated. Herein we demonstrated for the first time that alteration of mitochondrial dynamics contributed to SFN-induced apoptosis in human non-small cell lung cancer (NSCLC) cells. Reports showed that protein Bim not only induced apoptosis but also promoted proliferation under certain circumstances. We found that Bim was related to cell growth in NSCLC cells. Pro-survival Bim downregulation was shown to induce apoptosis in response to SFN. Further, Using the ERK1/2 inhibitor, PD98059, we found that SFN upregulated Bax and downregulated Bim through the ERK1/2-dependent signaling pathway. Furthermore, SFN activated ERK1/2 to increase 26S proteasome activity to degrade Bim, while the proteasome inhibitor MG132 reversed this effect. Therefore, SFN phosphorylated ERK1/2 and activated the proteasome system leading to the degradation of Bim, which contributed to apoptosis in NSCLC cells. These findings provided a novel insight into SFN-related therapeutics in cancer treatment.

  16. Transgenic Carrot Expressing Fusion Protein Comprising M. tuberculosis Antigens Induces Immune Response in Mice

    Directory of Open Access Journals (Sweden)

    Natalia V. Permyakova

    2015-01-01

    Full Text Available Tuberculosis remains one of the major infectious diseases, which continues to pose a major global health problem. Transgenic plants may serve as bioreactors to produce heterologous proteins including antibodies, antigens, and hormones. In the present study, a genetic construct has been designed that comprises the Mycobacterium tuberculosis genes cfp10, esat6 and dIFN gene, which encode deltaferon, a recombinant analog of the human γ-interferon designed for expression in plant tissues. This construct was transferred to the carrot (Daucus carota L. genome by Agrobacterium-mediated transformation. This study demonstrates that the fusion protein CFP10-ESAT6-dIFN is synthesized in the transgenic carrot storage roots. The protein is able to induce both humoral and cell-mediated immune responses in laboratory animals (mice when administered either orally or by injection. It should be emphasized that M. tuberculosis antigens contained in the fusion protein have no cytotoxic effect on peripheral blood mononuclear cells.

  17. Nuclear fusion induced by Coulomb explosion of heteronuclear clusters.

    Science.gov (United States)

    Last, I; Jortner, J

    2001-07-16

    We propose a new mechanism for the production of high-energy ( E>3 keV) deuterons, suitable to induce dd nuclear fusion, based on multielectron ionization and Coulomb explosion of heteronuclear deuterium containing molecular clusters, e.g., (D2O)n, in intense ( 10(16)-2x10(18) W/cm2) laser fields. Cluster size equations for E, in conjunction with molecular dynamics simulations, reveal important advantages of Coulomb explosion of (D2O)n heteronuclear clusters, as compared with (D)n clusters. These involve the considerably increased D+ kinetic energy and a narrow, high-energy distribution of deuterons.

  18. HCV-induced autophagosomes are generated via homotypic fusion of phagophores that mediate HCV RNA replication.

    Directory of Open Access Journals (Sweden)

    Linya Wang

    2017-09-01

    Full Text Available Hepatitis C virus (HCV induces autophagy to promote its replication, including its RNA replication, which can take place on double-membrane vesicles known as autophagosomes. However, how HCV induces the biogenesis of autophagosomes and how HCV RNA replication complex may be assembled on autophagosomes were largely unknown. During autophagy, crescent membrane structures known as phagophores first appear in the cytoplasm, which then progress to become autophagosomes. By conducting electron microscopy and in vitro membrane fusion assay, we found that phagophores induced by HCV underwent homotypic fusion to generate autophagosomes in a process dependent on the SNARE protein syntaxin 7 (STX7. Further analyses by live-cell imaging and fluorescence microscopy indicated that HCV-induced phagophores originated from the endoplasmic reticulum (ER. Interestingly, comparing with autophagy induced by nutrient starvation, the progression of phagophores to autophagosomes induced by HCV took significantly longer time, indicating fundamental differences in the biogenesis of autophagosomes induced by these two different stimuli. As the knockdown of STX7 to inhibit the formation of autophagosomes did not affect HCV RNA replication, and purified phagophores could mediate HCV RNA replication, the assembly of the HCV RNA replication complex on autophagosomes apparently took place during the formative stage of phagophores. These findings provided important information for understanding how HCV controlled and modified this important cellular pathway for its own replication.

  19. Genomic instability and telomere fusion of canine osteosarcoma cells.

    Directory of Open Access Journals (Sweden)

    Junko Maeda

    Full Text Available Canine osteosarcoma (OSA is known to present with highly variable and chaotic karyotypes, including hypodiploidy, hyperdiploidy, and increased numbers of metacentric chromosomes. The spectrum of genomic instabilities in canine OSA has significantly augmented the difficulty in clearly defining the biological and clinical significance of the observed cytogenetic abnormalities. In this study, eight canine OSA cell lines were used to investigate telomere fusions by fluorescence in situ hybridization (FISH using a peptide nucleotide acid probe. We characterized each cell line by classical cytogenetic studies and cellular phenotypes including telomere associated factors and then evaluated correlations from this data. All eight canine OSA cell lines displayed increased abnormal metacentric chromosomes and exhibited numerous telomere fusions and interstitial telomeric signals. Also, as evidence of unstable telomeres, colocalization of γ-H2AX and telomere signals in interphase cells was observed. Each cell line was characterized by a combination of data representing cellular doubling time, DNA content, chromosome number, metacentric chromosome frequency, telomere signal level, cellular radiosensitivity, and DNA-PKcs protein expression level. We have also studied primary cultures from 10 spontaneous canine OSAs. Based on the observation of telomere aberrations in those primary cell cultures, we are reasonably certain that our observations in cell lines are not an artifact of prolonged culture. A correlation between telomere fusions and the other characteristics analyzed in our study could not be identified. However, it is important to note that all of the canine OSA samples exhibiting telomere fusion utilized in our study were telomerase positive. Pending further research regarding telomerase negative canine OSA cell lines, our findings may suggest telomere fusions can potentially serve as a novel marker for canine OSA.

  20. Effective donor cell fusion conditions for production of cloned dogs by somatic cell nuclear transfer.

    Science.gov (United States)

    Park, JungEun; Oh, HyunJu; Hong, SoGun; Kim, MinJung; Kim, GeonA; Koo, OkJae; Kang, SungKeun; Jang, Goo; Lee, ByeongChun

    2011-03-01

    As shown by the birth of the first cloned dog 'Snuppy', a protocol to produce viable cloned dogs has been reported. In order to evaluate optimum fusion conditions for improving dog cloning efficiency, in vivo matured oocytes were reconstructed with adult somatic cells from a female Pekingese using different fusion conditions. Fusion with needle vs chamber methods, and with low vs high pulse strength was compared by evaluating fusion rate and in vivo development of canine cloned embryos. The fusion rates in the high voltage groups were significantly higher than in the low voltage groups regardless of fusion method (83.5 vs 66.1% for the needle fusion method, 67.4 vs 37.9% for the fusion chamber method). After embryo transfer, one each pregnancy was detected after using the needle fusion method with high and low voltage and in the chamber fusion method with high voltage, whereas no pregnancy was detected using the chamber method with low voltage. However, only the pregnancy from the needle fusion method with high voltage was maintained to term and one healthy puppy was delivered. The results of the present study demonstrated that two DC pulses of 3.8 to 4.0 kV/cm for 15 μsec using the needle fusion method were the most effective method for the production of cloned dogs under the conditions of this experiment. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. Stem cells regenerative properties on new rat spinal fusion model

    Czech Academy of Sciences Publication Activity Database

    Klíma, K.; Vaněček, Václav; Kohout, A.; Jiroušek, Ondřej; Foltán, R.; Štulík, J.; Machoň, V.; Pavlíková, G.; Jendelová, Pavla; Syková, Eva; Šedý, Jiří

    2015-01-01

    Roč. 64, č. 1 (2015), s. 119-128 ISSN 0862-8408 R&D Projects: GA MZd(CZ) NT13477; GA ČR(CZ) GAP304/10/0320 Institutional support: RVO:67985823 ; RVO:68378297 ; RVO:68378041 Keywords : mesenchymal stem cells * bone graft substitute * spinal fusion Subject RIV: FH - Neurology Impact factor: 1.643, year: 2015

  2. Peptides and membrane fusion : Towards an understanding of the molecular mechanism of protein-induced fusion

    NARCIS (Netherlands)

    Pecheur, EI; Sainte-Marie, J; Bienvenue, A; Hoekstra, D

    1999-01-01

    Processes such as endo- or exocytosis, membrane recycling, fertilization and enveloped viruses infection require one or more critical membrane fusion reactions. A key feature in viral and cellular fusion phenomena is the involvement of specific fusion proteins. Among the few well-characterized

  3. Cell-fusion method to visualize interphase nuclear pore formation.

    Science.gov (United States)

    Maeshima, Kazuhiro; Funakoshi, Tomoko; Imamoto, Naoko

    2014-01-01

    In eukaryotic cells, the nucleus is a complex and sophisticated organelle that organizes genomic DNA to support essential cellular functions. The nuclear surface contains many nuclear pore complexes (NPCs), channels for macromolecular transport between the cytoplasm and nucleus. It is well known that the number of NPCs almost doubles during interphase in cycling cells. However, the mechanism of NPC formation is poorly understood, presumably because a practical system for analysis does not exist. The most difficult obstacle in the visualization of interphase NPC formation is that NPCs already exist after nuclear envelope formation, and these existing NPCs interfere with the observation of nascent NPCs. To overcome this obstacle, we developed a novel system using the cell-fusion technique (heterokaryon method), previously also used to analyze the shuttling of macromolecules between the cytoplasm and the nucleus, to visualize the newly synthesized interphase NPCs. In addition, we used a photobleaching approach that validated the cell-fusion method. We recently used these methods to demonstrate the role of cyclin-dependent protein kinases and of Pom121 in interphase NPC formation in cycling human cells. Here, we describe the details of the cell-fusion approach and compare the system with other NPC formation visualization methods. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Fusion of CCL21 non-migratory active breast epithelial and breast cancer cells give rise to CCL21 migratory active tumor hybrid cell lines.

    Directory of Open Access Journals (Sweden)

    Benjamin Berndt

    Full Text Available The biological phenomenon of cell fusion has been linked to tumor progression because several data provided evidence that fusion of tumor cells and normal cells gave rise to hybrid cell lines exhibiting novel properties, such as increased metastatogenic capacity and an enhanced drug resistance. Here we investigated M13HS hybrid cell lines, derived from spontaneous fusion events between M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics and HS578T-Hyg breast cancer cells, concerning CCL21/CCR7 signaling. Western Blot analysis showed that all cell lines varied in their CCR7 expression levels as well as differed in the induction and kinetics of CCR7 specific signal transduction cascades. Flow cytometry-based calcium measurements revealed that a CCL21 induced calcium influx was solely detected in M13HS hybrid cell lines. Cell migration demonstrated that only M13HS hybrid cell lines, but not parental derivatives, responded to CCL21 stimulation with an increased migratory activity. Knockdown of CCR7 expression by siRNA completely abrogated the CCL21 induced migration of hybrid cell lines indicating the necessity of CCL21/CCR7 signaling. Because the CCL21/CCR7 axis has been linked to metastatic spreading of breast cancer to lymph nodes we conclude from our data that cell fusion could be a mechanism explaining the origin of metastatic cancer (hybrid cells.

  5. [Adenovirus mediated expression of recombinant human single chain interleukin-27(rhscIL-27) fusion gene in hepatoma cells].

    Science.gov (United States)

    Cheng, Xiao-gang; Zhang, Ya-qing; Ye, Wan; Zou, Qiang; Chen, Wei; Jin, Hong; Xu, Yan; Zhang, Shao-lan

    2010-06-01

    To explore the adenovirus mediated expression of recombinant human single chain interleukin-27(rhscIL-27) fusion gene in hepatoma cells. The rhscIL-27 fusion gene was subcloned into the shuttle plasmid pAdTrack-CMV and then clone the homologous recombinant adenovirus genomic plasmid pAdEasy in bacteria. The identified recombinant plasmid AdIL-27 was tranfected into 293 cells, and then the adenovirus did the package and amplification. The HepG2 cells were infected with AdIL-27 and the target gene expression was determined by RT-PCR and ELISA. The biological activity of rhscIL-27 was detected by IFN-gamma inducing assay. Restriction endonuclease and gene sequencing confirmed that the recombinant adenovirus vector of rhscIL-27 fusion gene was successfully constructed. The expression of rhscIL-27 fusion gene was observed at 48 h after the transfection of the HepG2 cells with AdIL-27. The IFN-gamma inducing assay showed that the rhscIL-27 protein has the ability inducing IFN-gamma secretion. By using adenovirus expression system, rhscIL-27 fusion gene with biological activity is expressed successfully in hepatoma cells. This experiment laid a foundation for gene therapy of hepatoma with IL-27.

  6. Reprogramming by cell fusion: boosted by Tets.

    Science.gov (United States)

    Ficz, Gabriella; Reik, Wolf

    2013-03-28

    Pluripotent cells, when fused with somatic cells, have the dominant ability to reprogram the somatic genome. Work by Piccolo et al. (2013) shows that the Tet1 and Tet2 hydroxylases are important for DNA methylation reprogramming of pluripotency genes and parental imprints. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Fusion

    Science.gov (United States)

    Herman, Robin

    1990-10-01

    The book abounds with fascinating anecdotes about fusion's rocky path: the spurious claim by Argentine dictator Juan Peron in 1951 that his country had built a working fusion reactor, the rush by the United States to drop secrecy and publicize its fusion work as a propaganda offensive after the Russian success with Sputnik; the fortune Penthouse magazine publisher Bob Guccione sank into an unconventional fusion device, the skepticism that met an assertion by two University of Utah chemists in 1989 that they had created "cold fusion" in a bottle. Aimed at a general audience, the book describes the scientific basis of controlled fusion--the fusing of atomic nuclei, under conditions hotter than the sun, to release energy. Using personal recollections of scientists involved, it traces the history of this little-known international race that began during the Cold War in secret laboratories in the United States, Great Britain and the Soviet Union, and evolved into an astonishingly open collaboration between East and West.

  8. Characterization of protective immune responses induced by pneumococcal surface protein A in fusion with pneumolysin derivatives.

    Directory of Open Access Journals (Sweden)

    Cibelly Goulart

    Full Text Available Pneumococcal surface protein A (PspA and Pneumolysin derivatives (Pds are important vaccine candidates, which can confer protection in different models of pneumococcal infection. Furthermore, the combination of these two proteins was able to increase protection against pneumococcal sepsis in mice. The present study investigated the potential of hybrid proteins generated by genetic fusion of PspA fragments to Pds to increase cross-protection against fatal pneumococcal infection. Pneumolisoids were fused to the N-terminus of clade 1 or clade 2 pspA gene fragments. Mouse immunization with the fusion proteins induced high levels of antibodies against PspA and Pds, able to bind to intact pneumococci expressing a homologous PspA with the same intensity as antibodies to rPspA alone or the co-administered proteins. However, when antibody binding to pneumococci with heterologous PspAs was examined, antisera to the PspA-Pds fusion molecules showed stronger antibody binding and C3 deposition than antisera to co-administered proteins. In agreement with these results, antisera against the hybrid proteins were more effective in promoting the phagocytosis of bacteria bearing heterologous PspAs in vitro, leading to a significant reduction in the number of bacteria when compared to co-administered proteins. The respective antisera were also capable of neutralizing the lytic activity of Pneumolysin on sheep red blood cells. Finally, mice immunized with fusion proteins were protected against fatal challenge with pneumococcal strains expressing heterologous PspAs. Taken together, the results suggest that PspA-Pd fusion proteins comprise a promising vaccine strategy, able to increase the immune response mediated by cross-reactive antibodies and complement deposition to heterologous strains, and to confer protection against fatal challenge.

  9. Influenza Hemifusion Phenotype Depends on Membrane Context: Differences in Cell-Cell and Virus-Cell Fusion.

    Science.gov (United States)

    Zawada, Katarzyna E; Okamoto, Kenta; Kasson, Peter M

    2018-03-02

    Influenza viral entry into the host cell cytoplasm is accomplished by a process of membrane fusion mediated by the viral hemagglutinin protein. Hemagglutinin acts in a pH-triggered fashion, inserting a short fusion peptide into the host membrane followed by refolding of a coiled-coil structure to draw the viral envelope and host membranes together. Mutations to this fusion peptide provide an important window into viral fusion mechanisms and protein-membrane interactions. Here, we show that a well-described fusion peptide mutant, G1S, has a phenotype that depends strongly on the viral membrane context. The G1S mutant is well known to cause a "hemifusion" phenotype based on experiments in transfected cells, where cells expressing G1S hemagglutinin can undergo lipid mixing in a pH-triggered fashion similar to virus but will not support fusion pores. We compare fusion by the G1S hemagglutinin mutant expressed either in cells or in influenza virions and show that this hemifusion phenotype occurs in transfected cells but that native virions are able to support full fusion, albeit at a slower rate and 10-100× reduced infectious titer. We explain this with a quantitative model where the G1S mutant, instead of causing an absolute block of fusion, alters the protein stoichiometry required for fusion. This change slightly slows fusion at high hemagglutinin density, as on the viral surface, but at lower hemagglutinin density produces a hemifusion phenotype. The quantitative model thus reproduces the observed virus-cell and cell-cell fusion phenotypes, yielding a unified explanation where membrane context can control the observed viral fusion phenotype. Copyright © 2018 Elsevier Ltd. All rights reserved.

  10. Evaluation of Posterolateral Lumbar Fusion in Sheep Using Mineral Scaffolds Seeded with Cultured Bone Marrow Cells

    Science.gov (United States)

    Cuenca-López, María D.; Andrades, José A.; Gómez, Santiago; Zamora-Navas, Plácido; Guerado, Enrique; Rubio, Nuria; Blanco, Jerónimo; Becerra, José

    2014-01-01

    The objective of this study is to investigate the efficacy of hybrid constructs in comparison to bone grafts (autograft and allograft) for posterolateral lumbar fusion (PLF) in sheep, instrumented with transpedicular screws and bars. Hybrid constructs using cultured bone marrow (BM) mesenchymal stem cells (MSCs) have shown promising results in several bone healing models. In particular, hybrid constructs made by calcium phosphate-enriched cells have had similar fusion rates to bone autografts in posterolateral lumbar fusion in sheep. In our study, four experimental spinal fusions in two animal groups were compared in sheep: autograft and allograft (reference group), hydroxyapatite scaffold, and hydroxyapatite scaffold seeded with cultured and osteoinduced bone marrow MSCs (hybrid construct). During the last three days of culture, dexamethasone (dex) and beta-glycerophosphate (β-GP) were added to potentiate osteoinduction. The two experimental situations of each group were tested in the same spinal segment (L4–L5). Spinal fusion and bone formation were studied by clinical observation, X-ray, computed tomography (CT), histology, and histomorphometry. Lumbar fusion rates assessed by CT scan and histology were higher for autograft and allograft (70%) than for mineral scaffold alone (22%) and hybrid constructs (35%). The quantity of new bone formation was also higher for the reference group, quite similar in both (autograft and allograft). Although the hybrid scaffold group had a better fusion rate than the non-hybrid scaffold group, the histological analysis revealed no significant differences between them in terms of quantity of bone formation. The histology results suggested that mineral scaffolds were partly resorbed in an early phase, and included in callus tissues. Far from the callus area the hydroxyapatite alone did not generate bone around it, but the hybrid scaffold did. In nude mice, labeled cells were induced to differentiate in vivo and monitored by

  11. Evaluation of Posterolateral Lumbar Fusion in Sheep Using Mineral Scaffolds Seeded with Cultured Bone Marrow Cells

    Directory of Open Access Journals (Sweden)

    María D. Cuenca-López

    2014-12-01

    Full Text Available The objective of this study is to investigate the efficacy of hybrid constructs in comparison to bone grafts (autograft and allograft for posterolateral lumbar fusion (PLF in sheep, instrumented with transpedicular screws and bars. Hybrid constructs using cultured bone marrow (BM mesenchymal stem cells (MSCs have shown promising results in several bone healing models. In particular, hybrid constructs made by calcium phosphate-enriched cells have had similar fusion rates to bone autografts in posterolateral lumbar fusion in sheep. In our study, four experimental spinal fusions in two animal groups were compared in sheep: autograft and allograft (reference group, hydroxyapatite scaffold, and hydroxyapatite scaffold seeded with cultured and osteoinduced bone marrow MSCs (hybrid construct. During the last three days of culture, dexamethasone (dex and beta-glycerophosphate (β-GP were added to potentiate osteoinduction. The two experimental situations of each group were tested in the same spinal segment (L4–L5. Spinal fusion and bone formation were studied by clinical observation, X-ray, computed tomography (CT, histology, and histomorphometry. Lumbar fusion rates assessed by CT scan and histology were higher for autograft and allograft (70% than for mineral scaffold alone (22% and hybrid constructs (35%. The quantity of new bone formation was also higher for the reference group, quite similar in both (autograft and allograft. Although the hybrid scaffold group had a better fusion rate than the non-hybrid scaffold group, the histological analysis revealed no significant differences between them in terms of quantity of bone formation. The histology results suggested that mineral scaffolds were partly resorbed in an early phase, and included in callus tissues. Far from the callus area the hydroxyapatite alone did not generate bone around it, but the hybrid scaffold did. In nude mice, labeled cells were induced to differentiate in vivo and monitored

  12. Cell Fusion in the War on Cancer: A Perspective on the Inception of Malignancy

    Directory of Open Access Journals (Sweden)

    Jeffrey L. Platt

    2016-07-01

    Full Text Available Cell fusion occurs in development and in physiology and rarely in those settings is it associated with malignancy. However, deliberate fusion of cells and possibly untoward fusion of cells not suitably poised can eventuate in aneuploidy, DNA damage and malignant transformation. How often cell fusion may initiate malignancy is unknown. However, cell fusion could explain the high frequency of cancers in tissues with low underlying rates of cell proliferation and mutation. On the other hand, cell fusion might also engage innate and adaptive immune surveillance, thus helping to eliminate or retard malignancies. Here we consider whether and how cell fusion might weigh on the overall burden of cancer in modern societies.

  13. Hemi-fused structure mediates and controls fusion and fission in live cells.

    Science.gov (United States)

    Zhao, Wei-Dong; Hamid, Edaeni; Shin, Wonchul; Wen, Peter J; Krystofiak, Evan S; Villarreal, Seth A; Chiang, Hsueh-Cheng; Kachar, Bechara; Wu, Ling-Gang

    2016-06-23

    Membrane fusion and fission are vital for eukaryotic life. For three decades, it has been proposed that fusion is mediated by fusion between the proximal leaflets of two bilayers (hemi-fusion) to produce a hemi-fused structure, followed by fusion between the distal leaflets, whereas fission is via hemi-fission, which also produces a hemi-fused structure, followed by full fission. This hypothesis remained unsupported owing to the lack of observation of hemi-fusion or hemi-fission in live cells. A competing fusion hypothesis involving protein-lined pore formation has also been proposed. Here we report the observation of a hemi-fused Ω-shaped structure in live neuroendocrine chromaffin cells and pancreatic β-cells, visualized using confocal and super-resolution stimulated emission depletion microscopy. This structure is generated from fusion pore opening or closure (fission) at the plasma membrane. Unexpectedly, the transition to full fusion or fission is determined by competition between fusion and calcium/dynamin-dependent fission mechanisms, and is notably slow (seconds to tens of seconds) in a substantial fraction of the events. These results provide key missing evidence in support of the hemi-fusion and hemi-fission hypothesis in live cells, and reveal the hemi-fused intermediate as a key structure controlling fusion and fission, as fusion and fission mechanisms compete to determine the transition to fusion or fission.

  14. Multimodality imaging of reporter gene expression using a novel fusion vector in living cells and animals

    Science.gov (United States)

    Gambhir, Sanjiv [Portola Valley, CA; Pritha, Ray [Mountain View, CA

    2011-06-07

    Novel double and triple fusion reporter gene constructs harboring distinct imagable reporter genes are provided, as well as applications for the use of such double and triple fusion constructs in living cells and in living animals using distinct imaging technologies.

  15. Candidate genes for expansion and transformation of hematopoietic stem cells by NUP98-HOX fusion genes.

    Directory of Open Access Journals (Sweden)

    Lars Palmqvist

    Full Text Available BACKGROUND: Hox genes are implicated in hematopoietic stem cell (HSC regulation as well as in leukemia development through translocation with the nucleoporin gene NUP98. Interestingly, an engineered NUP98-HOXA10 (NA10 fusion can induce a several hundred-fold expansion of HSCs in vitro and NA10 and the AML-associated fusion gene NUP98-HOXD13 (ND13 have a virtually indistinguishable ability to transform myeloid progenitor cells in vitro and to induce leukemia in collaboration with MEIS1 in vivo. METHODOLOGY/PRINCIPAL FINDINGS: These findings provided a potentially powerful approach to identify key pathways mediating Hox-induced expansion and transformation of HSCs by identifying gene expression changes commonly induced by ND13 and NA10 but not by a NUP98-Hox fusion with a non-DNA binding homedomain mutation (N51S. The gene expression repertoire of purified murine bone marrow Sca-1+Lin- cells transduced with retroviral vectors encoding for these genes was established using the Affymetrix GeneChip MOE430A. Approximately seventy genes were differentially expressed in ND13 and NA10 cells that were significantly changed by both compared to the ND13(N51S mutant. Intriguingly, several of these potential Hox target genes have been implicated in HSC expansion and self-renewal, including the tyrosine kinase receptor Flt3, the prion protein, Prnp, hepatic leukemia factor, Hlf and Jagged-2, Jag2. Consistent with these results, FLT3, HLF and JAG2 expression correlated with HOX A cluster gene expression in human leukemia samples. CONCLUSIONS: In conclusion this study has identified several novel Hox downstream target genes and provides important new leads to key regulators of the expansion and transformation of hematopoietic stem cells by Hox.

  16. Membrane fusion inducers, chloroquine and spermidine increase lipoplex-mediated gene transfection

    International Nuclear Information System (INIS)

    Wong-Baeza, Carlos; Bustos, Israel; Serna, Manuel; Tescucano, Alonso; Alcantara-Farfan, Veronica; Ibanez, Miguel; Montanez, Cecilia; Wong, Carlos; Baeza, Isabel

    2010-01-01

    Gene transfection into mammalian cells can be achieved with viral and non-viral vectors. Non-viral vectors, such as cationic lipids that form lipoplexes with DNA, are safer and more stable than viral vectors, but their transfection efficiencies are lower. Here we describe that the simultaneous treatment with a membrane fusion inducer (chlorpromazine or procainamide) plus the lysosomotropic agent chloroquine increases lipoplex-mediated gene transfection in human (HEK293 and C-33 A) and rat (PC12) cell lines (up to 9.2-fold), as well as in situ in BALB/c mice spleens and livers (up to 6-fold); and that the polyamine spermidine increases lipoplex-mediated gene transfection and expression in cell cultures. The use of these four drugs provides a novel, safe and relatively inexpensive way to considerably increase lipoplex-mediated gene transfection efficiency.

  17. Cell-to-Cell Measles Virus Spread between Human Neurons Is Dependent on Hemagglutinin and Hyperfusogenic Fusion Protein.

    Science.gov (United States)

    Sato, Yuma; Watanabe, Shumpei; Fukuda, Yoshinari; Hashiguchi, Takao; Yanagi, Yusuke; Ohno, Shinji

    2018-03-15

    Measles virus (MV) usually causes acute infection but in rare cases persists in the brain, resulting in subacute sclerosing panencephalitis (SSPE). Since human neurons, an important target affected in the disease, do not express the known MV receptors (signaling lymphocyte activation molecule [SLAM] and nectin 4), how MV infects neurons and spreads between them is unknown. Recent studies have shown that many virus strains isolated from SSPE patients possess substitutions in the extracellular domain of the fusion (F) protein which confer enhanced fusion activity. Hyperfusogenic viruses with such mutations, unlike the wild-type MV, can induce cell-cell fusion even in SLAM- and nectin 4-negative cells and spread efficiently in human primary neurons and the brains of animal models. We show here that a hyperfusogenic mutant MV, IC323-F(T461I)-EGFP (IC323 with a fusion-enhancing T461I substitution in the F protein and expressing enhanced green fluorescent protein), but not the wild-type MV, spreads in differentiated NT2 cells, a widely used human neuron model. Confocal time-lapse imaging revealed the cell-to-cell spread of IC323-F(T461I)-EGFP between NT2 neurons without syncytium formation. The production of virus particles was strongly suppressed in NT2 neurons, also supporting cell-to-cell viral transmission. The spread of IC323-F(T461I)-EGFP was inhibited by a fusion inhibitor peptide as well as by some but not all of the anti-hemagglutinin antibodies which neutralize SLAM- or nectin-4-dependent MV infection, suggesting the presence of a distinct neuronal receptor. Our results indicate that MV spreads in a cell-to-cell manner between human neurons without causing syncytium formation and that the spread is dependent on the hyperfusogenic F protein, the hemagglutinin, and the putative neuronal receptor for MV. IMPORTANCE Measles virus (MV), in rare cases, persists in the human central nervous system (CNS) and causes subacute sclerosing panencephalitis (SSPE) several

  18. Somatic Cell Fusions Reveal Extensive Heterogeneity in Basal-like Breast Cancer

    DEFF Research Database (Denmark)

    Su, Ying; Subedee, Ashim; Bloushtain-Qimron, Noga

    2015-01-01

    Basal-like and luminal breast tumors have distinct clinical behavior and molecular profiles, yet the underlying mechanisms are poorly defined. To interrogate processes that determine these distinct phenotypes and their inheritance pattern, we generated somatic cell fusions and performed integrate...... for luminal-basal fusions, and we identified EN1, TBX18, and TCF4 as candidate transcriptional regulators of the luminal-to-basal switch. Our findings highlight the remarkable epigenetic plasticity of breast cancer cells....... of heterogeneity in basal-like breast cancers that correlates with clinical outcome. We also found that protein extracts of basal-like cells are sufficient to induce a luminal-to-basal phenotypic switch, implying a trigger of basal-like autoregulatory circuits. We determined that KDM6A might be required......Basal-like and luminal breast tumors have distinct clinical behavior and molecular profiles, yet the underlying mechanisms are poorly defined. To interrogate processes that determine these distinct phenotypes and their inheritance pattern, we generated somatic cell fusions and performed integrated...

  19. p-Glycoprotein ABCB5 and YB-1 expression plays a role in increased heterogeneity of breast cancer cells: correlations with cell fusion and doxorubicin resistance

    International Nuclear Information System (INIS)

    Yang, Ji Yeon; Ha, Seon-Ah; Yang, Yun-Sik; Kim, Jin Woo

    2010-01-01

    Cancer cells recurrently develop into acquired resistance to the administered drugs. The iatrogenic mechanisms of induced chemotherapy-resistance remain elusive and the degree of drug resistance did not exclusively correlate with reductions of drug accumulation, suggesting that drug resistance may involve additional mechanisms. Our aim is to define the potential targets, that makes drug-sensitive MCF-7 breast cancer cells turn to drug-resistant, for the anti-cancer drug development against drug resistant breast cancer cells. Doxorubicin resistant human breast MCF-7 clones were generated. The doxorubicin-induced cell fusion events were examined. Heterokaryons were identified and sorted by FACS. In the development of doxorubicin resistance, cell-fusion associated genes, from the previous results of microarray, were verified using dot blot array and quantitative RT-PCR. The doxorubicin-induced expression patterns of pro-survival and pro-apoptotic genes were validated. YB-1 and ABCB5 were up regulated in the doxorubicin treated MCF-7 cells that resulted in certain degree of genomic instability that accompanied by the drug resistance phenotype. Cell fusion increased diversity within the cell population and doxorubicin resistant MCF-7 cells emerged probably through clonal selection. Most of the drug resistant hybrid cells were anchorage independent. But some of the anchorage dependent MCF-7 cells exhibited several unique morphological appearances suggesting minor population of the fused cells maybe de-differentiated and have progenitor cell like characteristics. Our work provides valuable insight into the drug induced cell fusion event and outcome, and suggests YB-1, GST, ABCB5 and ERK3 could be potential targets for the anti-cancer drug development against drug resistant breast cancer cells. Especially, the ERK-3 serine/threonine kinase is specifically up-regulated in the resistant cells and known to be susceptible to synthetic antagonists

  20. Hybrid cells derived from breast epithelial cell/breast cancer cell fusion events show a differential RAF-AKT crosstalk

    Directory of Open Access Journals (Sweden)

    Özel Cem

    2012-04-01

    Full Text Available Abstract Background The biological phenomenon of cell fusion has been linked to several characteristics of tumour progression, including an enhanced metastatogenic capacity and an enhanced drug resistance of hybrid cells. We demonstrated recently that M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics spontaneously fused with MDA-MB-435-Hyg breast cancer cells, thereby giving rise to stable M13MDA435 hybrid cells, which are characterised by a unique gene expression profile and migratory behaviour. Here we investigated the involvement of the PLC-β/γ1, PI3K/AKT and RAS-RAF-ERK signal transduction cascades in the EGF and SDF-1α induced migration of two M13MDA435 hybrid cell clones in comparison to their parental cells. Results Analysis of the migratory behaviour by using the three-dimensional collagen matrix migration assay showed that M13SV1-EGFP-Neo cells as well as M13MDA435 hybrid cells, but not the breast cancer cell line, responded to EGF stimulation with an increased locomotory activity. By contrast, SDF-1α solely stimulated the migration of M13SV1-EGFP-Neo cells, whereas the migratory activity of the other cell lines was blocked. Analysis of signal transduction cascades revealed a putative differential RAF-AKT crosstalk in M13MDA435-1 and -3 hybrid cell clones. The PI3K inhibitor Ly294002 effectively blocked the EGF induced migration of M13MDA435-3 hybrid cells, whereas the EGF induced locomotion of M13MDA435-1 hybrid cells was markedly increased. Analysis of RAF-1 S259 phosphorylation, being a major mediator of the negative regulation of RAF-1 by AKT, showed decreased pRAF-1 S259 levels in LY294002 treated M13MDA435-1 hybrid cells. By contrast, pRAF-1 S259 levels remained unaltered in the other cell lines. Inhibition of PI3K/AKT signalling by Ly294002 relieves the AKT mediated phosphorylation of RAF-1, thereby restoring MAPK signalling. Conclusions Here we show that hybrid cells could evolve exhibiting a

  1. Search for a nuclear signature of the electrolytically induced cold fusion

    International Nuclear Information System (INIS)

    Wojtkowska, J.; Glowacki, S.; Jaskola, M.; Moroz, Z.; Ratynski, W.; Kedzierzawski, P.; Zoltowski, P.

    1990-01-01

    The neutron flux from the Pd-Pt electrolytic cell filled with D 2 O+D 2 SO 4 and D 2 O+LiOD was measured to search for the ''cold fusion'' induced in the deuterium stored in the palladium electrode. The fast-neutron ''long counter'', the scintillation detector with the NE - 213 liquid scintillator and with the PSD gamma-neutron distinguishing system and the scintillation detector of fast-neutron and/or gamma-ray bursts were used. No direct correlation was observed between the neutron flux measured in the presence or absence of the electrolysis. The upper limit is given for the neutron flux induced by the process under study. (author)

  2. Prm3p is a pheromone-induced peripheral nuclear envelope protein required for yeast nuclear fusion.

    Science.gov (United States)

    Shen, Shu; Tobery, Cynthia E; Rose, Mark D

    2009-05-01

    Nuclear membrane fusion is the last step in the mating pathway of the yeast Saccharomyces cerevisiae. We adapted a bioinformatics approach to identify putative pheromone-induced membrane proteins potentially required for nuclear membrane fusion. One protein, Prm3p, was found to be required for nuclear membrane fusion; disruption of PRM3 caused a strong bilateral defect, in which nuclear congression was completed but fusion did not occur. Prm3p was localized to the nuclear envelope in pheromone-responding cells, with significant colocalization with the spindle pole body in zygotes. A previous report, using a truncated protein, claimed that Prm3p is localized to the inner nuclear envelope. Based on biochemistry, immunoelectron microscopy and live cell microscopy, we find that functional Prm3p is a peripheral membrane protein exposed on the cytoplasmic face of the outer nuclear envelope. In support of this, mutations in a putative nuclear localization sequence had no effect on full-length protein function or localization. In contrast, point mutations and deletions in the highly conserved hydrophobic carboxy-terminal domain disrupted both protein function and localization. Genetic analysis, colocalization, and biochemical experiments indicate that Prm3p interacts directly with Kar5p, suggesting that nuclear membrane fusion is mediated by a protein complex.

  3. Spatial focalization of pheromone/MAPK signaling triggers commitment to cell–cell fusion

    Science.gov (United States)

    Merlini, Laura

    2016-01-01

    Cell fusion is universal in eukaryotes for fertilization and development, but what signals this process is unknown. Here, we show in Schizosaccharomyces pombe that fusion does not require a dedicated signal but is triggered by spatial focalization of the same pheromone–GPCR (G-protein-coupled receptor)–MAPK signaling cascade that drives earlier mating events. Autocrine cells expressing the receptor for their own pheromone trigger fusion attempts independently of cell–cell contact by concentrating pheromone release at the fusion focus, a dynamic actin aster underlying the secretion of cell wall hydrolases. Pheromone receptor and MAPK cascade are similarly enriched at the fusion focus, concomitant with fusion commitment in wild-type mating pairs. This focalization promotes cell fusion by immobilizing the fusion focus, thus driving local cell wall dissolution. We propose that fusion commitment is imposed by a local increase in MAPK concentration at the fusion focus, driven by a positive feedback between fusion focus formation and focalization of pheromone release and perception. PMID:27798845

  4. Membrane fusion-competent virus-like proteoliposomes and proteinaceous supported bilayers made directly from cell plasma membranes.

    Science.gov (United States)

    Costello, Deirdre A; Hsia, Chih-Yun; Millet, Jean K; Porri, Teresa; Daniel, Susan

    2013-05-28

    Virus-like particles are useful materials for studying virus-host interactions in a safe manner. However, the standard production of pseudovirus based on the vesicular stomatitis virus (VSV) backbone is an intricate procedure that requires trained laboratory personnel. In this work, a new strategy for creating virus-like proteoliposomes (VLPLs) and virus-like supported bilayers (VLSBs) is presented. This strategy uses a cell blebbing technique to induce the formation of nanoscale vesicles from the plasma membrane of BHK cells expressing the hemagglutinin (HA) fusion protein of influenza X-31. These vesicles and supported bilayers contain HA and are used to carry out single particle membrane fusion events, monitored using total internal reflection fluorescence microscopy. The results of these studies show that the VLPLs and VLSBs contain HA proteins that are fully competent to carry out membrane fusion, including the formation of a fusion pore and the release of fluorophores loaded into vesicles. This new strategy for creating spherical and planar geometry virus-like membranes has many potential applications. VLPLs could be used to study fusion proteins of virulent viruses in a safe manner, or they could be used as therapeutic delivery particles to transport beneficial proteins coexpressed in the cells to a target cell. VLSBs could facilitate high throughput screening of antiviral drugs or pathogen-host cell interactions.

  5. Influenza-virus membrane fusion by cooperative fold-back of stochastically induced hemagglutinin intermediates

    NARCIS (Netherlands)

    Ivanovic, Tijana; Choi, Jason L.; Whelan, Sean P.; Oijen, Antoine M. van; Harrison, Stephen C.

    2013-01-01

    Influenza virus penetrates cells by fusion of viral and endosomal membranes catalyzed by the viral hemagglutinin (HA). Structures of the initial and final states of the HA trimer define the fusion endpoints, but do not specify intermediates. We have characterized these transitions by analyzing

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

    International Nuclear Information System (INIS)

    Wu Liguo; Hutt-Fletcher, Lindsey M.

    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 recognized by an antibody that differentially inhibits epithelial and B cell fusion significantly impact both the core fusion machinery and cell-specific events. Substitution of alanine for glycine at residue 594 completely abrogates fusion with either B cells or epithelial cells. Substitution of alanine for glutamic acid at residue 595 reduces fusion with epithelial cells, greatly enhances fusion with B cells and allows low levels of B cell fusion even in the absence of gL

  7. Construction and Characterization of Insect Cell-Derived Influenza VLP: Cell Binding, Fusion, and EGFP Incorporation

    Directory of Open Access Journals (Sweden)

    Yi-Shin Pan

    2010-01-01

    Full Text Available We have constructed virus-like particles (VLPs harboring hemagglutinin (HA, neuraminidase (NA, matrix protein 1 (M1 ,and proton channel protein (M2 using baculovirus as a vector in the SF9 insect cell. The size of the expressed VLP was estimated to be ~100 nm by light scattering experiment and transmission electron microscopy. Recognition of HA on the VLP surface by the HA2-specific monoclonal antibody IIF4 at acidic pH, as probed by surface plasmon resonance, indicated the pH-induced structural rearrangement of HA. Uptake of the particle by A549 mediated by HA-sialylose receptor interaction was visualized by the fluorescent-labeled VLP. The HA-promoted cell-virus fusion activity was illustrated by fluorescence imaging on the Jurkat cells incubated with rhodamine-loaded VLP performed at fusogenic pH. Furthermore, the green fluorescence protein (GFP was fused to NA to produce VLP with a pH-sensitive probe, expanding the use of VLP as an antigen carrier and a tool for viral tracking.

  8. Dysregulated Glycoprotein B-Mediated Cell-Cell Fusion Disrupts Varicella-Zoster Virus and Host Gene Transcription during Infection.

    Science.gov (United States)

    Oliver, Stefan L; Yang, Edward; Arvin, Ann M

    2017-01-01

    The highly conserved herpesvirus glycoprotein complex gB/gH-gL mediates membrane fusion during virion entry and cell-cell fusion. Varicella-zoster virus (VZV) characteristically forms multinucleated cells, or syncytia, during the infection of human tissues, but little is known about this process. The cytoplasmic domain of VZV gB (gBcyt) has been implicated in cell-cell fusion regulation because a gB[Y881F] substitution causes hyperfusion. gBcyt regulation is necessary for VZV pathogenesis, as the hyperfusogenic mutant gB[Y881F] is severely attenuated in human skin xenografts. In this study, gBcyt-regulated fusion was investigated by comparing melanoma cells infected with wild-type-like VZV or hyperfusogenic mutants. The gB[Y881F] mutant exhibited dramatically accelerated syncytium formation in melanoma cells caused by fusion of infected cells with many uninfected cells, increased cytoskeleton reorganization, and rapid displacement of nuclei to dense central structures compared to pOka using live-cell confocal microscopy. VZV and human transcriptomes were concurrently investigated using whole transcriptome sequencing (RNA-seq) to identify viral and cellular responses induced when gBcyt regulation was disrupted by the gB[Y881F] substitution. The expression of four vital VZV genes, ORF61 and the genes for glycoproteins gC, gE, and gI, was significantly reduced at 36 h postinfection for the hyperfusogenic mutants. Importantly, hierarchical clustering demonstrated an association of differential gene expression with dysregulated gBcyt-mediated fusion. A subset of Ras GTPase genes linked to membrane remodeling were upregulated in cells infected with the hyperfusogenic mutants. These data implicate gBcyt in the regulation of gB fusion function that, if unmodulated, triggers cellular processes leading to hyperfusion that attenuates VZV infection. The highly infectious, human-restricted pathogen varicella-zoster virus (VZV) causes chickenpox and shingles. Postherpetic

  9. RhoE is regulated by cyclic AMP and promotes fusion of human BeWo choriocarcinoma cells.

    Directory of Open Access Journals (Sweden)

    Gavin P Collett

    Full Text Available Fusion of placental villous cytotrophoblasts with the overlying syncytiotrophoblast is essential for the maintenance of successful pregnancy, and disturbances in this process have been implicated in pathological conditions such as pre-eclampsia and intra-uterine growth retardation. In this study we examined the role of the Rho GTPase family member RhoE in trophoblast differentiation and fusion using the BeWo choriocarcinoma cell line, a model of villous cytotrophoblast fusion. Treatment of BeWo cells with the cell permeable cyclic AMP analogue dibutyryl cyclic AMP (dbcAMP resulted in a strong upregulation of RhoE at 24 h, coinciding with the onset of fusion. Using the protein kinase A (PKA-specific cAMP analogue N(6-phenyl-cAMP, and a specific inhibitor of PKA (14-22 amide, PKI, we found that upregulation of RhoE by cAMP was mediated through activation of PKA signalling. Silencing of RhoE expression by RNA interference resulted in a significant decrease in dbcAMP-induced fusion. However, expression of differentiation markers human chorionic gonadotrophin and placental alkaline phosphatase was unaffected by RhoE silencing. Finally, we found that RhoE upregulation by dbcAMP was significantly reduced under hypoxic conditions in which cell fusion is impaired. These results show that induction of RhoE by cAMP is mediated through PKA and promotes BeWo cell fusion but has no effect on functional differentiation, supporting evidence that these two processes may be controlled by separate or diverging pathways.

  10. Investigation of the influence of incomplete fusion on complete fusion of 16O induced reactions at moderate excitation energies

    Directory of Open Access Journals (Sweden)

    Ahamad Tauseef

    2012-12-01

    Full Text Available An attempt has been made to investigate for the reaction dynamics leading to incomplete fusion (ICF of heavy ions at moderate excitation energies, especially the influence of incomplete fusion on complete fusion (CF of 16O induced reactions at specific energies. Excitation functions (EFs of various reaction products populated via CF and/or ICF of 16O projectile with 45Sc target were measured at energies ≈3-7 MeV/nucleon, using recoil catcher technique followed by offline γ-ray spectroscopy. The measured EFs were compared with theoretical values obtained using the statistical model code PACE4. The experimentally measured EFs were in general found to be in good agreement with the theoretical predictions for non α-emitting channels in the present target projectile system. However, for α-emitting channels the measured EFs were higher than the predictions of the theoretical model codes, which may be credited to incomplete fusion reactions at these energies.

  11. Mesenchymal stem cells expressing baculovirus-engineered BMP-2 and VEGF enhance posterolateral spine fusion in a rabbit model.

    Science.gov (United States)

    Fu, Tsai-Sheng; Chang, Yu-Han; Wong, Chak-Bor; Wang, I-Chun; Tsai, Tsung-Ting; Lai, Po-Liang; Chen, Lih-Huei; Chen, Wen-Jer

    2015-09-01

    were significantly greater in the MSC/Bac/TCP group. The results indicate the potential of using baculovirus as a vector for gene/cell therapy approaches to improve bone healing and support the feasibility of using allogeneic MSCs for inducing bone formation and intertransverse process fusion. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Recurrent Fusion Genes in Gastric Cancer: CLDN18-ARHGAP26 Induces Loss of Epithelial Integrity

    Directory of Open Access Journals (Sweden)

    Fei Yao

    2015-07-01

    Full Text Available Genome rearrangements, a hallmark of cancer, can result in gene fusions with oncogenic properties. Using DNA paired-end-tag (DNA-PET whole-genome sequencing, we analyzed 15 gastric cancers (GCs from Southeast Asians. Rearrangements were enriched in open chromatin and shaped by chromatin structure. We identified seven rearrangement hot spots and 136 gene fusions. In three out of 100 GC cases, we found recurrent fusions between CLDN18, a tight junction gene, and ARHGAP26, a gene encoding a RHOA inhibitor. Epithelial cell lines expressing CLDN18-ARHGAP26 displayed a dramatic loss of epithelial phenotype and long protrusions indicative of epithelial-mesenchymal transition (EMT. Fusion-positive cell lines showed impaired barrier properties, reduced cell-cell and cell-extracellular matrix adhesion, retarded wound healing, and inhibition of RHOA. Gain of invasion was seen in cancer cell lines expressing the fusion. Thus, CLDN18-ARHGAP26 mediates epithelial disintegration, possibly leading to stomach H+ leakage, and the fusion might contribute to invasiveness once a cell is transformed.

  13. [Targeted tumor suppression by a secreted fusion protein consisting of anti- erbB2 antibody and reversed caspase-3 to SKBr3 cells].

    Science.gov (United States)

    Zhang, Li-hong; Jia, Lin-tao; Yu, Cui-juan; Qu, Ping; Dong, Hai-long; Zhao, Jing; Xu, Yan-ming; Wang, Cheng-Ji; Yang, An-gang

    2003-04-10

    To investigate the targeted killing effect to SKBr3 cells due to the expression of a secreted fusion protein consisting of anti-erbB2 antibody and reversed caspase-3. A recombinant plasmid pCMV-e23scFv-PEII-revcasp 3 was constructed by subcloning reversed caspase-3 gene to the downstream of anti-erbB2 antibody and transfected into Jurkat cells. The cell lines which secreted expressing fusion protein stably were selected. The fusion protein in media was detected by ELISA and the media was used to culture human breast cancer SKBr3 cells. The recombinant plasmids with liposomes was administrated to BALB/C nude mouses bearing SKBr3 tumor by intramuscular injection. The targetting effect of the recombinant fusion protein caspase-3 was detected by indirect immunofluorescence staining. Fusion protein can be expressed and secreted by Jurkat cells stably and kill SKBr3 cells. Significant prolonged survival time (prolonged by 72%) and inhibition of tumor growth in vivo (within inhibition ratio of 77%) were seen in the group administered with recombinant plasmids. Indirect immunofluorescence staining showed that the recombinant fusion protein caspase-3 has targetting effect. Secreted expression of the fusion protein consisting of anti-erbB2 antibody and reversed caspase-3 can targetedly induce SKBr3 cells to death.

  14. `Full fusion' is not ineluctable during vesicular exocytosis of neurotransmitters by endocrine cells

    Science.gov (United States)

    Oleinick, Alexander; Svir, Irina; Amatore, Christian

    2017-01-01

    Vesicular exocytosis is an essential and ubiquitous process in neurons and endocrine cells by which neurotransmitters are released in synaptic clefts or extracellular fluids. It involves the fusion of a vesicle loaded with chemical messengers with the cell membrane through a nanometric fusion pore. In endocrine cells, unless it closes after some flickering (`Kiss-and-Run' events), this initial pore is supposed to expand exponentially, leading to a full integration of the vesicle membrane into the cell membrane-a stage called `full fusion'. We report here a compact analytical formulation that allows precise measurements of the fusion pore expansion extent and rate to be extracted from individual amperometric spike time courses. These data definitively establish that, during release of catecholamines, fusion pores enlarge at most to approximately one-fifth of the radius of their parent vesicle, hence ruling out the ineluctability of `full fusion'.

  15. Detecting ALK, ROS1 and RET Fusion Genes in Cell Block Samples.

    Science.gov (United States)

    Zhao, Chao; Li, Xuefei; Li, Jiayu; Zhang, Yishi; Ren, Shengxiang; Chen, Xiaoxia; Zhou, Caicun

    2014-06-01

    Whether Cell block (CB) samples are applicable to detect anaplastic lymphoma kinase (ALK), c-ros oncogene 1 (ROS1) and ret proto-oncogene (RET) fusion genes in lung adenocarcinoma is still unknown. In this study, 108 cytological samples that contained lung adenocarcinoma cells were collected, and made into CB. The CB samples all contained at least 30% lung adenocarcinoma cells. In these patients, 48 harbored EGFR mutation. Among the 50 EGFR wild type patients who detected fusion genes, 14 carried EML4-ALK fusion (28%), 2 had TPM3-ROS1 fusion (4%), and 3 harbored KIF5B-RET fusion (6%). No double fusions were found in one sample. Patients with fusion genes were younger than those without fusion genes (p = 0.032), but no significant difference was found in sex and smoking status (p > 0.05). In the thirty-five patients who received first-line chemotherapy, patients with fusion gene positive had disease control rate (DCR) (72.7% VS 50%, p > 0.05) and objective response rate (ORR) (9.1% VS 4.2%, p > 0.05) compared with those having fusion gene negative. The median progression free survival (mPFS) were 4.0 and 2.7 months in patients harbored fusion mutations and wild type, respectively (p > 0.05). We conclude that CB samples could be used to detect ALK, ROS1 and RET fusions in NSCLC. The frequency distribution of three fusion genes is higher in lung adenocarcinoma with wild-type EGFR, compared with unselected NSCLC patient population. Patients with fusion genes positive are younger than those with fusion gene negative, but they had no significantly different PFS in first-line chemotherapy.

  16. Dynamic clustering and dispersion of lipid rafts contribute to fusion competence of myogenic cells

    International Nuclear Information System (INIS)

    Mukai, Atsushi; Kurisaki, Tomohiro; Sato, Satoshi B.; Kobayashi, Toshihide; Kondoh, Gen; Hashimoto, Naohiro

    2009-01-01

    Recent research indicates that the leading edge of lamellipodia of myogenic cells (myoblasts and myotubes) contains presumptive fusion sites, yet the mechanisms that render the plasma membrane fusion-competent remain largely unknown. Here we show that dynamic clustering and dispersion of lipid rafts contribute to both cell adhesion and plasma membrane union during myogenic cell fusion. Adhesion-complex proteins including M-cadherin, β-catenin, and p120-catenin accumulated at the leading edge of lamellipodia, which contains the presumptive fusion sites of the plasma membrane, in a lipid raft-dependent fashion prior to cell contact. In addition, disruption of lipid rafts by cholesterol depletion directly prevented the membrane union of myogenic cell fusion. Time-lapse recording showed that lipid rafts were laterally dispersed from the center of the lamellipodia prior to membrane fusion. Adhesion proteins that had accumulated at lipid rafts were also removed from the presumptive fusion sites when lipid rafts were laterally dispersed. The resultant lipid raft- and adhesion complex-free area at the leading edge fused with the opposing plasma membrane. These results demonstrate a key role for dynamic clustering/dispersion of lipid rafts in establishing fusion-competent sites of the myogenic cell membrane, providing a novel mechanistic insight into the regulation of myogenic cell fusion.

  17. Dynamic clustering and dispersion of lipid rafts contribute to fusion competence of myogenic cells

    Energy Technology Data Exchange (ETDEWEB)

    Mukai, Atsushi [Department of Regenerative Medicine, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, 36-3 Gengo, Morioka, Oobu, Aichi 474-8522 (Japan); Kurisaki, Tomohiro [Department of Growth Regulation, Institute for Frontier Medical Sciences, Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507 (Japan); Sato, Satoshi B. [Research Center for Low Temperature and Material Sciences, Kyoto University, Yoshida-honmachi, Kyoto 606-8501 (Japan); Kobayashi, Toshihide [Lipid Biology Laboratory, Discovery Research Institute, RIKEN, Wako, Saitama 351-0198 (Japan); Kondoh, Gen [Laboratory of Animal Experiments for Regeneration, Institute for Frontier Medical Sciences, Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507 (Japan); Hashimoto, Naohiro, E-mail: nao@nils.go.jp [Department of Regenerative Medicine, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, 36-3 Gengo, Morioka, Oobu, Aichi 474-8522 (Japan)

    2009-10-15

    Recent research indicates that the leading edge of lamellipodia of myogenic cells (myoblasts and myotubes) contains presumptive fusion sites, yet the mechanisms that render the plasma membrane fusion-competent remain largely unknown. Here we show that dynamic clustering and dispersion of lipid rafts contribute to both cell adhesion and plasma membrane union during myogenic cell fusion. Adhesion-complex proteins including M-cadherin, {beta}-catenin, and p120-catenin accumulated at the leading edge of lamellipodia, which contains the presumptive fusion sites of the plasma membrane, in a lipid raft-dependent fashion prior to cell contact. In addition, disruption of lipid rafts by cholesterol depletion directly prevented the membrane union of myogenic cell fusion. Time-lapse recording showed that lipid rafts were laterally dispersed from the center of the lamellipodia prior to membrane fusion. Adhesion proteins that had accumulated at lipid rafts were also removed from the presumptive fusion sites when lipid rafts were laterally dispersed. The resultant lipid raft- and adhesion complex-free area at the leading edge fused with the opposing plasma membrane. These results demonstrate a key role for dynamic clustering/dispersion of lipid rafts in establishing fusion-competent sites of the myogenic cell membrane, providing a novel mechanistic insight into the regulation of myogenic cell fusion.

  18. Near-maximal expansions of hematopoietic stem cells in culture using NUP98-HOX fusions

    Science.gov (United States)

    Ohta, Hideaki; Sekulovic, Sanja; Bakovic, Silvia; Eaves, Connie J.; Pineault, Nicolas; Gasparetto, Maura; Smith, Clayton; Sauvageau, Guy; Humphries, R. Keith

    2009-01-01

    Objective Strategies to expand hematopoietic stem cells (HSCs) ex vivo are of key interest. The objective of this study was to resolve if ability of HOXB4, previously documented to induce a significant expansion of HSCs in culture, may extend to other HOX genes and also to further analyze the HOX sequence requirements to achieve this effect. Methods To investigate the ability of Nucleoporin98-Homeobox fusion genes to stimulate HSC self-renewal, we evaluated their presence in 10- to 20-day cultures of transduced mouse bone marrow cells. Stem cell recovery was measured by limiting-dilution assay for long-term competitive repopulating cells (CRU Assay). Results These experiments revealed remarkable expansions of Nucleoporin98-Homeobox–transduced HSCs (1000-fold to 10,000-fold over input) in contrast to the expected decline of HSCs in control cultures. Nevertheless, the Nucleoporin98-Homeobox-expanded HSCs displayed no proliferative senescence and retained normal lympho-myeloid differentiation activity and a controlled pool size in vivo. Analysis of proviral integration patterns showed the cells regenerated in vivo were highly polyclonal, indicating they had derived from a large proportion of the initially targeted HSCs. Importantly, these effects were preserved when all HOX sequences flanking the homeodomain were removed, thus defining the homeodomain as a key and independent element in the fusion. Conclusion These findings create new possibilities for investigating HSCs biochemically and genetically and for achieving clinically significant expansion of human HSCs. PMID:17577930

  19. Remotely controlled fusion of selected vesicles and living cells: a key issue review

    Science.gov (United States)

    Bahadori, Azra; Moreno-Pescador, Guillermo; Oddershede, Lene B.; Bendix, Poul M.

    2018-03-01

    Remote control over fusion of single cells and vesicles has a great potential in biological and chemical research allowing both transfer of genetic material between cells and transfer of molecular content between vesicles. Membrane fusion is a critical process in biology that facilitates molecular transport and mixing of cellular cytoplasms with potential formation of hybrid cells. Cells precisely regulate internal membrane fusions with the aid of specialized fusion complexes that physically provide the energy necessary for mediating fusion. Physical factors like membrane curvature, tension and temperature, affect biological membrane fusion by lowering the associated energy barrier. This has inspired the development of physical approaches to harness the fusion process at a single cell level by using remotely controlled electromagnetic fields to trigger membrane fusion. Here, we critically review various approaches, based on lasers or electric pulses, to control fusion between individual cells or between individual lipid vesicles and discuss their potential and limitations for present and future applications within biochemistry, biology and soft matter.

  20. Microfilament dynamics during cell movement and chemotaxis monitored using a GFP-actin fusion protein.

    Science.gov (United States)

    Westphal, M; Jungbluth, A; Heidecker, M; Mühlbauer, B; Heizer, C; Schwartz, J M; Marriott, G; Gerisch, G

    1997-03-01

    The microfilament system in the cortex of highly motile cells, such as neutrophils and cells of the eukaryotic microorganism Dictyostelium discoideum, is subject to rapid re-organization, both spontaneously and in response to external signals. In particular, actin polymerization induced by a gradient of chemoattractant leads to local accumulation of filamentous actin and protrusion of a 'leading edge' of the cell in the direction of the gradient. In order to study the dynamics of actin in these processes, actin was tagged at its amino terminus with green fluorescent protein (GFP) and observed with fluorescence microscopy in living cells of D. discoideum. Purified GFP-actin was capable of copolymerizing with actin. In the transfected cells of D. discoideum studied, GFP-actin made up 10-20% of the total actin. Microfilaments containing GFP-actin were capable of generating force with myosin in an in vitro assay. Observations of single living cells using fluorescence microscopy showed that the fusion protein was enriched in cell projections, including filopodia and leading edges, and that the fusion protein reflected the dynamics of the microfilament system in cells that were freely moving, being chemotactically stimulated, or aggregated. When confocal sections of fixed cells containing GFP-actin were labeled with fluorescent phalloidin, which binds only to filamentous actin, there was a correlation between the areas of GFP-actin and phalloidin fluorescence, but there were distinct sites in which GFP-actin was more prominent. Double labeling with GFP-actin and other probes provides an indication of the various states of actin in motile cells. A major portion of the actin assemblies visualized using GFP-actin are networks or bundles of filamentous actin. Other clusters of GFP-actin might represent stores of monomeric actin in the form of complexes with actin-sequestering proteins.

  1. A Unique Opportunity To Test Whether Cell Fusion Is a Mechanism of Breast Cancer Metastasis

    Science.gov (United States)

    2016-08-01

    cells of a melanoma clone ( wild -type for tyrosinase,C/C) implanted into BALB/c nu/numice (homozygous mutation for albino tyrosinase, c/c) devel- oped...polymorphism and forensic genetic techniques to suggest that ametastaticmelanoma lesion inapatientarose fromthe fusion between a bone marrow–derived cell the...Spontaneous fusion between cancer cells and endothelial cells. Cell. Mol. Life Sci. 61, 2125–2131 41. Rappa, G., Mercapide, J., and Lorico, A. (2012

  2. Effect of lipid molecule headgroup mismatch on non steroidal anti-inflammatory drugs induced membrane fusion.

    Science.gov (United States)

    Mondal Roy, Sutapa; Sarkar, Munna

    2011-12-20

    Membrane fusion is an essential process guiding many important biological events, which most commonly requires the aid of proteins and peptides as fusogenic agents. Small drug induced fusion at low drug concentration is a rare event. Only three drugs, namely, meloxicam (Mx), piroxicam (Px), and tenoxicam (Tx), belonging to the oxicam group of non steroidal anti-inflammatory drugs (NSAIDs) have been shown by us to induce membrane fusion successfully at low drug concentration. A better elucidation of the mechanism and the effect of different parameters in modulating the fusion process will allow the use of these common drugs to induce and control membrane fusion in various biochemical processes. In this study, we monitor the effect of lipid headgroup size mismatch in the bilayer on oxicam NSAIDs induced membrane fusion, by introducing dimyristoylphosphatidylethanolamine (DMPE) in dimyristoylphosphatidylcholine (DMPC) small unilamellar vesicles (SUVs). Such headgroup mismatch affects various lipid parameters which includes inhibition of trans-bilayer motion, domain formation, decrease in curvature, etc. Changes in various lipidic parameters introduce defects in the membrane bilayer and thereby modulate membrane fusion. SUVs formed by DMPC with increasing DMPE content (10, 20, and 30 mol %) were used as simple model membranes. Transmission electron microscopy (TEM) and differential scanning calorimetry (DSC) were used to characterize the DMPC-DMPE mixed vesicles. Fluorescence assays were used to probe the time dependence of lipid mixing, content mixing, and leakage and also used to determine the partitioning of the drugs in the membrane bilayer. How the inhibition of trans-bilayer motion, heterogeneous distribution of lipids, decrease in vesicle curvature, etc., arising due to headgroup mismatch affect the fusion process has been isolated and identified here. Mx amplifies these effects maximally followed by Px and Tx. This has been correlated to the enhanced

  3. Fusion of intestinal epithelial cells with bone marrow derived cells is dispensable for tissue homeostasis

    OpenAIRE

    de Jong, Joan H.; Rodermond, Hans M.; Zimberlin, Cheryl D.; Lascano, Valeria; De Sousa E Melo, Felipe; Richel, Dick J.; Medema, Jan Paul; Vermeulen, Louis

    2012-01-01

    The epithelial lining of the intestine is characterized by an immense cellular turn-over ascertaining an extensive regenerative capacity. Multiple reports suggest that besides the local intestinal stem cell pool, circulating cells of bone marrow origin (BMDCs) contribute to this process by fusing with the epithelial lineage. However, the functional relevance of these observations is unknown. In the present study we employ a model system in which we cannot only detect cell fusion but also exam...

  4. Mechanical Stimulation of C2C12 Cells Increases m-Calpain Expression and Activity, Focal Adhesion Plaque Degradation and Cell Fusion

    DEFF Research Database (Denmark)

    Grossi, Alberto; Karlsson, Anders H; Lawson, Moira Ann

    2005-01-01

    to stretch- or load-induced signaling is now beginning to be understood as a factor which affects gene sequences, protein synthesis and an increase in Ca2+ infux in myocytes. Evidence of the involvement of Ca2+ dependent activity in myoblast fusion, cell membrane and cytoskeleton component reorganization due......Abstract Mechanical Stimulation of C2C12 Cells Increases m-calpain Expression and Activity, Focal Adhesion Plaque Degradation and Cell Fusion A. Grossi, A. H. Karlsson, M. A. Lawson; Department of Dairy and Food Science, Royal Veterinary and Agricultural University, Frederiksberg C, Denmark...... to the activity of ubiquitous proteolytic enzymes known as calpains has been reported. Whether there is a link between stretch- or load induced signaling and calpain expression and activation is not known. Using a magnetic bead stimulation assay and C2C12 mouse myoblasts cell population, we have demonstrated...

  5. Site-specific modification of genome with cell-permeable Cre fusion protein in preimplantation mouse embryo

    International Nuclear Information System (INIS)

    Kim, Kyoungmi; Kim, Hwain; Lee, Daekee

    2009-01-01

    Site-specific recombination (SSR) by Cre recombinase and its target sequence, loxP, is a valuable tool in genetic analysis of gene function. Recently, several studies reported successful application of Cre fusion protein containing protein transduction peptide for inducing gene modification in various mammalian cells including ES cell as well as in the whole animal. In this study, we show that a short incubation of preimplantation mouse embryos with purified cell-permeable Cre fusion protein results in efficient SSR. X-Gal staining of preimplantation embryos, heterozygous for Gtrosa26 tm1Sor , revealed that treatment of 1-cell or 2-cell embryos with 3 μM of Cre fusion protein for 2 h leads to Cre-mediated excision in 70-85% of embryos. We have examined the effect of the concentration of the Cre fusion protein and the duration of the treatment on embryonic development, established a condition for full term development and survival to adulthood, and demonstrated the germ line transmission of excised Gtrosa26 allele. Potential applications and advantages of the highly efficient technique described here are discussed.

  6. Dual-specificity tyrosine phosphorylation-regulated kinase 2 regulates osteoclast fusion in a cell heterotypic manner.

    Science.gov (United States)

    Guterman-Ram, Gali; Pesic, Milena; Orenbuch, Ayelet; Czeiger, Tal; Aflalo, Anastasia; Levaot, Noam

    2018-01-01

    Monocyte fusion into osteoclasts, bone resorbing cells, plays a key role in bone remodeling and homeostasis; therefore, aberrant cell fusion may be involved in a variety of debilitating bone diseases. Research in the last decade has led to the discovery of genes that regulate osteoclast fusion, but the basic molecular and cellular regulatory mechanisms underlying the fusion process are not completely understood. Here, we reveal a role for Dyrk2 in osteoclast fusion. We demonstrate that Dyrk2 down regulation promotes osteoclast fusion, whereas its overexpression inhibits fusion. Moreover, Dyrk2 also promotes the fusion of foreign-body giant cells, indicating that Dyrk2 plays a more general role in cell fusion. In an earlier study, we showed that fusion is a cell heterotypic process initiated by fusion-founder cells that fuse to fusion-follower cells, the latter of which are unable to initiate fusion. Here, we show that Dyrk2 limits the expansion of multinucleated founder cells through the suppression of the fusion competency of follower cells. © 2017 Wiley Periodicals, Inc.

  7. Development and characterization of a Rift Valley fever virus cell-cell fusion assay using alphavirus replicon vectors

    International Nuclear Information System (INIS)

    Filone, Claire Marie; Heise, Mark; Doms, Robert W.; Bertolotti-Ciarlet, Andrea

    2006-01-01

    Rift Valley fever virus (RVFV), a member of the Phlebovirus genus in the Bunyaviridae family, is transmitted by mosquitoes and infects both humans and domestic animals, particularly cattle and sheep. Since primary RVFV strains must be handled in BSL-3+ or BSL-4 facilities, a RVFV cell-cell fusion assay will facilitate the investigation of RVFV glycoprotein function under BSL-2 conditions. As for other members of the Bunyaviridae family, RVFV glycoproteins are targeted to the Golgi, where the virus buds, and are not efficiently delivered to the cell surface. However, overexpression of RVFV glycoproteins using an alphavirus replicon vector resulted in the expression of the glycoproteins on the surface of multiple cell types. Brief treatment of RVFV glycoprotein expressing cells with mildly acidic media (pH 6.2 and below) resulted in rapid and efficient syncytia formation, which we quantified by β-galactosidase α-complementation. Fusion was observed with several cell types, suggesting that the receptor(s) for RVFV is widely expressed or that this acid-dependent virus does not require a specific receptor to mediate cell-cell fusion. Fusion occurred over a broad temperature range, as expected for a virus with both mosquito and mammalian hosts. In contrast to cell fusion mediated by the VSV-G glycoprotein, RVFV glycoprotein-dependent cell fusion could be prevented by treating target cells with trypsin, indicating that one or more proteins (or protein-associated carbohydrate) on the host cell surface are needed to support membrane fusion. The cell-cell fusion assay reported here will make it possible to study the membrane fusion activity of RVFV glycoproteins in a high-throughput format and to screen small molecule inhibitors for the ability to block virus-specific membrane fusion

  8. Normal microspore production after cell fusion in Brachiaria jubata (Gramineae

    Directory of Open Access Journals (Sweden)

    Andréa Beatriz Mendes-Bonato

    2003-12-01

    Full Text Available Cytogenetic studies were carried out on 22 accessions of Brachiaria jubata from the Embrapa Beef Cattle Brachiaria collection. One accession was diploid (2n = 2x = 18 and the remaining 21 were tetraploid (2n = 4x = 36. Among five tetraploid accessions, a specific and constant pattern of cell fusion involving only two microsporocytes was recorded. Meiosis proceeded normally from prophase I to the end, giving rise to an octad with normal microspores that developed into fertile pollen grains. Regular octad formation was possible because each cellular chromosome set was maintained in its proper domain, spindles were correctly positioned, and cytokinesis planes were formed in the correct places. Such behavior of meiosis in syncytes has never been reported in any other plant species.

  9. Development of a lentivirus vector-based assay for non-destructive monitoring of cell fusion activity.

    Directory of Open Access Journals (Sweden)

    Zeinab Neshati

    Full Text Available Cell-to-cell fusion can be quantified by endowing acceptor and donor cells with latent reporter genes/proteins and activators of these genes/proteins, respectively. One way to accomplish this goal is by using a bipartite lentivirus vector (LV-based cell fusion assay system in which the cellular fusion partners are transduced with a flippase-activatable Photinus pyralis luciferase (PpLuc expression unit (acceptor cells or with a recombinant gene encoding FLPeNLS+, a nuclear-targeted and molecularly evolved version of flippase (donor cells. Fusion of both cell populations will lead to the FLPe-dependent generation of a functional PpLuc gene. PpLuc activity is typically measured in cell lysates, precluding consecutive analysis of one cell culture. Therefore, in this study the PpLuc-coding sequence was replaced by that of Gaussia princeps luciferase (GpLuc, a secretory protein allowing repeated analysis of the same cell culture. In myotubes the spread of FLPeNLS+ may be limited due to its nuclear localization signal (NLS causing low signal outputs. To test this hypothesis, myoblasts were transduced with LVs encoding either FLPeNLS+ or an NLS-less version of FLPe (FLPeNLS- and subsequently co-cultured in different ratios with myoblasts containing the FLPe-activatable GpLuc expression cassette. At different times after induction of cell-to-cell fusion the GpLuc activity in the culture medium was determined. FLPeNLS+ and FLPeNLS- both activated the latent GpLuc gene but when the percentage of FLPe-expressing myoblasts was limiting, FLPeNLS+ generally yielded slightly higher signals than FLPeNLS- while at low acceptor-to-donor cell ratios FLPeNLS- was usually superior. The ability of FLPeNLS+ to spread through myofibers and to induce reporter gene expression is thus not limited by its NLS. However, at high FLPe concentrations the presence of the NLS negatively affected reporter gene expression. In summary, a rapid and simple chemiluminescence assay for

  10. 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

    chromaffin cells. Simultaneous expression of wild-type SNAP-25 and a mutant unable to support exocytosis progressively altered fusion kinetics and fusion pore opening, indicating that both proteins assemble into heteromeric fusion complexes. Expressing different wild-type:mutant ratios revealed a third power......Exocytosis requires formation of SNARE complexes between vesicle- and target-membranes. Recent assessments in reduced model systems have produced divergent estimates of the number of SNARE complexes needed for fusion. Here, we used a titration approach to answer this question in intact, cultured...... 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...

  11. Nuclear fusion induced by x rays in a crystal

    Science.gov (United States)

    Belyaev, V. B.; Miller, M. B.; Otto, J.; Rakityansky, S. A.

    2016-03-01

    The nuclei that constitute a crystalline lattice oscillate relative to each other with a very low energy that is not sufficient to penetrate through the Coulomb barriers separating them. An additional energy, which is needed to tunnel through the barrier and fuse, can be supplied by external electromagnetic waves (x rays or synchrotron radiation). Exposing the solid compound LiD (lithium deuteride) to x rays for the duration of 111 h, we detect 88 events of nuclear fusion d +6Li→8Be* . Our theoretical estimate agrees with what we observed. One possible application of the phenomenon we found is in measurements of the rates of various nuclear reactions (not necessarily fusion) at extremely low energies inaccessible in accelerator experiments.

  12. Controlling cell position in complex heterotypic 3D microtissues by tissue fusion.

    Science.gov (United States)

    Rago, Adam P; Dean, Dylan M; Morgan, Jeffrey R

    2009-03-01

    Tissue fusion and cell sorting are processes fundamental to developmental biology with applications in tissue engineering. We have designed a fusion assay to investigate the factors governing the fusion of microtissues and the cell sorting that occurs after fusion. Normal human fibroblast (NHF) spheroids were self-assembled and cultured for 1, 4, or 7 days, then combined in trough shaped recesses. Over a 24-h period the spheroids fused to become a rod shaped microtissue and the kinetics and extent of fusion could be quantified by assessing rod contraction. By varying the amount of spheroid culture time prior to fusion (1-7 days), the rate of fusion, the coherence of the building units (as measured by fusion angle) and the steady state length of the structure could be easily controlled. Longer pre-culture times for the spheroids resulted in slower fusion, less coherence and increased length of rod microtissues. The fusion kinetics and steady length of rods formed by smaller versus larger spheroids ( approximately 100 vs. 300 microm diameter) were indistinguishable, even though smaller spheroids had twice the surface area and greater numbers of contacts between units. Both small and large spheroids were strongly influenced by spheroid pre-culture time. Pre-culture time could also be used to control cell sorting and cell position when combinations of NHFs and H35s, a rat hepatocyte cell line, were fused to form heterotypic microtissues. Control of fusion and cell position are important parameters for creating functional heterotypic microtissues as well as the use of microtissues as building units to create larger tissue structures.

  13. Tracking fusion of human mesenchymal stem cells after transplantation to the heart.

    Science.gov (United States)

    Freeman, Brian T; Kouris, Nicholas A; Ogle, Brenda M

    2015-06-01

    Evidence suggests that transplanted mesenchymal stem cells (MSCs) can aid recovery of damaged myocardium caused by myocardial infarction. One possible mechanism for MSC-mediated recovery is reprogramming after cell fusion between transplanted MSCs and recipient cardiac cells. We used a Cre/LoxP-based luciferase reporter system coupled to biophotonic imaging to detect fusion of transplanted human pluripotent stem cell-derived MSCs to cells of organs of living mice. Human MSCs, with transient expression of a viral fusogen, were delivered to the murine heart via a collagen patch. At 2 days and 1 week later, living mice were probed for bioluminescence indicative of cell fusion. Cell fusion was detected at the site of delivery (heart) and in distal tissues (i.e., stomach, small intestine, liver). Fusion was confirmed at the cellular scale via fluorescence in situ hybridization for human-specific and mouse-specific centromeres. Human cells in organs distal to the heart were typically located near the vasculature, suggesting MSCs and perhaps MSC fusion products have the ability to migrate via the circulatory system to distal organs and engraft with local cells. The present study reveals previously unknown migratory patterns of delivered human MSCs and associated fusion products in the healthy murine heart. The study also sets the stage for follow-on studies to determine the functional effects of cell fusion in a model of myocardial damage or disease. Mesenchymal stem cells (MSCs) are transplanted to the heart, cartilage, and other tissues to recover lost function or at least limit overactive immune responses. Analysis of tissues after MSC transplantation shows evidence of fusion between MSCs and the cells of the recipient. To date, the biologic implications of cell fusion remain unclear. A newly developed in vivo tracking system was used to identify MSC fusion products in living mice. The migratory patterns of fusion products were determined both in the target organ (i

  14. Assessing cell fusion and cytokinesis failure as mechanisms of clone 9 hepatocyte multinucleation in vitro.

    Science.gov (United States)

    Simic, Damir; Euler, Catherine; Thurby, Christina; Peden, Mike; Tannehill-Gregg, Sarah; Bunch, Todd; Sanderson, Thomas; Van Vleet, Terry

    2012-08-01

    In this in vitro model of hepatocyte multinucleation, separate cultures of rat Clone 9 cells are labeled with either red or green cell tracker dyes (Red Cell Tracker CMPTX or Vybrant CFDA SE Cell Tracer), plated together in mixed-color colonies, and treated with positive or negative control agents for 4 days. The fluorescent dyes become cell-impermeant after entering cells and are not transferred to adjacent cells in a population, but are inherited by daughter cells after fusion. The mixed-color cultures are then evaluated microscopically for multinucleation and analysis of the underlying mechanism (cell fusion/cytokinesis). Multinucleated cells containing only one dye have undergone cytokinesis failure, whereas dual-labeled multinucleated cells have resulted from fusion. © 2012 by John Wiley & Sons, Inc.

  15. Analysis of mammary specific gene locus regulation in differentiated cells derived by somatic cell fusion

    International Nuclear Information System (INIS)

    Robinson, Claire; Kolb, Andreas F.

    2009-01-01

    The transcriptional regulation of a gene is best analysed in the context of its normal chromatin surroundings. However, most somatic cells, in contrast to embryonic stem cells, are refractory to accurate modification by homologous recombination. We show here that it is possible to introduce precise genomic modifications in ES cells and to analyse the phenotypic consequences in differentiated cells by using a combination of gene targeting, site-specific recombination and somatic cell fusion. To provide a proof of principle, we have analysed the regulation of the casein gene locus in mammary gland cells derived from modified murine ES cells by somatic cell fusion. A β-galactosidase reporter gene was inserted in place of the β-casein gene and the modified ES cells, which do not express the reporter gene, were fused with the mouse mammary gland cell line HC11. The resulting cell clones expressed the β-galactosidase gene to a similar extent and with similar hormone responsiveness as the endogenous gene. However, a reporter gene under the control of a minimal β-casein promoter (encompassing the two consensus STAT5 binding sites which mediate the hormone response of the casein genes) was unable to replicate expression levels or hormone responsiveness of the endogenous gene when inserted into the same site of the casein locus. As expected, these results implicate sequences other than the STAT5 sites in the regulation of the β-casein gene

  16. ER/K linked GPCR-G protein fusions systematically modulate second messenger response in cells.

    Science.gov (United States)

    Malik, Rabia U; Dysthe, Matthew; Ritt, Michael; Sunahara, Roger K; Sivaramakrishnan, Sivaraj

    2017-08-10

    FRET and BRET approaches are well established for detecting ligand induced GPCR-G protein interactions in cells. Currently, FRET/BRET assays rely on co-expression of GPCR and G protein, and hence depend on the stoichiometry and expression levels of the donor and acceptor probes. On the other hand, GPCR-G protein fusions have been used extensively to understand the selectivity of GPCR signaling pathways. However, the signaling properties of fusion proteins are not consistent across GPCRs. In this study, we describe and characterize novel sensors based on the Systematic Protein Affinity Strength Modulation (SPASM) technique. Sensors consist of a GPCR and G protein tethered by an ER/K linker flanked by FRET probes. SPASM sensors are tested for the β2-, α1-, and α2- adrenergic receptors, and adenosine type 1 receptor (A 1 R), tethered to Gαs-XL, Gαi 2 , or Gαq subunits. Agonist stimulation of β2-AR and α2-AR increases FRET signal comparable to co-expressed FRET/BRET sensors. SPASM sensors also retain signaling through the endogenous G protein milieu. Importantly, ER/K linker length systematically tunes the GPCR-G protein interaction, with consequent modulation of second messenger signaling for cognate interactions. SPASM GPCR sensors serve the dual purpose of detecting agonist-induced changes in GPCR-G protein interactions, and linking these changes to downstream signaling.

  17. A soluble form of Epstein-Barr virus gH/gL inhibits EBV-induced membrane fusion and does not function in fusion

    Energy Technology Data Exchange (ETDEWEB)

    Rowe, Cynthia L. [Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611 (United States); Connolly, Sarah A. [Department of Health Sciences, DePaul University, Chicago, IL 60614 (United States); Chen, Jia [Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611 (United States); Jardetzky, Theodore S. [Department of Structural Biology, Stanford University School of Medicine, 371 Serra Mall, Stanford, CA 94305 (United States); Longnecker, Richard, E-mail: r-longnecker@northwestern.edu [Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611 (United States)

    2013-02-05

    We investigated whether soluble EBV gH/gL (sgH/gL) functions in fusion and made a series of truncations of gH/gL domains based on the gH/gL crystal structure. We found sgH/gL failed to mediate cell-cell fusion both when co-expressed with the other entry glycoproteins and when added exogenously to fusion assays. Interestingly, sgH/gL inhibited cell-cell fusion in a dose dependent manner when co-expressed. sgH/gL from HSV was unable to inhibit EBV fusion, suggesting the inhibition was specific to EBV gH/gL. sgH/gL stably binds gp42, but not gB nor gH/gL. The domain mutants, DI/gL, DI-II/gL and DI-II-III/gL were unable to bind gp42. Instead, DI-II/gL, DI-II-III/gL and sgH/gL but not DI/gL decreased the expression of gp42, resulting in decreased overall fusion. Overall, our results suggest that domain IV may be required for proper folding and the transmembrane domain and cytoplasmic tail of EBV gH/gL are required for the most efficient fusion.

  18. Photoactivatable green fluorescent protein-based visualization and quantification of mitochondrial fusion and mitochondrial network complexity in living cells.

    Science.gov (United States)

    Karbowski, Mariusz; Cleland, Megan M; Roelofs, Brian A

    2014-01-01

    Technological improvements in microscopy and the development of mitochondria-specific imaging molecular tools have illuminated the dynamic rearrangements of these essential organelles. These rearrangements are mainly the result of two opposing processes: mitochondrial fusion and mitochondrial fission. Consistent with this, in addition to mitochondrial motility, these two processes are major factors determining the overall degree of continuity of the mitochondrial network, as well as the average size of mitochondria within the cell. In this chapter, we detail the use of advanced confocal microscopy and mitochondrial matrix-targeted photoactivatable green fluorescent protein (mito-PAGFP) for the investigation of mitochondrial dynamics. We focus on direct visualization and quantification of mitochondrial fusion and mitochondrial network complexity in living mammalian cells. These assays were instrumental in important recent discoveries within the field of mitochondrial biology, including the role of mitochondrial fusion in the activation of mitochondrial steps in apoptosis, participation of Bcl-2 family proteins in mitochondrial morphogenesis, and stress-induced mitochondrial hyperfusion. We present some basic directions that should be helpful in designing mito-PAGFP-based experiments. Furthermore, since analyses of mitochondrial fusion using mito-PAGFP-based assays rely on time-lapse imaging, critical parameters of time-lapse microscopy and cell preparation are also discussed.

  19. Enfuvirtide (T20)-Based Lipopeptide Is a Potent HIV-1 Cell Fusion Inhibitor: Implications for Viral Entry and Inhibition.

    Science.gov (United States)

    Ding, Xiaohui; Zhang, Xiujuan; Chong, Huihui; Zhu, Yuanmei; Wei, Huamian; Wu, Xiyuan; He, Jinsheng; Wang, Xinquan; He, Yuxian

    2017-09-15

    The peptide drug enfuvirtide (T20) is the only viral fusion inhibitor used in combination therapy for HIV-1 infection, but it has relatively low antiviral activity and easily induces drug resistance. Emerging studies demonstrate that lipopeptide-based fusion inhibitors, such as LP-11 and LP-19, which mainly target the gp41 pocket site, have greatly improved antiviral potency and in vivo stability. In this study, we focused on developing a T20-based lipopeptide inhibitor that lacks pocket-binding sequence and targets a different site. First, the C-terminal tryptophan-rich motif (TRM) of T20 was verified to be essential for its target binding and inhibition; then, a novel lipopeptide, termed LP-40, was created by replacing the TRM with a fatty acid group. LP-40 showed markedly enhanced binding affinity for the target site and dramatically increased inhibitory activity on HIV-1 membrane fusion, entry, and infection. Unlike LP-11 and LP-19, which required a flexible linker between the peptide sequence and the lipid moiety, addition of a linker to LP-40 sharply reduced its potency, implying different binding modes with the extended N-terminal helices of gp41. Also, interestingly, LP-40 showed more potent activity than LP-11 in inhibiting HIV-1 Env-mediated cell-cell fusion while it was less active than LP-11 in inhibiting pseudovirus entry, and the two inhibitors displayed synergistic antiviral effects. The crystal structure of LP-40 in complex with a target peptide revealed their key binding residues and motifs. Combined, our studies have not only provided a potent HIV-1 fusion inhibitor, but also revealed new insights into the mechanisms of viral inhibition. IMPORTANCE T20 is the only membrane fusion inhibitor available for treatment of viral infection; however, T20 requires high doses and has a low genetic barrier for resistance, and its inhibitory mechanism and structural basis remain unclear. Here, we report the design of LP-40, a T20-based lipopeptide inhibitor

  20. Topical and Targeted Delivery of siRNAs to Melanoma Cells Using a Fusion Peptide Carrier.

    Science.gov (United States)

    Ruan, Renquan; Chen, Ming; Sun, Sijie; Wei, Pengfei; Zou, Lili; Liu, Jing; Gao, Dayong; Wen, Longping; Ding, Weiping

    2016-07-04

    Topical application of siRNAs through the skin is a potentially effective strategy for the treatment of melanoma tumors. In this study, we designed a new and safe fusion peptide carrier SPACE-EGF to improve the skin and cell penetration function of the siRNAs and their targeting ability to B16 cells, such that the apoptosis of B16 cells can be induced. The results show that the carrier is stable and less toxic. The EGF motif does not affect the skin and cell penetration function of the SPACE. Because EGF can strongly bind EGFR, which is overexpressed in cancer cells, the targeting ability of the SPACE-EGF-siRNA complex is increased. In vitro experiments indicate that GAPDH siRNAs conjugated with SPACE-EGF can significantly reduce the GAPDH concentration in B16 cells, and c-Myc siRNAs can cause the gene silencing of c-Myc and thus the apoptosis of cells. In vivo experiments show that the topical application of c-Myc siRNAs delivered by SPACE-EGF through the skin can significantly inhibit the growth of melanoma tumors. This work may provide insight into the development of new transdermal drug carriers to treat a variety of skin disorders.

  1. Khz-cp (crude polysaccharide extract obtained from the fusion of Ganoderma lucidum and Polyporus umbellatus mycelia) induces apoptosis by increasing intracellular calcium levels and activating P38 and NADPH oxidase-dependent generation of reactive oxygen species in SNU-1 cells.

    Science.gov (United States)

    Kim, Tae Hwan; Kim, Ju Sung; Kim, Zoo Haye; Huang, Ren Bin; Chae, Young Lye; Wang, Ren Sheng

    2014-07-10

    Khz-cp is a crude polysaccharide extract that is obtained after nuclear fusion in Ganoderma lucidum and Polyporus umbellatus mycelia (Khz). It inhibits the growth of cancer cells. Khz-cp was extracted by solvent extraction. The anti-proliferative activity of Khz-cp was confirmed by using Annexin-V/PI-flow cytometry analysis. Intracellular calcium increase and measurement of intracellular reactive oxygen species (ROS) were performed by using flow cytometry and inverted microscope. SNU-1 cells were treated with p38, Bcl-2 and Nox family siRNA. siRNA transfected cells was employed to investigate the expression of apoptotic, growth and survival genes in SNU-1 cells. Western blot analysis was performed to confirm the expression of the genes. In the present study, Khz-cp induced apoptosis preferentially in transformed cells and had only minimal effects on non-transformed cells. Furthermore, Khz-cp was found to induce apoptosis by increasing the intracellular Ca2+ concentration ([Ca2+]i) and activating P38 to generate reactive oxygen species (ROS) via NADPH oxidase and the mitochondria. Khz-cp-induced apoptosis was caspase dependent and occurred via a mitochondrial pathway. ROS generation by NADPH oxidase was critical for Khz-cp-induced apoptosis, and although mitochondrial ROS production was also required, it appeared to occur secondary to ROS generation by NADPH oxidase. Activation of NADPH oxidase was shown by the translocation of the regulatory subunits p47phox and p67phox to the cell membrane and was necessary for ROS generation by Khz-cp. Khz-cp triggered a rapid and sustained increase in [Ca2+]i that activated P38. P38 was considered to play a key role in the activation of NADPH oxidase because inhibition of its expression or activity abrogated membrane translocation of the p47phox and p67phox subunits and ROS generation. In summary, these data indicate that Khz-cp preferentially induces apoptosis in cancer cells and that the signaling mechanisms involve an

  2. Palmitoylation of the cysteine-rich endodomain of the SARS-coronavirus spike glycoprotein is important for spike-mediated cell fusion

    International Nuclear Information System (INIS)

    Petit, Chad M.; Chouljenko, Vladimir N.; Iyer, Arun; Colgrove, Robin; Farzan, Michael; Knipe, David M.; Kousoulas, K.G.

    2007-01-01

    The SARS-coronavirus (SARS-CoV) is the etiological agent of the severe acute respiratory syndrome (SARS). The SARS-CoV spike (S) glycoprotein mediates membrane fusion events during virus entry and virus-induced cell-to-cell fusion. The cytoplasmic portion of the S glycoprotein contains four cysteine-rich amino acid clusters. Individual cysteine clusters were altered via cysteine-to-alanine amino acid replacement and the modified S glycoproteins were tested for their transport to cell-surfaces and ability to cause cell fusion in transient transfection assays. Mutagenesis of the cysteine cluster I, located immediately proximal to the predicted transmembrane, domain did not appreciably reduce cell-surface expression, although S-mediated cell fusion was reduced by more than 50% in comparison to the wild-type S. Similarly, mutagenesis of the cysteine cluster II located adjacent to cluster I reduced S-mediated cell fusion by more than 60% compared to the wild-type S, while cell-surface expression was reduced by less than 20%. Mutagenesis of cysteine clusters III and IV did not appreciably affect S cell-surface expression or S-mediated cell fusion. The wild-type S was palmitoylated as evidenced by the efficient incorporation of 3 H-palmitic acid in wild-type S molecules. S glycoprotein palmitoylation was significantly reduced for mutant glycoproteins having cluster I and II cysteine changes, but was largely unaffected for cysteine cluster III and IV mutants. These results show that the S cytoplasmic domain is palmitoylated and that palmitoylation of the membrane proximal cysteine clusters I and II may be important for S-mediated cell fusion

  3. Fusion between Intestinal epithelial cells and macrophages in a cancer context results in nuclear reprogramming.

    Science.gov (United States)

    Powell, Anne E; Anderson, Eric C; Davies, Paige S; Silk, Alain D; Pelz, Carl; Impey, Soren; Wong, Melissa H

    2011-02-15

    The most deadly phase in cancer progression is attributed to the inappropriate acquisition of molecular machinery leading to metastatic transformation and spread of disease to distant organs. Although it is appreciated that metastasis involves epithelial-mesenchymal interplay, the underlying mechanism defining this process is poorly understood. Specifically, how cancer cells evade immune surveillance and gain the ability to navigate the circulatory system remains a focus. One possible mechanism underlying metastatic conversion is fusion between blood-derived immune cells and cancer cells. While this notion is a century old, in vivo evidence that cell fusion occurs within tumors and imparts genetic or physiologic changes remains controversial. We have previously demonstrated in vivo cell fusion between blood cells and intestinal epithelial cells in an injury setting. Here, we hypothesize that immune cells, such as macrophages, fuse with tumor cells imparting metastatic capabilities by transferring their cellular identity. We used parabiosis to introduce fluorescent-labeled bone marrow-derived cells to mice with intestinal tumors, finding that fusion between circulating blood-derived cells and tumor epithelium occurs during the natural course of tumorigenesis. Moreover, we identify the macrophage as a key cellular partner for this process. Interestingly, cell fusion hybrids retain a transcriptome identity characteristic of both parental derivatives, while also expressing a unique subset of transcripts. Our data supports the novel possibility that tumorigenic cell fusion may impart physical behavior attributed to migratory macrophages, including navigation of circulation and immune evasion. As such, cell fusion may represent a promising novel mechanism underlying the metastatic conversion of cancer cells. ©2011 AACR.

  4. Multiple sampling ionization chamber (MUSIC) for investigation of fusion induced by halo nuclei

    International Nuclear Information System (INIS)

    Petrascu, H.; Kumagai, H.; Tanihata, I.; Fueloep, Zs.; Petrascu, M.

    1999-01-01

    A high resolution MUSIC for low and medium energy ions up to ∼ 20 AMeV, for investigation of fusion processes induced by halo nuclei, has been achieved. The chamber was used in the first experiments, aiming at investigating fusion processes induced by 9,11 Li with light targets. In these experiments MUSIC was used for the identification of the inclusive evaporation residues produced in the Si detector target, mounted inside the chamber. By using MUSIC it was possible to separate the inclusive spectra corresponding to the fusion processes, from the background due to the energy degraded beam particles. In principle such a chamber could be also used for investigation of particular fusion channels produced in the entrance window. In this case one could obtain the fusion product trajectory angle with the horizontal plane, by coupling each anode pad to a TDC. The chamber was also provided by a position grid, mounted between the Frisch grid and the anode pads. The energy loss distribution widths were measured using α particles. The chamber was filled with P-10 gas at pressures between 200 and 300 torr. The obtained resolution corresponding to a single pad, is close to the limit derived from the theory of Badhwar. (authors)

  5. Excitation functions for some Ne induced reactions with Holmium: incomplete fusion vs complete fusion

    International Nuclear Information System (INIS)

    Agarwal, Avinash; Kumar, Munish; Sharma, Anjali; Rizvi, I.A.; Ahamad, Tauseef; Ghugre, S.S.; Sinha, A.K.; Chaubey, A.K.

    2010-01-01

    Reactions induced by 20 Ne are expected to be considerably more complex than those of 12 C, and 16 O. As a part of the ongoing program to understand CF and ICF reaction mechanisms, it is of great interest to see whether the same experimental technique yield similarly valuable information for 20 Ne induced reactions. In this present work an attempt has been made to measure the excitation functions for fifteen evaporation residues (ERs) identified in the interaction of 20 Ne + 165 Ho system in the energy range 4 -7 MeV/A

  6. Fibroblast growth factor receptor signaling plays a key role in transformation induced by the TMPRSS2/ERG fusion gene and decreased PTEN.

    Science.gov (United States)

    Shao, Longjiang; Wang, Jianghua; Karatas, Omer Faruk; Feng, Shu; Zhang, Yiqun; Creighton, Chad J; Ittmann, Michael

    2018-03-06

    Prostate cancer is the most common visceral malignancy and the second leading cause of cancer deaths in US men. Correlative studies in human prostate cancers reveal a frequent association of the TMPRSS2/ERG (TE) fusion gene with loss of PTEN and studies in mouse models reveal that ERG expression and PTEN loss synergistically promote prostate cancer progression. To determine the mechanism by which ERG overexpression and PTEN loss leads to transformation, we overexpressed the TE fusion gene and knocked down PTEN in an immortalized but non-transformed prostate epithelial cell line. We show that ERG overexpression in combination with PTEN loss can transform these immortalized but non-tumorigenic cells, while either alteration alone was not sufficient to fully transform these cells. Expression microarray analysis revealed extensive changes in gene expression in cells expressing the TE fusion with loss of PTEN. Among these gene expression changes was increased expression of multiple FGF ligands and receptors. We show that activation of fibroblast growth factor receptor signaling plays a key role in transformation induced by TE fusion gene expression in association with PTEN loss. In addition, in vitro and in silico analysis reveals PTEN loss is associated with widespread increases in FGF ligands and receptors in prostate cancer. Inhibitors of FGF receptor signaling are currently entering the clinic and our results suggests that FGF receptor signaling is a therapeutic target in cancers with TE fusion gene expression and PTEN loss.

  7. Identification of target genes of synovial sarcoma-associated fusion oncoprotein using human pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Hayakawa, Kazuo [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Ikeya, Makoto [Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Fukuta, Makoto [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Woltjen, Knut [Department of Reprogramming Sciences, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Tamaki, Sakura; Takahara, Naoko; Kato, Tomohisa; Sato, Shingo [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Otsuka, Takanobu [Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Toguchida, Junya, E-mail: togjun@frontier.kyoto-u.ac.jp [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University, Kyoto (Japan)

    2013-03-22

    Highlights: ► We tried to identify targets of synovial sarcoma (SS)-associated SYT–SSX fusion gene. ► We established pluripotent stem cell (PSC) lines with inducible SYT–SSX gene. ► SYT–SSX responsive genes were identified by the induction of SYT–SSX in PSC. ► SS-related genes were selected from database by in silico analyses. ► 51 genes were finally identified among SS-related genes as targets of SYT–SSX in PSC. -- Abstract: Synovial sarcoma (SS) is a malignant soft tissue tumor harboring chromosomal translocation t(X; 18)(p11.2; q11.2), which produces SS-specific fusion gene, SYT–SSX. Although precise function of SYT–SSX remains to be investigated, accumulating evidences suggest its role in gene regulation via epigenetic mechanisms, and the product of SYT–SSX target genes may serve as biomarkers of SS. Lack of knowledge about the cell-of-origin of SS, however, has placed obstacle in the way of target identification. Here we report a novel approach to identify SYT–SSX2 target genes using human pluripotent stem cells (hPSCs) containing a doxycycline-inducible SYT–SSX2 gene. SYT–SSX2 was efficiently induced both at mRNA and protein levels within three hours after doxycycline administration, while no morphological change of hPSCs was observed until 24 h. Serial microarray analyses identified genes of which the expression level changed more than twofold within 24 h. Surprisingly, the majority (297/312, 95.2%) were up-regulated genes and a result inconsistent with the current concept of SYT–SSX as a transcriptional repressor. Comparing these genes with SS-related genes which were selected by a series of in silico analyses, 49 and 2 genes were finally identified as candidates of up- and down-regulated target of SYT–SSX, respectively. Association of these genes with SYT–SSX in SS cells was confirmed by knockdown experiments. Expression profiles of SS-related genes in hPSCs and human mesenchymal stem cells (hMSCs) were strikingly

  8. Antibody-Induced Internalization of the Human Respiratory Syncytial Virus Fusion Protein.

    Science.gov (United States)

    Leemans, A; De Schryver, M; Van der Gucht, W; Heykers, A; Pintelon, I; Hotard, A L; Moore, M L; Melero, J A; McLellan, J S; Graham, B S; Broadbent, L; Power, U F; Caljon, G; Cos, P; Maes, L; Delputte, P

    2017-07-15

    Respiratory syncytial virus (RSV) infections remain a major cause of respiratory disease and hospitalizations among infants. Infection recurs frequently and establishes a weak and short-lived immunity. To date, RSV immunoprophylaxis and vaccine research is mainly focused on the RSV fusion (F) protein, but a vaccine remains elusive. The RSV F protein is a highly conserved surface glycoprotein and is the main target of neutralizing antibodies induced by natural infection. Here, we analyzed an internalization process of antigen-antibody complexes after binding of RSV-specific antibodies to RSV antigens expressed on the surface of infected cells. The RSV F protein and attachment (G) protein were found to be internalized in both infected and transfected cells after the addition of either RSV-specific polyclonal antibodies (PAbs) or RSV glycoprotein-specific monoclonal antibodies (MAbs), as determined by indirect immunofluorescence staining and flow-cytometric analysis. Internalization experiments with different cell lines, well-differentiated primary bronchial epithelial cells (WD-PBECs), and RSV isolates suggest that antibody internalization can be considered a general feature of RSV. More specifically for RSV F, the mechanism of internalization was shown to be clathrin dependent. All RSV F-targeted MAbs tested, regardless of their epitopes, induced internalization of RSV F. No differences could be observed between the different MAbs, indicating that RSV F internalization was epitope independent. Since this process can be either antiviral, by affecting virus assembly and production, or beneficial for the virus, by limiting the efficacy of antibodies and effector mechanism, further research is required to determine the extent to which this occurs in vivo and how this might impact RSV replication. IMPORTANCE Current research into the development of new immunoprophylaxis and vaccines is mainly focused on the RSV F protein since, among others, RSV F-specific antibodies are

  9. Color-coded Live Imaging of Heterokaryon Formation and Nuclear Fusion of Hybridizing Cancer Cells.

    Science.gov (United States)

    Suetsugu, Atsushi; Matsumoto, Takuro; Hasegawa, Kosuke; Nakamura, Miki; Kunisada, Takahiro; Shimizu, Masahito; Saji, Shigetoyo; Moriwaki, Hisataka; Bouvet, Michael; Hoffman, Robert M

    2016-08-01

    Fusion of cancer cells has been studied for over half a century. However, the steps involved after initial fusion between cells, such as heterokaryon formation and nuclear fusion, have been difficult to observe in real time. In order to be able to visualize these steps, we have established cancer-cell sublines from the human HT-1080 fibrosarcoma, one expressing green fluorescent protein (GFP) linked to histone H2B in the nucleus and a red fluorescent protein (RFP) in the cytoplasm and the other subline expressing RFP in the nucleus (mCherry) linked to histone H2B and GFP in the cytoplasm. The two reciprocal color-coded sublines of HT-1080 cells were fused using the Sendai virus. The fused cells were cultured on plastic and observed using an Olympus FV1000 confocal microscope. Multi-nucleate (heterokaryotic) cancer cells, in addition to hybrid cancer cells with single-or multiple-fused nuclei, including fused mitotic nuclei, were observed among the fused cells. Heterokaryons with red, green, orange and yellow nuclei were observed by confocal imaging, even in single hybrid cells. The orange and yellow nuclei indicate nuclear fusion. Red and green nuclei remained unfused. Cell fusion with heterokaryon formation and subsequent nuclear fusion resulting in hybridization may be an important natural phenomenon between cancer cells that may make them more malignant. The ability to image the complex processes following cell fusion using reciprocal color-coded cancer cells will allow greater understanding of the genetic basis of malignancy. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  10. Studies of OC-STAMP in Osteoclast Fusion: A New Knockout Mouse Model, Rescue of Cell Fusion, and Transmembrane Topology.

    Directory of Open Access Journals (Sweden)

    Hanna Witwicka

    Full Text Available The fusion of monocyte/macrophage lineage cells into fully active, multinucleated, bone resorbing osteoclasts is a complex cell biological phenomenon that utilizes specialized proteins. OC-STAMP, a multi-pass transmembrane protein, has been shown to be required for pre-osteoclast fusion and for optimal bone resorption activity. A previously reported knockout mouse model had only mononuclear osteoclasts with markedly reduced resorption activity in vitro, but with paradoxically normal skeletal micro-CT parameters. To further explore this and related questions, we used mouse ES cells carrying a gene trap allele to generate a second OC-STAMP null mouse strain. Bone histology showed overall normal bone form with large numbers of TRAP-positive, mononuclear osteoclasts. Micro-CT parameters were not significantly different between knockout and wild type mice at 2 or 6 weeks old. At 6 weeks, metaphyseal TRAP-positive areas were lower and mean size of the areas were smaller in knockout femora, but bone turnover markers in serum were normal. Bone marrow mononuclear cells became TRAP-positive when cultured with CSF-1 and RANKL, but they did not fuse. Expression levels of other osteoclast markers, such as cathepsin K, carbonic anhydrase II, and NFATc1, were not significantly different compared to wild type. Actin rings were present, but small, and pit assays showed a 3.5-fold decrease in area resorbed. Restoring OC-STAMP in knockout cells by lentiviral transduction rescued fusion and resorption. N- and C-termini of OC-STAMP were intracellular, and a predicted glycosylation site was shown to be utilized and to lie on an extracellular loop. The site is conserved in all terrestrial vertebrates and appears to be required for protein stability, but not for fusion. Based on this and other results, we present a topological model of OC-STAMP as a 6-transmembrane domain protein. We also contrast the osteoclast-specific roles of OC- and DC-STAMP with more generalized

  11. FOXO1 is a direct target of EWS-Fli1 oncogenic fusion protein in Ewing's sarcoma cells

    International Nuclear Information System (INIS)

    Yang, Liu; Hu, Hsien-Ming; Zielinska-Kwiatkowska, Anna; Chansky, Howard A.

    2010-01-01

    Research highlights: → Inducible and reversible siRNA knockdown of an oncogenic fusion protein such as EWS-Fli1 is feasible and more advantageous than other siRNA methods. → The tumor suppressor gene FOXO1 is a new EWS-Fli1 target. → While trans-activators are known for the FOXO1 gene, there has been no report on negative regulators of FOXO1 transcription. → This study provides first evidence that the EWS-Fli1 oncogenic fusion protein can function as a transcriptional repressor of the FOXO1 gene. -- Abstract: Ewing's family tumors are characterized by a specific t(11;22) chromosomal translocation that results in the formation of EWS-Fli1 oncogenic fusion protein. To investigate the effects of EWS-Fli1 on gene expression, we carried out DNA microarray analysis after specific knockdown of EWS-Fli1 through transfection of synthetic siRNAs. EWS-Fli1 knockdown increased expression of genes such as DKK1 and p57 that are known to be repressed by EWS-Fli1 fusion protein. Among other potential EWS-Fli1 targets identified by our microarray analysis, we have focused on the FOXO1 gene since it encodes a potential tumor suppressor and has not been previously reported in Ewing's cells. To better understand how EWS-Fli1 affects FOXO1 expression, we have established a doxycycline-inducible siRNA system to achieve stable and reversible knockdown of EWS-Fli1 in Ewing's sarcoma cells. Here we show that FOXO1 expression in Ewing's cells has an inverse relationship with EWS-Fli1 protein level, and FOXO1 promoter activity is increased after doxycycline-induced EWS-Fli1 knockdown. In addition, we have found that direct binding of EWS-Fli1 to FOXO1 promoter is attenuated after doxycycline-induced siRNA knockdown of the fusion protein. Together, these results suggest that suppression of FOXO1 function by EWS-Fli1 fusion protein may contribute to cellular transformation in Ewing's family tumors.

  12. An IL12-IL2-antibody fusion protein targeting Hodgkin's lymphoma cells potentiates activation of NK and T cells for an anti-tumor attack.

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    Tobias Jahn

    Full Text Available Successful immunotherapy of Hodgkin's disease is so far hampered by the striking unresponsiveness of lymphoma infiltrating immune cells. To mobilize both adoptive and innate immune cells for an anti-tumor attack we fused the pro-inflammatory cytokines IL2 and IL12 to an anti-CD30 scFv antibody in a dual cytokine fusion protein to accumulate both cytokines at the malignant CD30(+ Hodgkin/Reed-Sternberg cells in the lymphoma lesion. The tumor-targeted IL12-IL2 fusion protein was superior in activating resting T cells to amplify and secrete pro-inflammatory cytokines compared to targeted IL2 or IL12 alone. NK cells were also activated by the dual cytokine protein to secrete IFN-γ and to lyse target cells. The tumor-targeted IL12-IL2, when applied by i.v. injection to immune-competent mice with established antigen-positive tumors, accumulated at the tumor site and induced tumor regression. Data demonstrate that simultaneous targeting of two cytokines in a spatial and temporal simultaneous fashion to pre-defined tissues is feasible by a dual-cytokine antibody fusion protein. In the case of IL12 and IL2, this produced superior anti-tumor efficacy implying the strategy to muster a broader immune cell response in the combat against cancer.

  13. A fusion DNA vaccine that targets antigen-presenting cells increases protection from viral challenge

    Science.gov (United States)

    Deliyannis, Georgia; Boyle, Jefferey S.; Brady, Jamie L.; Brown, Lorena E.; Lew, Andrew M.

    2000-06-01

    Improving the immunological potency, particularly the Ab response, is a serious hurdle for the protective efficacy and hence broad application of DNA vaccines. We examined the immunogenicity and protective efficacy of a hemagglutinin-based influenza DNA vaccine that was targeted to antigen-presenting cells (APCs) by fusion to CTLA4. The targeted vaccine was shown to induce an accelerated and increased Ab response (as compared with those receiving the nontargeted control) that was predominated by IgG1 and recognized conformationally dependent viral epitopes. Moreover, mice receiving the APC-targeted DNA vaccine had significantly reduced viral titers (100-fold) after a nonlethal virus challenge. The increased protective efficacy was most likely because of increased Ab responses, as cytotoxic T lymphocyte responses were not enhanced. Targeting was demonstrated by direct binding studies of CTLA4 fusion proteins to the cognate ligand (B7; expressed on APCs in vivo). In addition, a targeted protein was detected at 4-fold higher levels in draining lymph nodes within 2-24 h of administration. Therefore, this study demonstrates that targeting DNA-encoded antigen to APCs results in enhanced immunity and strongly suggests that this approach may be useful in improving the protective efficacy of DNA vaccines.

  14. Identification of a novel SEPT9-ABL1 fusion gene in a patient with T-cell prolymphocytic leukemia

    Directory of Open Access Journals (Sweden)

    Rikio Suzuki

    2014-01-01

    Full Text Available T-cell prolymphocytic leukemia (T-PLL, a rare type of peripheral T-cell leukemia, is characterized by marked splenomegaly with rapidly progressive lymphocytosis and a poor prognosis. Nine kinds of ABL1 chimeric genes have been identified in various kinds of hematological malignancies, such as chronic myeloid leukemia and B- or T-lymphoblastic leukemia. However, there have been no reports describing T-PLL cases with ABL1 rearrangements. We herein report a case of T-PLL with a novel SEPT9-ABL1 fusion gene which induced strong resistance to tyrosine kinase inhibitors such as imatinib and dasatinib.

  15. Palmitoylation of SARS-CoV S protein is necessary for partitioning into detergent-resistant membranes and cell-cell fusion but not interaction with M protein

    International Nuclear Information System (INIS)

    McBride, Corrin E.; Machamer, Carolyn E.

    2010-01-01

    Coronaviruses are enveloped RNA viruses that generally cause mild disease in humans. However, the recently emerged coronavirus that caused severe acute respiratory syndrome (SARS-CoV) is the most pathogenic human coronavirus discovered to date. The SARS-CoV spike (S) protein mediates virus entry by binding cellular receptors and inducing fusion between the viral envelope and the host cell membrane. Coronavirus S proteins are palmitoylated, which may affect function. Here, we created a non-palmitoylated SARS-CoV S protein by mutating all nine cytoplasmic cysteine residues. Palmitoylation of SARS-CoV S was required for partitioning into detergent-resistant membranes and for cell-cell fusion. Surprisingly, however, palmitoylation of S was not required for interaction with SARS-CoV M protein. This contrasts with the requirement for palmitoylation of mouse hepatitis virus S protein for interaction with M protein and may point to important differences in assembly and infectivity of these two coronaviruses.

  16. Physiological and molecular triggers for SARS-CoV membrane fusion and entry into host cells.

    Science.gov (United States)

    Millet, Jean Kaoru; Whittaker, Gary R

    2018-04-01

    During viral entry, enveloped viruses require the fusion of their lipid envelope with host cell membranes. For coronaviruses, this critical step is governed by the virally-encoded spike (S) protein, a class I viral fusion protein that has several unique features. Coronavirus entry is unusual in that it is often biphasic in nature, and can occur at or near the cell surface or in late endosomes. Recent advances in structural, biochemical and molecular biology of the coronavirus S protein has shed light on the intricacies of coronavirus entry, in particular the molecular triggers of coronavirus S-mediated membrane fusion. Furthermore, characterization of the coronavirus fusion peptide (FP), the segment of the fusion protein that inserts to a target lipid bilayer during membrane fusion, has revealed its particular attributes which imparts some of the unusual properties of the S protein, such as Ca 2+ -dependency. These unusual characteristics can explain at least in part the biphasic nature of coronavirus entry. In this review, using severe acute respiratory syndrome coronavirus (SARS-CoV) as model virus, we give an overview of advances in research on the coronavirus fusion peptide with an emphasis on its role and properties within the biological context of host cell entry. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Selective Retention of Bone Marrow-Derived Cells to Enhance Spinal Fusion

    Science.gov (United States)

    Matsukura, Yoichi; Nitto, Hironori; Boehm, Cynthia A.; Valdevit, Antonio D.; Kambic, Helen E.; Davros, William J.; Easley, Kirk A.; Powell, Kimerly A.

    2005-01-01

    Connective tissue progenitors can be concentrated rapidly from fresh bone marrow aspirates using some porous matrices as a surface for cell attachment and selective retention, and for creating a cellular graft that is enriched with respect to the number of progenitor cells. We evaluated the potential value of this method using demineralized cortical bone powder as the matrix. Matrix alone, matrix plus marrow, and matrix enriched with marrow cells were compared in an established canine spinal fusion model. Fusions were compared based on union score, fusion mass, fusion volume, and by mechanical testing. Enriched matrix grafts delivered a mean of 2.3 times more cells and approximately 5.6 times more progenitors than matrix mixed with bone marrow. The union score with enriched matrix was superior to matrix alone and matrix plus marrow. Fusion volume and fusion area also were greater with the enriched matrix. These data suggest that the strategy of selective retention provides a rapid, simple, and effective method for concentration and delivery of marrow-derived cells and connective tissue progenitors that may improve the outcome of bone grafting procedures in various clinical settings. PMID:15738828

  18. [Adenovirus-mediated killing of hepatocellular carcinoma HepG2 cells by heterogeneous fusion gene NT4p53(N15)Ant].

    Science.gov (United States)

    Li, Yue-ping; Qiu, Shu-dong; Song, Li-ping; Wang, Quan-ying; Yang, Guang-xiao

    2007-07-01

    OBJECTIVE To construct a recombinant adenovirus Ad.NT4p53(N15)Ant and explore its cytotoxic effect against hepatocellular carcinoma HepG2 cells in vitro. The recombinant adenovirus containing the fusion gene of neurotrophin 4 (NT4)signal peptide, N-terminal residues (12-26) of p53 and 17 amino acid Drosophila homeobox protein Antennapedia (Ant) was constructed by gene cloning protocol. The effect of this fusion gene on HepG2 cells was evaluated by MTT assay, PI staining and flow cytometry. The fusion gene Ad.NT4p53(N15)Ant was successfully constructed, as verified by restriction endonuclease digestion and PCR. Ad.NT4p53(N15)Ant could strongly suppress the growth of HepG2 cells (with a growth inhibition rate of 63.3% 48 h after infection) without affecting NIH-3T3 cells. Flow cytometry showed that Ad.NT4p53(N15)Ant could induce obvious apoptosis of HepG2 cells. The recombinant adenovirus containing NT4p53(N15)Ant fusion gene can inhibit the growth the of HepG2 cells in vitro partially by inducing cell apoptosis.

  19. Frequent CTLA4-CD28 gene fusion in diverse types of T-cell lymphoma.

    Science.gov (United States)

    Yoo, Hae Yong; Kim, Pora; Kim, Won Seog; Lee, Seung Ho; Kim, Sangok; Kang, So Young; Jang, Hye Yoon; Lee, Jong-Eun; Kim, Jaesang; Kim, Seok Jin; Ko, Young Hyeh; Lee, Sanghyuk

    2016-06-01

    CTLA4 and CD28 are co-regulatory receptors with opposite roles in T-cell signaling. By RNA sequencing, we identified a fusion between the two genes from partial gene duplication in a case of angioimmunoblastic T-cell lymphoma. The fusion gene, which codes for the extracellular domain of CTLA4 and the cytoplasmic region of CD28, is likely capable of transforming inhibitory signals into stimulatory signals for T-cell activation. Ectopic expression of the fusion transcript in Jurkat and H9 cells resulted in enhanced proliferation and AKT and ERK phosphorylation, indicating activation of downstream oncogenic pathways. To estimate the frequency of this gene fusion in mature T-cell lymphomas, we examined 115 T-cell lymphoma samples of diverse subtypes using reverse transcriptase polymerase chain reaction analysis and Sanger sequencing. We identified the fusion in 26 of 45 cases of angioimmunoblastic T-cell lymphomas (58%), nine of 39 peripheral T-cell lymphomas, not otherwise specified (23%), and nine of 31 extranodal NK/T cell lymphomas (29%). We further investigated the mutation status of 70 lymphoma-associated genes using ultra-deep targeted resequencing for 74 mature T-cell lymphoma samples. The mutational landscape we obtained suggests that T-cell lymphoma results from diverse combinations of multiple gene mutations. The CTLA4-CD28 gene fusion is likely a major contributor to the pathogenesis of T-cell lymphomas and represents a potential target for anti-CTLA4 cancer immunotherapy. Copyright© Ferrata Storti Foundation.

  20. Macrophage-tumor cell fusions from peripheral blood of melanoma patients.

    Science.gov (United States)

    Clawson, Gary A; Matters, Gail L; Xin, Ping; Imamura-Kawasawa, Yuka; Du, Zhen; Thiboutot, Diane M; Helm, Klaus F; Neves, Rogerio I; Abraham, Thomas

    2015-01-01

    While the morbidity and mortality from cancer are largely attributable to its metastatic dissemination, the integral features of the cascade are not well understood. The widely accepted hypothesis is that the primary tumor microenvironment induces the epithelial-to-mesenchymal transition in cancer cells, facilitating their escape into the bloodstream, possibly accompanied by cancer stem cells. An alternative theory for metastasis involves fusion of macrophages with tumor cells (MTFs). Here we culture and characterize apparent MTFs from blood of melanoma patients. We isolated enriched CTC populations from peripheral blood samples from melanoma patients, and cultured them. We interrogated these cultured cells for characteristic BRAF mutations, and used confocal microscopy for immunophenotyping, motility, DNA content and chromatin texture analyses, and then conducted xenograft studies using nude mice. Morphologically, the cultured MTFs were generally large with many pseudopod extensions and lamellipodia. Ultrastructurally, the cultured MTFs appeared to be macrophages. They were rich in mitochondria and lysosomes, as well as apparent melanosomes. The cultured MTF populations were all heterogeneous with regard to DNA content, containing aneuploid and/or high-ploidy cells, and they typically showed large sheets (and/or clumps) of cytoplasmic chromatin. This cytoplasmic DNA was found within heterogeneously-sized autophagic vacuoles, which prominently contained chromatin and micronuclei. Cultured MTFs uniformly expressed pan-macrophage markers (CD14, CD68) and macrophage markers indicative of M2 polarization (CD163, CD204, CD206). They also expressed melanocyte-specific markers (ALCAM, MLANA), epithelial biomarkers (KRT, EpCAM), as well as the pro-carcinogenic cytokine MIF along with functionally related stem cell markers (CXCR4, CD44). MTF cultures from individual patients (5 of 8) contained melanoma-specific BRAF activating mutations. Chromatin texture analysis of

  1. Macrophage-tumor cell fusions from peripheral blood of melanoma patients.

    Directory of Open Access Journals (Sweden)

    Gary A Clawson

    Full Text Available While the morbidity and mortality from cancer are largely attributable to its metastatic dissemination, the integral features of the cascade are not well understood. The widely accepted hypothesis is that the primary tumor microenvironment induces the epithelial-to-mesenchymal transition in cancer cells, facilitating their escape into the bloodstream, possibly accompanied by cancer stem cells. An alternative theory for metastasis involves fusion of macrophages with tumor cells (MTFs. Here we culture and characterize apparent MTFs from blood of melanoma patients.We isolated enriched CTC populations from peripheral blood samples from melanoma patients, and cultured them. We interrogated these cultured cells for characteristic BRAF mutations, and used confocal microscopy for immunophenotyping, motility, DNA content and chromatin texture analyses, and then conducted xenograft studies using nude mice.Morphologically, the cultured MTFs were generally large with many pseudopod extensions and lamellipodia. Ultrastructurally, the cultured MTFs appeared to be macrophages. They were rich in mitochondria and lysosomes, as well as apparent melanosomes. The cultured MTF populations were all heterogeneous with regard to DNA content, containing aneuploid and/or high-ploidy cells, and they typically showed large sheets (and/or clumps of cytoplasmic chromatin. This cytoplasmic DNA was found within heterogeneously-sized autophagic vacuoles, which prominently contained chromatin and micronuclei. Cultured MTFs uniformly expressed pan-macrophage markers (CD14, CD68 and macrophage markers indicative of M2 polarization (CD163, CD204, CD206. They also expressed melanocyte-specific markers (ALCAM, MLANA, epithelial biomarkers (KRT, EpCAM, as well as the pro-carcinogenic cytokine MIF along with functionally related stem cell markers (CXCR4, CD44. MTF cultures from individual patients (5 of 8 contained melanoma-specific BRAF activating mutations. Chromatin texture analysis

  2. Epstein-Barr Virus Fusion with Epithelial Cells Triggered by gB Is Restricted by a gL Glycosylation Site.

    Science.gov (United States)

    Möhl, Britta S; Chen, Jia; Park, Seo Jin; Jardetzky, Theodore S; Longnecker, Richard

    2017-12-01

    Epstein-Barr virus (EBV) entry into epithelial cells is mediated by the conserved core fusion machinery, composed of the fusogen gB and the receptor-binding complex gH/gL. The heterodimeric gH/gL complex binds to the EBV epithelial cell receptor or gp42, which binds to the B-cell receptor, triggering gB-mediated fusion of the virion envelope with cellular membranes. Our previous study found that the gL glycosylation mutant N69L/S71V had an epithelial cell-specific hyperfusogenic phenotype. To study the influence of this gL mutant on the initiation and kinetics of gB-driven epithelial cell fusion, we established a virus-free split-green fluorescent protein cell-cell fusion assay that enables real-time measurements of membrane fusion using live cells. The gL_N69L/S71V mutant had a large increase in epithelial cell fusion activity of up to 300% greater than that of wild-type gL starting at early time points. The hyperfusogenicity of the gL mutant was not a result of alterations in complex formation with gH or alterations in cellular localization. Moreover, the hyperfusogenic phenotype of the gL mutant correlated with the formation of enlarged syncytia. In summary, our present findings highlight an important role of gL in the kinetics of gB-mediated epithelial cell fusion, adding to previous findings indicating a direct interaction between gL and gB in EBV membrane fusion. IMPORTANCE EBV predominantly infects epithelial cells and B lymphocytes, which are the cells of origin for the EBV-associated malignancies Hodgkin and Burkitt lymphoma as well as nasopharyngeal carcinoma. Contrary to the other key players of the core fusion machinery, gL has the most elusive role during EBV-induced membrane fusion. We found that the glycosylation site N69/S71 of gL is involved in restricting epithelial cell fusion activity, strongly correlating with syncytium size. Interestingly, our data showed that the gL glycosylation mutant increases the fusion activity of the hyperfusogenic g

  3. Binding and Fusion of Extracellular Vesicles to the Plasma Membrane of Their Cell Targets.

    Science.gov (United States)

    Prada, Ilaria; Meldolesi, Jacopo

    2016-08-09

    Exosomes and ectosomes, extracellular vesicles of two types generated by all cells at multivesicular bodies and the plasma membrane, respectively, play critical roles in physiology and pathology. A key mechanism of their function, analogous for both types of vesicles, is the fusion of their membrane to the plasma membrane of specific target cells, followed by discharge to the cytoplasm of their luminal cargo containing proteins, RNAs, and DNA. Here we summarize the present knowledge about the interactions, binding and fusions of vesicles with the cell plasma membrane. The sequence initiates with dynamic interactions, during which vesicles roll over the plasma membrane, followed by the binding of specific membrane proteins to their cell receptors. Membrane binding is then converted rapidly into fusion by mechanisms analogous to those of retroviruses. Specifically, proteins of the extracellular vesicle membranes are structurally rearranged, and their hydrophobic sequences insert into the target cell plasma membrane which undergoes lipid reorganization, protein restructuring and membrane dimpling. Single fusions are not the only process of vesicle/cell interactions. Upon intracellular reassembly of their luminal cargoes, vesicles can be regenerated, released and fused horizontally to other target cells. Fusions of extracellular vesicles are relevant also for specific therapy processes, now intensely investigated.

  4. Cell Fusion along the Anterior-Posterior Neuroaxis in Mice with Experimental Autoimmune Encephalomyelitis.

    Directory of Open Access Journals (Sweden)

    Sreenivasa R Sankavaram

    Full Text Available It is well documented that bone marrow-derived cells can fuse with a diverse range of cells, including brain cells, under normal or pathological conditions. Inflammation leads to robust fusion of bone marrow-derived cells with Purkinje cells and the formation of binucleate heterokaryons in the cerebellum. Heterokaryons form through the fusion of two developmentally differential cells and as a result contain two distinct nuclei without subsequent nuclear or chromosome loss.In the brain, fusion of bone marrow-derived cells appears to be restricted to the complex and large Purkinje cells, raising the question whether the size of the recipient cell is important for cell fusion in the central nervous system. Purkinje cells are among the largest neurons in the central nervous system and accordingly can harbor two nuclei.Using a well-characterized model for heterokaryon formation in the cerebellum (experimental autoimmune encephalomyelitis - a mouse model of multiple sclerosis, we report for the first time that green fluorescent protein-labeled bone marrow-derived cells can fuse and form heterokaryons with spinal cord motor neurons. These spinal cord heterokaryons are predominantly located in or adjacent to an active or previously active inflammation site, demonstrating that inflammation and infiltration of immune cells are key for cell fusion in the central nervous system. While some motor neurons were found to contain two nuclei, co-expressing green fluorescent protein and the neuronal marker, neuron-specific nuclear protein, a number of small interneurons also co-expressed green fluorescent protein and the neuronal marker, neuron-specific nuclear protein. These small heterokaryons were scattered in the gray matter of the spinal cord.This novel finding expands the repertoire of neurons that can form heterokaryons with bone marrow-derived cells in the central nervous system, albeit in low numbers, possibly leading to a novel therapy for spinal cord

  5. Significances of RET Fusion Gene in Non-small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Jingjing LIU

    2013-11-01

    Full Text Available Lung cancer is the leading cause of cancer-related death worldwide, molecular target therapy has become a hot research direction of non-small cell lung cancer (NSCLC treatment. RET fusion gene with an identifiable clinical pathological features, is present in some subsets of lung cancer, and its treatment is effective by RET inhibitor, suggesting that RET fusion gene may be a new target for individualized treatment to the subgroup of NSCLC. This article reviews the structural characteristics of RET fusion gene and expression model in clinical samples, and treatment of NSCLC.

  6. Novel insights into the role of mitochondrial fusion and fission in cardiomyocyte apoptosis induced by ischemia/reperfusion.

    Science.gov (United States)

    Li, YuZhen; Liu, XiuHua

    2018-03-12

    As the main source of energy in the body, mitochondria are highly dynamic organelles, which are constantly going through fusion and fission. The fine balance of mitochondrial fusion and fission plays an important role in maintaining the stability of cardiomyocyte homeostasis. The processes of mitochondrial fusion and fission are very complex, which is mediated by fusion and fission proteins. Disruptions in these processes through controlling fusion and fission proteins affect mitochondrial functions and cardiomyocyte survival. Ischemia/reperfusion (I/R) can regulate the expression and post-translational modifications of fusion and fission proteins thereby inducing the abnormality of mitochondrial fusion and fission and cardiomyocyte apoptosis. Furthermore, intervention with the expression and function of fusion and fission proteins influences on cardiomyocyte apoptosis under I/R conditions. In this review, we focus on the current developments in the effects of mitochondrial fusion and fission on cardiomyocyte functions, the implications for cardiomyocyte apoptosis in response to I/R, and possible mechanisms. And we review their roles as a potential therapeutic target for treating I/R-induced cardiomyocyte injury. © 2018 Wiley Periodicals, Inc.

  7. Integrin αvβ1 Modulation Affects Subtype B Avian Metapneumovirus Fusion Protein-mediated Cell-Cell Fusion and Virus Infection.

    Science.gov (United States)

    Yun, Bing-Ling; Guan, Xiao-Lu; Liu, Yong-Zhen; Zhang, Yao; Wang, Yong-Qiang; Qi, Xiao-Le; Cui, Hong-Yu; Liu, Chang-Jun; Zhang, Yan-Ping; Gao, Hong-Lei; Gao, Li; Li, Kai; Gao, Yu-Long; Wang, Xiao-Mei

    2016-07-08

    Avian metapneumovirus (aMPV) fusion (F) protein mediates virus-cell membrane fusion to initiate viral infection, which requires F protein binding to its receptor(s) on the host cell surface. However, the receptor(s) for aMPV F protein is still not identified. All known subtype B aMPV (aMPV/B) F proteins contain a conserved Arg-Asp-Asp (RDD) motif, suggesting that the aMPV/B F protein may mediate membrane fusion via the binding of RDD to integrin. When blocked with integrin-specific peptides, aMPV/B F protein fusogenicity and viral replication were significantly reduced. Specifically we identified integrin αv and/or β1-mediated F protein fusogenicity and viral replication using antibody blocking, small interfering RNAs (siRNAs) knockdown, and overexpression. Additionally, overexpression of integrin αv and β1 in aMPV/B non-permissive cells conferred aMPV/B F protein binding and aMPV/B infection. When RDD was altered to RAE (Arg-Ala-Glu), aMPV/B F protein binding and fusogenic activity were profoundly impaired. These results suggest that integrin αvβ1 is a functional receptor for aMPV/B F protein-mediated membrane fusion and virus infection, which will provide new insights on the fusogenic mechanism and pathogenesis of aMPV. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Integrin αvβ1 Modulation Affects Subtype B Avian Metapneumovirus Fusion Protein-mediated Cell-Cell Fusion and Virus Infection*

    Science.gov (United States)

    Yun, Bing-Ling; Guan, Xiao-Lu; Liu, Yong-Zhen; Zhang, Yao; Wang, Yong-Qiang; Qi, Xiao-Le; Cui, Hong-Yu; Liu, Chang-Jun; Zhang, Yan-Ping; Gao, Hong-Lei; Gao, Li; Li, Kai; Gao, Yu-Long; Wang, Xiao-Mei

    2016-01-01

    Avian metapneumovirus (aMPV) fusion (F) protein mediates virus-cell membrane fusion to initiate viral infection, which requires F protein binding to its receptor(s) on the host cell surface. However, the receptor(s) for aMPV F protein is still not identified. All known subtype B aMPV (aMPV/B) F proteins contain a conserved Arg-Asp-Asp (RDD) motif, suggesting that the aMPV/B F protein may mediate membrane fusion via the binding of RDD to integrin. When blocked with integrin-specific peptides, aMPV/B F protein fusogenicity and viral replication were significantly reduced. Specifically we identified integrin αv and/or β1-mediated F protein fusogenicity and viral replication using antibody blocking, small interfering RNAs (siRNAs) knockdown, and overexpression. Additionally, overexpression of integrin αv and β1 in aMPV/B non-permissive cells conferred aMPV/B F protein binding and aMPV/B infection. When RDD was altered to RAE (Arg-Ala-Glu), aMPV/B F protein binding and fusogenic activity were profoundly impaired. These results suggest that integrin αvβ1 is a functional receptor for aMPV/B F protein-mediated membrane fusion and virus infection, which will provide new insights on the fusogenic mechanism and pathogenesis of aMPV. PMID:27226547

  9. Protective antitumor activity induced by a fusion vaccine with murine ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-08-04

    Aug 4, 2009 ... Briefly, alginate beads formed and contained about 1×105 tumor cells per bead. Four beads were implanted ... cad in coating buffer (carbonate bicarbonate, pH 9.6) overnight at. 4ºC. Diluted mice sera were added ... into a 96-well microtiter plate in different E: T ratios. The CTL acti- vity was calculated by the ...

  10. Laboratory feasibility study of fusion vessel inner wall chemical analysis by Laser Induced Breakdown Spectroscopy

    International Nuclear Information System (INIS)

    Almaviva, Salvatore; Caneve, Luisa; Colao, Francesco; Fantoni, Roberta; Maddaluno, Giorgio

    2012-01-01

    Graphical abstract: Laser-Induced-Breakdown-Spectroscopy was used for the determination of the atomic composition of multilayered samples simulating the tiles of plasma facing components in the next generation fusion machines. Highlights: ► Description and characterization of an LIBS set-up for diagnostics in fusion machines. ► Identification of atomic composition of multilayered tiles simulating plasma facing components. ► Qualitative applicability of the Calibration Free method for quantitative analysis. ► Feasibility of large scale application in the processes of control during the tiles fabrication. ► Feasibility of erosion monitoring during operation of fusion machines. - Abstract: Laser Induced Breakdown Spectroscopy (LIBS) is nowadays a well established tool for qualitative, semi-quantitative and quantitative analyses of surfaces, with micro-destructive characteristics and capabilities for stratigraphy. LIBS is an appealing technique compared with many other types of elemental analysis thanks to the set up versatility facilitating non-invasive and remote analyses, as well as suitability to diagnostics in harsh environments. In this work, LIBS capabilities were used for the determination of the atomic composition of multilayered samples simulating the tiles of plasma facing components in the next generation fusion machines such as ITER. A new experimental setup was designed and realized in order to optimize the characteristics of an LIBS system working at low pressure and remotely, as it should be for an in situ system to be applied in monitoring the erosion and redeposition phenomena occurring on the inner walls of a fusion device. The effects of time delay and laser fluence on LIBS sensitivity at reduced pressure were examined, looking for operational conditions suitable to analytical applications. The quantitative analysis of some atomic species in the superficial layer has been carried out using a Calibration Free (CF) approach in the time

  11. Expanding the molecular toolbox for Lactococcus lactis: construction of an inducible thioredoxin gene fusion expression system

    LENUS (Irish Health Repository)

    Douillard, Francois P

    2011-08-09

    Abstract Background The development of the Nisin Inducible Controlled Expression (NICE) system in the food-grade bacterium Lactococcus lactis subsp. cremoris represents a cornerstone in the use of Gram-positive bacterial expression systems for biotechnological purposes. However, proteins that are subjected to such over-expression in L. lactis may suffer from improper folding, inclusion body formation and\\/or protein degradation, thereby significantly reducing the yield of soluble target protein. Although such drawbacks are not specific to L. lactis, no molecular tools have been developed to prevent or circumvent these recurrent problems of protein expression in L. lactis. Results Mimicking thioredoxin gene fusion systems available for E. coli, two nisin-inducible expression vectors were constructed to over-produce various proteins in L. lactis as thioredoxin fusion proteins. In this study, we demonstrate that our novel L. lactis fusion partner expression vectors allow high-level expression of soluble heterologous proteins Tuc2009 ORF40, Bbr_0140 and Tuc2009 BppU\\/BppL that were previously insoluble or not expressed using existing L. lactis expression vectors. Over-expressed proteins were subsequently purified by Ni-TED affinity chromatography. Intact heterologous proteins were detected by immunoblotting analyses. We also show that the thioredoxin moiety of the purified fusion protein was specifically and efficiently cleaved off by enterokinase treatment. Conclusions This study is the first description of a thioredoxin gene fusion expression system, purposely developed to circumvent problems associated with protein over-expression in L. lactis. It was shown to prevent protein insolubility and degradation, allowing sufficient production of soluble proteins for further structural and functional characterization.

  12. Evaluation of inducible promoters on the secretion of a ZZ-proinsulin fusion protein in Escherichia coli.

    Science.gov (United States)

    Mergulhão, Filipe J M; Monteiro, Gabriel A; Larsson, Gen; Bostrom, Maria; Farewell, Anne; Nyström, Thomas; Cabral, Joaquim M S; Taipa, M Angela

    2003-08-01

    Four inducible promoters, uspA, uspB, lacUV5 and malK, were evaluated in the expression of the fusion protein ZZ-proinsulin by Escherichia coli. The aim was to select for their effects on the most appropriate expression system (promoter and culture medium) for secretion of ZZ-proinsulin to the periplasmic space and culture medium. All the expression vectors contained the RNase III cleavage site to ensure that the mRNA translation rate remained independent of 5'-untranslated regions thus making promoter strength comparisons more accurate. The highest ZZ-proinsulin secretion yields were 6.2 mg/g of dry cell weight in the periplasmic space and 2.6 mg/g of dry cell weight in the culture medium using the malK promoter. It was also demonstrated that the use of M9 minimal medium favours secretion.

  13. Long-Term Endurance Exercise in Humans Stimulates Cell Fusion of Myoblasts along with Fusogenic Endogenous Retroviral Genes In Vivo

    OpenAIRE

    Frese, Sebastian; Ruebner, Matthias; Suhr, Frank; Konou, Thierry M; Tappe, Kim A; Toigo, Marco; Jung, Hans H; Henke, Christine; Steigleder, Ruth; Strissel, Pamela L; Huebner, Hanna; Beckmann, Matthias W; van der Keylen, Piet; Schoser, Benedikt; Schiffer, Thorsten

    2015-01-01

    Myogenesis is defined as growth, differentiation and repair of muscles where cell fusion of myoblasts to multinucleated myofibers is one major characteristic. Other cell fusion events in humans are found with bone resorbing osteoclasts and placental syncytiotrophoblasts. No unifying gene regulation for natural cell fusions has been found. We analyzed skeletal muscle biopsies of competitive cyclists for muscle-specific attributes and expression of human endogenous retrovirus (ERV) envelope gen...

  14. AMPK Activation Prevents and Reverses Drug-Induced Mitochondrial and Hepatocyte Injury by Promoting Mitochondrial Fusion and Function.

    Directory of Open Access Journals (Sweden)

    Sun Woo Sophie Kang

    Full Text Available Mitochondrial damage is the major factor underlying drug-induced liver disease but whether conditions that thwart mitochondrial injury can prevent or reverse drug-induced liver damage is unclear. A key molecule regulating mitochondria quality control is AMP activated kinase (AMPK. When activated, AMPK causes mitochondria to elongate/fuse and proliferate, with mitochondria now producing more ATP and less reactive oxygen species. Autophagy is also triggered, a process capable of removing damaged/defective mitochondria. To explore whether AMPK activation could potentially prevent or reverse the effects of drug-induced mitochondrial and hepatocellular damage, we added an AMPK activator to collagen sandwich cultures of rat and human hepatocytes exposed to the hepatotoxic drugs, acetaminophen or diclofenac. In the absence of AMPK activation, the drugs caused hepatocytes to lose polarized morphology and have significantly decreased ATP levels and viability. At the subcellular level, mitochondria underwent fragmentation and had decreased membrane potential due to decreased expression of the mitochondrial fusion proteins Mfn1, 2 and/or Opa1. Adding AICAR, a specific AMPK activator, at the time of drug exposure prevented and reversed these effects. The mitochondria became highly fused and ATP production increased, and hepatocytes maintained polarized morphology. In exploring the mechanism responsible for this preventive and reversal effect, we found that AMPK activation prevented drug-mediated decreases in Mfn1, 2 and Opa1. AMPK activation also stimulated autophagy/mitophagy, most significantly in acetaminophen-treated cells. These results suggest that activation of AMPK prevents/reverses drug-induced mitochondrial and hepatocellular damage through regulation of mitochondrial fusion and autophagy, making it a potentially valuable approach for treatment of drug-induced liver injury.

  15. AMPK Activation Prevents and Reverses Drug-Induced Mitochondrial and Hepatocyte Injury by Promoting Mitochondrial Fusion and Function

    Science.gov (United States)

    Taniane, Caitlin; Farrell, Geoffrey; Arias, Irwin M.; Lippincott-Schwartz, Jennifer; Fu, Dong

    2016-01-01

    Mitochondrial damage is the major factor underlying drug-induced liver disease but whether conditions that thwart mitochondrial injury can prevent or reverse drug-induced liver damage is unclear. A key molecule regulating mitochondria quality control is AMP activated kinase (AMPK). When activated, AMPK causes mitochondria to elongate/fuse and proliferate, with mitochondria now producing more ATP and less reactive oxygen species. Autophagy is also triggered, a process capable of removing damaged/defective mitochondria. To explore whether AMPK activation could potentially prevent or reverse the effects of drug-induced mitochondrial and hepatocellular damage, we added an AMPK activator to collagen sandwich cultures of rat and human hepatocytes exposed to the hepatotoxic drugs, acetaminophen or diclofenac. In the absence of AMPK activation, the drugs caused hepatocytes to lose polarized morphology and have significantly decreased ATP levels and viability. At the subcellular level, mitochondria underwent fragmentation and had decreased membrane potential due to decreased expression of the mitochondrial fusion proteins Mfn1, 2 and/or Opa1. Adding AICAR, a specific AMPK activator, at the time of drug exposure prevented and reversed these effects. The mitochondria became highly fused and ATP production increased, and hepatocytes maintained polarized morphology. In exploring the mechanism responsible for this preventive and reversal effect, we found that AMPK activation prevented drug-mediated decreases in Mfn1, 2 and Opa1. AMPK activation also stimulated autophagy/mitophagy, most significantly in acetaminophen-treated cells. These results suggest that activation of AMPK prevents/reverses drug-induced mitochondrial and hepatocellular damage through regulation of mitochondrial fusion and autophagy, making it a potentially valuable approach for treatment of drug-induced liver injury. PMID:27792760

  16. Reovirus FAST Proteins Drive Pore Formation and Syncytiogenesis Using a Novel Helix-Loop-Helix Fusion-Inducing Lipid Packing Sensor

    Science.gov (United States)

    Sarker, Muzaddid; de Antueno, Roberto; Langelaan, David N.; Parmar, Hiren B.; Shin, Kyungsoo; Rainey, Jan K.; Duncan, Roy

    2015-01-01

    Pore formation is the most energy-demanding step during virus-induced membrane fusion, where high curvature of the fusion pore rim increases the spacing between lipid headgroups, exposing the hydrophobic interior of the membrane to water. How protein fusogens breach this thermodynamic barrier to pore formation is unclear. We identified a novel fusion-inducing lipid packing sensor (FLiPS) in the cytosolic endodomain of the baboon reovirus p15 fusion-associated small transmembrane (FAST) protein that is essential for pore formation during cell-cell fusion and syncytiogenesis. NMR spectroscopy and mutational studies indicate the dependence of this FLiPS on a hydrophobic helix-loop-helix structure. Biochemical and biophysical assays reveal the p15 FLiPS preferentially partitions into membranes with high positive curvature, and this partitioning is impeded by bis-ANS, a small molecule that inserts into hydrophobic defects in membranes. Most notably, the p15 FLiPS can be functionally replaced by heterologous amphipathic lipid packing sensors (ALPS) but not by other membrane-interactive amphipathic helices. Furthermore, a previously unrecognized amphipathic helix in the cytosolic domain of the reptilian reovirus p14 FAST protein can functionally replace the p15 FLiPS, and is itself replaceable by a heterologous ALPS motif. Anchored near the cytoplasmic leaflet by the FAST protein transmembrane domain, the FLiPS is perfectly positioned to insert into hydrophobic defects that begin to appear in the highly curved rim of nascent fusion pores, thereby lowering the energy barrier to stable pore formation. PMID:26061049

  17. Reovirus FAST Proteins Drive Pore Formation and Syncytiogenesis Using a Novel Helix-Loop-Helix Fusion-Inducing Lipid Packing Sensor.

    Directory of Open Access Journals (Sweden)

    Jolene Read

    2015-06-01

    Full Text Available Pore formation is the most energy-demanding step during virus-induced membrane fusion, where high curvature of the fusion pore rim increases the spacing between lipid headgroups, exposing the hydrophobic interior of the membrane to water. How protein fusogens breach this thermodynamic barrier to pore formation is unclear. We identified a novel fusion-inducing lipid packing sensor (FLiPS in the cytosolic endodomain of the baboon reovirus p15 fusion-associated small transmembrane (FAST protein that is essential for pore formation during cell-cell fusion and syncytiogenesis. NMR spectroscopy and mutational studies indicate the dependence of this FLiPS on a hydrophobic helix-loop-helix structure. Biochemical and biophysical assays reveal the p15 FLiPS preferentially partitions into membranes with high positive curvature, and this partitioning is impeded by bis-ANS, a small molecule that inserts into hydrophobic defects in membranes. Most notably, the p15 FLiPS can be functionally replaced by heterologous amphipathic lipid packing sensors (ALPS but not by other membrane-interactive amphipathic helices. Furthermore, a previously unrecognized amphipathic helix in the cytosolic domain of the reptilian reovirus p14 FAST protein can functionally replace the p15 FLiPS, and is itself replaceable by a heterologous ALPS motif. Anchored near the cytoplasmic leaflet by the FAST protein transmembrane domain, the FLiPS is perfectly positioned to insert into hydrophobic defects that begin to appear in the highly curved rim of nascent fusion pores, thereby lowering the energy barrier to stable pore formation.

  18. Detection of fusion gene in cell-free DNA of a gastric synovial sarcoma.

    Science.gov (United States)

    Ogino, Shinpei; Konishi, Hirotaka; Ichikawa, Daisuke; Hamada, Junichi; Shoda, Katsutoshi; Arita, Tomohiro; Komatsu, Shuhei; Shiozaki, Atsushi; Okamoto, Kazuma; Yamazaki, Sanae; Yasukawa, Satoru; Konishi, Eiichi; Otsuji, Eigo

    2018-02-28

    Synovial sarcoma (SS) is genetically characterized by chromosomal translocation, which generates SYT-SSX fusion transcripts. Although SS can occur in any body part, primary gastric SS is substantially rare. Here we describe a detection of the fusion gene sequence of gastric SS in plasma cell-free DNA (cfDNA). A gastric submucosal tumor was detected in the stomach of a 27-year-old woman and diagnosed as SS. Candidate intronic primers were designed to detect the intronic fusion breakpoint and this fusion sequence was confirmed in intron 10 of SYT and intron 5 of SSX2 by genomic polymerase chain reaction (PCR) and direct sequencing. A locked nucleic acid (LNA) probe specific to the fusion sequence was designed for detecting the fusion sequence in plasma and the fusion sequence was detected in preoperative plasma cfDNA, while not detected in postoperative plasma cfDNA. This technique will be useful for monitoring translocation-derived diseases such as SS.

  19. Cold fusion, Alchemist's dream

    International Nuclear Information System (INIS)

    Clayton, E.D.

    1989-09-01

    In this report the following topics relating to cold fusion are discussed: muon catalysed cold fusion; piezonuclear fusion; sundry explanations pertaining to cold fusion; cosmic ray muon catalysed cold fusion; vibrational mechanisms in excited states of D 2 molecules; barrier penetration probabilities within the hydrogenated metal lattice/piezonuclear fusion; branching ratios of D 2 fusion at low energies; fusion of deuterons into 4 He; secondary D+T fusion within the hydrogenated metal lattice; 3 He to 4 He ratio within the metal lattice; shock induced fusion; and anomalously high isotopic ratios of 3 He/ 4 He

  20. Cold fusion, Alchemist's dream

    Energy Technology Data Exchange (ETDEWEB)

    Clayton, E.D.

    1989-09-01

    In this report the following topics relating to cold fusion are discussed: muon catalysed cold fusion; piezonuclear fusion; sundry explanations pertaining to cold fusion; cosmic ray muon catalysed cold fusion; vibrational mechanisms in excited states of D{sub 2} molecules; barrier penetration probabilities within the hydrogenated metal lattice/piezonuclear fusion; branching ratios of D{sub 2} fusion at low energies; fusion of deuterons into {sup 4}He; secondary D+T fusion within the hydrogenated metal lattice; {sup 3}He to {sup 4}He ratio within the metal lattice; shock induced fusion; and anomalously high isotopic ratios of {sup 3}He/{sup 4}He.

  1. Molecular Mechanisms Regulating Cell Fusion and Heterokaryon Formation in Filamentous Fungi.

    Science.gov (United States)

    Daskalov, Asen; Heller, Jens; Herzog, Stephanie; Fleißner, André; Glass, N Louise

    2017-03-01

    For the majority of fungal species, the somatic body of an individual is a network of interconnected cells sharing a common cytoplasm and organelles. This syncytial organization contributes to an efficient distribution of resources, energy, and biochemical signals. Cell fusion is a fundamental process for fungal development, colony establishment, and habitat exploitation and can occur between hyphal cells of an individual colony or between colonies of genetically distinct individuals. One outcome of cell fusion is the establishment of a stable heterokaryon, culminating in benefits for each individual via shared resources or being of critical importance for the sexual or parasexual cycle of many fungal species. However, a second outcome of cell fusion between genetically distinct strains is formation of unstable heterokaryons and the induction of a programmed cell death reaction in the heterokaryotic cells. This reaction of nonself rejection, which is termed heterokaryon (or vegetative) incompatibility, is widespread in the fungal kingdom and acts as a defense mechanism against genome exploitation and mycoparasitism. Here, we review the currently identified molecular players involved in the process of somatic cell fusion and its regulation in filamentous fungi. Thereafter, we summarize the knowledge of the molecular determinants and mechanism of heterokaryon incompatibility and place this phenomenon in the broader context of biotropic interactions and immunity.

  2. A Unique Opportunity to Test Whether Cell Fusion is a Mechanism of Breast Cancer Metastasis

    Science.gov (United States)

    2012-07-01

    cancer cell cocultures such as inactivated monocytes and nontumorigenic mammary epithelial cells, we sought to subclone our BiFC DNA constructs into a...human immunodeficiency virus type-1 ( HIV -1) envelope protein (Env) use a hydrophobic fusion peptide that is only exposed after specific receptor

  3. Evaluation of cell binding peptide (p15) with silk fibre enhanced hydroxyappatite bone substitute for posterolateral spinal fusion in sheep

    DEFF Research Database (Denmark)

    Axelsen, M.; Jespersen, Stig; Overgaard, Søren

    2015-01-01

    's gold standard are highly desired. Uninstrumented posterolateral fusion (PLF) is one of the most challenging bone graft procedures because of large graft size and lack of external stability. P15 is a synthetic 15 amino acid peptide sequence, identical to the biding site for alpha2-beta1 integrin...... on the surface of bone forming cells. The binding initiates natural intra- and extracellular signalling pathways, inducing production of growth factors, bone morphogenic proteins and cytokines. P15 peptide has previously shown to improve osteoinductive properties when coated on graft materials. Purpose...

  4. Characterization of an immunomodulatory Der p 2-FIP-fve fusion protein produced in transformed rice suspension cell culture.

    Science.gov (United States)

    Su, Chin-Fen; Kuo, I-Chun; Chen, Peng-Wen; Huang, Chiung-Hui; Seow, See Voon; Chua, Kaw Yan; Yu, Su-May

    2012-02-01

    Der p 2, a major allergen of Dermatophagoides pteronyssinus mites, is one of the most clinically relevant allergens to allergic patients worldwide. FIP-fve protein (Fve) from the golden needle mushroom (Flammulina velutipes) is an immunomodulatory protein with potential Th1-skewed adjuvant properties. Here, we produced and immunologically evaluated a Der p 2-Fve fusion protein as a potential immunotherapeutic for allergic diseases. Using an inducible expression system in cultured rice suspension cells, the recombinant Der p 2-Fve fusion protein (designated as OsDp2Fve) was expressed in rice cells under the control of an α-amylase gene (αAmy8) promoter and secreted under sucrose starvation. OsDp2Fve was partially purified from the cultured medium. The conformation of Der p 2 in OsDp2Fve remains intact as reflected by its unaltered allergenicity, as assessed by human IgE ELISA and histamine release assays, compared to non-fusion Der p 2 protein. Furthermore, the Fve protein expressed in OsDp2Fve retains its in vitro lymphoproliferative activity but loses its hemagglutination and lymphoagglutination effects compared to the native protein. Notably, in vivo evaluation showed that mice administered with OsDp2Fve possessed an enhanced production of Der p 2-specific IgG antibodies without potentiating the production of Der p 2-specific IgE and Th2 effector cytokines in comparison with mice co-administered with native Fve and Der p 2 proteins. These results suggest that the recombinant Der p 2-Fve fusion protein produced in rice suspension cell cultures has a great potential for allergy immunotherapy.

  5. Heat generation above break-even from laser-induced fusion in ultra-dense deuterium

    Directory of Open Access Journals (Sweden)

    Leif Holmlid

    2015-08-01

    Full Text Available Previous results from laser-induced processes in ultra-dense deuterium D(0 give conclusive evidence for ejection of neutral massive particles with energy >10 MeV u−1. Such particles can only be formed from nuclear processes like nuclear fusion at the low laser intensity used. Heat generation is of interest for future fusion energy applications and has now been measured by a small copper (Cu cylinder surrounding the laser target. The temperature rise of the Cu cylinder is measured with an NTC resistor during around 5000 laser shots per measured point. No heating in the apparatus or the gas feed is normally used. The fusion process is suboptimal relative to previously published studies by a factor of around 10. The small neutral particles HN(0 of ultra-dense hydrogen (size of a few pm escape with a substantial fraction of the energy. Heat loss to the D2 gas (at <1 mbar pressure is measured and compensated for under various conditions. Heat release of a few W is observed, at up to 50% higher energy than the total laser input thus a gain of 1.5. This is uniquely high for the use of deuterium as fusion fuel. With a slightly different setup, a thermal gain of 2 is reached, thus clearly above break-even for all neutronicity values possible. Also including the large kinetic energy which is directly measured for MeV particles leaving through a small opening gives a gain of 2.3. Taking into account the lower efficiency now due to the suboptimal fusion process, previous studies indicate a gain of at least 20 during long periods.

  6. Effect of TheraCyte-encapsulated parathyroid cells on lumbar fusion in a rat model.

    Science.gov (United States)

    Chen, Sung-Hsiung; Huang, Shun-Chen; Lui, Chun-Chung; Lin, Tzu-Ping; Chou, Fong-Fu; Ko, Jih-Yang

    2012-09-01

    Implantation of TheraCyte 4 × 10(6) live parathyroid cells can increase the bone marrow density of the spine of ovariectomized rats. There has been no published study examining the effect of such implantation on spinal fusion outcomes. The purpose of this study was to examine the effect of TheraCyte-encapsulated parathyroid cells on posterolateral lumbar fusions in a rat model. Forty Sprague-Dawley rats underwent single-level, intertransverse process spinal fusions using iliac crest autograft. The rats were randomly assigned to two groups: Group 1 rats received sham operations on their necks (control; N = 20); Group 2 rats were implanted with TheraCyte-encapsulated 4 × 10(6) live parathyroid cells into the subcutis of their necks (TheraCyte; N = 20). Six weeks after surgery the rats were killed. Fusion was assessed by inspection, manual palpation, radiography, and histology. Blood was drawn to measure the serum levels of calcium, phosphorus, and intact parathyroid hormone (iPTH). Based on manual palpation, the control group had a fusion rate of 33 % (6/18) and the TheraCyte group had a fusion rate of 72 % (13/18) (P = 0.044). Histology confirmed the manual palpation results. Serum iPTH levels were significantly higher in the TheraCyte group compared with the control group (P animal study revealed that there were more fusions in rats that received TheraCyte-encapsulated 4 × 10(6) live parathyroid cells than in control rats without significant change in serum calcium or phosphorus concentrations. As with any animal study, the results may not extrapolate to a higher species. Further studies are needed to determine if these effects are clinically significant.

  7. Evaluation of EML4-ALK Fusion Proteins in Non–Small Cell Lung Cancer Using Small Molecule Inhibitors

    Directory of Open Access Journals (Sweden)

    Yongjun Li

    2011-01-01

    Full Text Available The echinoderm microtubule–associated protein-like 4–anaplastic lymphoma kinase (EML4-ALK fusion gene resulting from an inversion within chromosome 2p occurs in approximately 5% of non–small cell lung cancer and is mu-tually exclusive with Ras and EGFR mutations. In this study, we have used a potent and selective ALK small molecule inhibitor, NPV-TAE684, to assess the oncogenic role of EML4-ALK in non–small cell lung cancer (NSCLC. We show here that TAE684 inhibits proliferation and induces cell cycle arrest, apoptosis, and tumor regression in two NSCLC models that harbor EML4-ALK fusions. TAE684 inhibits EML4-ALK activation and its downstream signaling including ERK, AKT, and STAT3. We used microarray analysis to carry out targeted pathway studies of gene expression changes in H2228 NSCLC xenograft model after TAE684 treatment and identified a gene signature of EML4-ALK inhibition. The gene signature represents 1210 known human genes, and the top biologic processes represented by these genes are cell cycle, DNA synthesis, cell proliferation, and cell death. We also compared the effect of TAE684 with PF2341066, a c-Met and ALK small molecule inhibitor currently in clinical trial in cancers harboring ALK fusions, and demonstrated that TAE684 is a much more potent inhibitor of EML4-ALK. Our data demonstrate that EML4-ALK plays an important role in the pathogenesis of a subset of NSCLC and provides insight into the mech-anism of EML4-ALK inhibition by a small molecule inhibitor.

  8. Fusion of Selected Cells and Vesicles Mediated by Optically Trapped Plasmonic Nanoparticles

    DEFF Research Database (Denmark)

    Bahadori, Azra

    . In this work, we introduce a novel and extremely flexible physical method which can trigger membrane fusion in a highly selective manner not only between synthetic GUVs of different compositions, but also between live cells which remain viable after fusion. Optical tweezers’ laser (1064 nm) is used to position......Selective fusion of two membrane surrounded volumes is of great interest in nanochemistry and nanomedicine as it can pave the way for performing controlled nanoscale chemical reactions and for delivering a cargo (e.g., chemicals, genetic regulatory factors, etc.) to a desired living cell...... the two desired cells and/or GUVs next to each other and in immediate contact. Then, the same laser is placed in the contact zone between the two adjacent membranes until one or more gold nanoparticles diffuse into the focus. Gold nanoparticles absorb part of near infrared light and dissipate the absorbed...

  9. The 2-Body Cytoskeleton Problem: Studying Cell-Cell Fusion Mechanics in Osteoclasts with Multiscale Imaging

    Science.gov (United States)

    Silverberg, Jesse; Ng, Pei Ying; Baron, Roland; Yin, Peng

    Most research on in vivocytoskeletal mechanics focuses on what happens in a single cell context. This foundational work has opened up new avenues to study higher-order problems, such as what happens when cells physically interact. For example, osteoclasts, one of the cell types responsible for maintaining healthy skeletal structure, are formed when 10 or more mononuclear cells fuse into a multinuclear behemoth. But how does the cytoskeleton of two or more cells fuse? And what is the role of mechanics in understanding the resulting cytoskeletal organization? In this work, we use the multiscale multiplexed Molecular Atlas Platform to image and study the cytoskeletal mechanics of cell-cell fusion. Our work documents the processes involved and uses observed structures to infer mechanical events during these interactions. Broadly this work takes a technology-driven approach to perform fundamental exploratory work, and uses current state-of-the-art cytoskeletal mechanical modeling to interpret our observations. National Cancer Institute of the National Institutes of Health under Award Number F32CA204038.

  10. Novel fusion proteins for the antigen-specific staining and elimination of B cell receptor-positive cell populations demonstrated by a tetanus toxoid fragment C (TTC) model antigen.

    Science.gov (United States)

    Klose, Diana; Saunders, Ute; Barth, Stefan; Fischer, Rainer; Jacobi, Annett Marita; Nachreiner, Thomas

    2016-02-17

    In an earlier study we developed a unique strategy allowing us to specifically eliminate antigen-specific murine B cells via their distinct B cell receptors using a new class of fusion proteins. In the present work we elaborated our idea to demonstrate the feasibility of specifically addressing and eliminating human memory B cells. The present study reveals efficient adaptation of the general approach to selectively target and eradicate human memory B cells. In order to demonstrate the feasibility we engineered a fusion protein following the principle of recombinant immunotoxins by combining a model antigen (tetanus toxoid fragment C, TTC) for B cell receptor targeting and a truncated version of Pseudomonas aeruginosa exotoxin A (ETA') to induce apoptosis after cellular uptake. The TTC-ETA' fusion protein not only selectively bound to a TTC-reactive murine B cell hybridoma cell line in vitro but also to freshly isolated human memory B cells from immunized donors ex vivo. Specific toxicity was confirmed on an antigen-specific population of human CD27(+) memory B cells. This protein engineering strategy can be used as a generalized platform approach for the construction of therapeutic fusion proteins with disease-relevant antigens as B cell receptor-binding domains, offering a promising approach for the specific depletion of autoreactive B-lymphocytes in B cell-driven autoimmune diseases.

  11. Doping-Induced Isotopic Mg11B2 Bulk Superconductor for Fusion Application

    Directory of Open Access Journals (Sweden)

    Qi Cai

    2017-03-01

    Full Text Available Superconducting wires are widely used for fabricating magnetic coils in fusion reactors. Superconducting magnet system represents a key determinant of the thermal efficiency and the construction/operating costs of such a reactor. In consideration of the stability of 11B against fast neutron irradiation and its lower induced radioactivation properties, MgB2 superconductor with 11B serving as the boron source is an alternative candidate for use in fusion reactors with a severe high neutron flux environment. In the present work, the glycine-doped Mg11B2 bulk superconductor was synthesized from isotopic 11B powder to enhance the high field properties. The critical current density was enhanced (103 A·cm−2 at 20 K and 5 T over the entire field in contrast with the sample prepared from natural boron.

  12. pH Optimum of Hemagglutinin-Mediated Membrane Fusion Determines Sensitivity of Influenza A Viruses to the Interferon-Induced Antiviral State and IFITMs.

    Science.gov (United States)

    Gerlach, Thomas; Hensen, Luca; Matrosovich, Tatyana; Bergmann, Janina; Winkler, Michael; Peteranderl, Christin; Klenk, Hans-Dieter; Weber, Friedemann; Herold, Susanne; Pöhlmann, Stefan; Matrosovich, Mikhail

    2017-06-01

    The replication and pathogenicity of influenza A viruses (IAVs) critically depend on their ability to tolerate the antiviral interferon (IFN) response. To determine a potential role for the IAV hemagglutinin (HA) in viral sensitivity to IFN, we studied the restriction of IAV infection in IFN-β-treated human epithelial cells by using 2:6 recombinant IAVs that shared six gene segments of A/Puerto Rico/8/1934 virus (PR8) and contained HAs and neuraminidases of representative avian, human, and zoonotic H5N1 and H7N9 viruses. In A549 and Calu-3 cells, viruses displaying a higher pH optimum of HA-mediated membrane fusion, H5N1-PR8 and H7N9-PR8, were less sensitive to the IFN-induced antiviral state than their counterparts with HAs from duck and human viruses, which fused at a lower pH. The association between a high pH optimum of fusion and reduced IFN sensitivity was confirmed by using HA point mutants of A/Hong Kong/1/1968-PR8 that differed solely by their fusion properties. Furthermore, similar effects of the viral fusion pH on IFN sensitivity were observed in experiments with (i) primary human type II alveolar epithelial cells and differentiated cultures of human airway epithelial cells, (ii) nonrecombinant zoonotic and pandemic IAVs, and (iii) preparations of IFN-α and IFN-λ1. A higher pH of membrane fusion and reduced sensitivity to IFN correlated with lower restriction of the viruses in MDCK cells stably expressing the IFN-inducible transmembrane proteins IFITM2 and IFITM3, which are known to inhibit viral fusion. Our results reveal that the pH optimum of HA-driven membrane fusion of IAVs is a determinant of their sensitivity to IFN and IFITM proteins. IMPORTANCE The IFN system constitutes an important innate defense against viral infection. Substantial information is available on how IAVs avoid detection by sensors of the IFN system and disable IFN signaling pathways. Much less is known about the ability of IAVs to tolerate the antiviral activity of IFN-induced

  13. A fusion cytokine coupling GMCSF to IL9 induces heterologous receptor clustering and STAT1 hyperactivation through JAK2 promiscuity.

    Directory of Open Access Journals (Sweden)

    Pingxin Li

    Full Text Available Cytokine receptors are randomly distributed on the cell surface membrane and are activated upon binding of their extracellular ligands to mediate downstream cellular activities. We hypothesized that pharmaceutical clustering of ligand-bound, activated receptors may lead to heretofore unrealized gain-of-function with therapeutically desirable properties. We here describe an engineered bifunctional cytokine borne of the fusion of Granulocyte Macrophage Colony Stimulating Factor (GMCSF and Interleukin-9 (IL9 (hereafter GIFT9 fusokine and demonstrate that it chaperones co-clustering of the functionally unrelated GMCSF receptor (GMCSFR and IL9 receptor (IL9R on cell surface of target cells. We demonstrate that GIFT9 treatment of MC/9 cells leads to transhyperphosphorylation of IL9R-associated STAT1 by GMCSFR-associated JAK2. We also show that IL9R-associated JAK1 and JAK3 augment phosphorylation of GMCSFR-linked STAT5. The functional relevance of these synergistic JAK/STAT transphosphorylation events translates to an increased mitogenic response by GMCSFR/IL9R-expressing primary marrow mast cells. The notion of inducing heterologous receptor clustering by engineered fusokines such as GIFT9 opens the door to a novel type of biopharmaceutical platform where designer fusokines modulate cell physiology through clustering of targeted receptor complexes.

  14. Expression,purification and cell penetrativity of fusion protein PDT/GR-ΔLBD

    Directory of Open Access Journals (Sweden)

    Fang ZHANG

    2011-01-01

    Full Text Available Objective To construct the fusion gene expression vector of penetrating peptide(PDT and the glucocorticoid receptor lack of ligand binding domain(GR-ΔLBD,and evaluate the prokaryotic expression,purification and cell penetrativity of fusion protein PDT/GR-ΔLBD.Methods The target gene fragment GR-ΔLBD was obtained from plasmid pEGFP-GR-ΔLBD by double digestion,and sub-cloned into the prokaryotic expression vector pGEX-PDT to construct the fusion gene expression vector pGEX-PDT/GR-ΔLBD.PDT/GR-ΔLBD fusion protein was obtained after the expression vector was transformed into E.coli,followed by sequential induction with IPTG,treatment with glutathione-agarose resin and elution with glutathione.SDS-PAGE was performed to determine the expression of PDT/GR-ΔLBD fusion protein,and it which was diluted into a final concentration of 0,500 and 1000nmol/L,labeled with fluorescein FITC and co-cultivated with TC-1 cells for 2 hours,and the penetrativity was observed by fluorescence microscopy.Results The successfully constructed prokaryotic expression vector pPDT/GR-ΔLBD had the capacity of expressing protein,and it was 78.6kD in molecular weight,which was consistent with the theoretical value(80kD of the fusion protein PDT/GR-ΔLBD.PDT-GR-ΔLBD,penetrating the nuclear membrane in a concentration-dependent manner,was concentrated within nuclei.Conclusion PDT/GR-ΔLBD fusion protein,with good solubility and cell penetrativity,paves the way for further research on its anti-inflammatory effects.

  15. ROS1 protein-tyrosine kinase inhibitors in the treatment of ROS1 fusion protein-driven non-small cell lung cancers.

    Science.gov (United States)

    Roskoski, Robert

    2017-07-01

    ROS1 protein-tyrosine kinase fusion proteins are expressed in 1-2% of non-small cell lung cancers. The ROS1 fusion partners include CD74, CCDC6, EZR, FIG, KDELR2, LRIG3, MSN, SDC4, SLC34A2, TMEM106B, TMP3, and TPD52L1. Physiological ROS1 is closely related to the ALK, LTK, and insulin receptor protein-tyrosine kinases. ROS1 is a so-called orphan receptor because the identity of its activating ligand, if any, is unknown. The receptor is expressed during development, but little is expressed in adults and its physiological function is unknown. The human ROS1 gene encodes 2347 amino acid residues and ROS1 is the largest protein-tyrosine kinase receptor protein. Unlike the ALK fusion proteins that are activated by the dimerization induced by their amino-terminal portions, the amino-terminal domains of several of its fusion proteins including CD74 apparently lack the ability to induce dimerization so that the mechanism of constitutive protein kinase activation is unknown. Downstream signaling from the ROS1 fusion protein leads to the activation of the Ras/Raf/MEK/ERK1/2 cell proliferation module, the phosphatidyl inositol 3-kinase cell survival pathway, and the Vav3 cell migration pathway. Moreover, several of the ROS1 fusion proteins are implicated in the pathogenesis of a very small proportion of other cancers including glioblastoma, angiosarcoma, and cholangiocarcinoma as well as ovarian, gastric, and colorectal carcinomas. The occurrence of oncogenic ROS1 fusion proteins, particularly in non-small cell lung cancer, has fostered considerable interest in the development of ROS1 inhibitors. Although the percentage of lung cancers driven by ROS1 fusion proteins is low, owing to the large number of new cases of non-small cell lung cancer per year, the number of new cases of ROS1-positive lung cancers is significant and ranges from 2000 to 4000 per year in the United States and 10,000-15,000 worldwide. Crizotinib was the first inhibitor approved by the US Food and Drug

  16. A specific flagellum beating mode for inducing fusion in mammalian fertilization and kinetics of sperm internalization

    Science.gov (United States)

    Ravaux, Benjamin; Garroum, Nabil; Perez, Eric; Willaime, Hervé; Gourier, Christine

    2016-01-01

    The salient phases of fertilization are gamete adhesion, membrane fusion, and internalization of the spermatozoon into the oocyte but the precise timeline and the molecular, membrane and cell mechanisms underlying these highly dynamical events are far from being established. The high motility of the spermatozoa and the unpredictable location of sperm/egg fusion dramatically hinder the use of real time imaging optical techniques that should directly provide the dynamics of cell events. Using an approach based on microfluidics technology, the sperm/egg interaction zone was imaged with the best front view, and the timeline of the fertilization events was established with an unparalleled temporal accuracy from the onset of gamete contact to full sperm DNA decondensation. It reveals that a key element of the adhesion phase to initiate fusion is the oscillatory motion of the sperm head on the oocyte plasma membrane generated by a specific flagellum-beating mode. It also shows that the incorporation of the spermatozoon head is a two steps process that includes simultaneous diving, tilt, and plasma membrane degradation of the sperm head into the oocyte and subsequent DNA decondensation. PMID:27539564

  17. Horizontal gene transfers with or without cell fusions in all categories of the living matter.

    Science.gov (United States)

    Sinkovics, Joseph G

    2011-01-01

    This article reviews the history of widespread exchanges of genetic segments initiated over 3 billion years ago, to be part of their life style, by sphero-protoplastic cells, the ancestors of archaea, prokaryota, and eukaryota. These primordial cells shared a hostile anaerobic and overheated environment and competed for survival. "Coexist with, or subdue and conquer, expropriate its most useful possessions, or symbiose with it, your competitor" remain cellular life's basic rules. This author emphasizes the role of viruses, both in mediating cell fusions, such as the formation of the first eukaryotic cell(s) from a united crenarchaeon and prokaryota, and the transfer of host cell genes integrated into viral (phages) genomes. After rising above the Darwinian threshold, rigid rules of speciation and vertical inheritance in the three domains of life were established, but horizontal gene transfers with or without cell fusions were never abolished. The author proves with extensive, yet highly selective documentation, that not only unicellular microorganisms, but the most complex multicellular entities of the highest ranks resort to, and practice, cell fusions, and donate and accept horizontally (laterally) transferred genes. Cell fusions and horizontally exchanged genetic materials remain the fundamental attributes and inherent characteristics of the living matter, whether occurring accidentally or sought after intentionally. These events occur to cells stagnating for some 3 milliard years at a lower yet amazingly sophisticated level of evolution, and to cells achieving the highest degree of differentiation, and thus functioning in dependence on the support of a most advanced multicellular host, like those of the human brain. No living cell is completely exempt from gene drains or gene insertions.

  18. Nuclear envelope breakdown induced by herpes simplex virus type 1 involves the activity of viral fusion proteins

    Energy Technology Data Exchange (ETDEWEB)

    Maric, Martina; Haugo, Alison C. [Department of Microbiology, University of Iowa, Iowa City, IA 52242 (United States); Dauer, William [Department of Neurology, University of Michigan, Ann Arbor, MI 48109 (United States); Johnson, David [Department of Microbiology and Immunology, Oregon Health Sciences University, Portland, OR 97201 (United States); Roller, Richard J., E-mail: richard-roller@uiowa.edu [Department of Microbiology, University of Iowa, Iowa City, IA 52242 (United States)

    2014-07-15

    Herpesvirus infection reorganizes components of the nuclear lamina usually without loss of integrity of the nuclear membranes. We report that wild-type HSV infection can cause dissolution of the nuclear envelope in transformed mouse embryonic fibroblasts that do not express torsinA. Nuclear envelope breakdown is accompanied by an eight-fold inhibition of virus replication. Breakdown of the membrane is much more limited during infection with viruses that lack the gB and gH genes, suggesting that breakdown involves factors that promote fusion at the nuclear membrane. Nuclear envelope breakdown is also inhibited during infection with virus that does not express UL34, but is enhanced when the US3 gene is deleted, suggesting that envelope breakdown may be enhanced by nuclear lamina disruption. Nuclear envelope breakdown cannot compensate for deletion of the UL34 gene suggesting that mixing of nuclear and cytoplasmic contents is insufficient to bypass loss of the normal nuclear egress pathway. - Highlights: • We show that wild-type HSV can induce breakdown of the nuclear envelope in a specific cell system. • The viral fusion proteins gB and gH are required for induction of nuclear envelope breakdown. • Nuclear envelope breakdown cannot compensate for deletion of the HSV UL34 gene.

  19. Cells with dysfunctional telomeres are susceptible to reactive oxygen species hydrogen peroxide via generation of multichromosomal fusions and chromosomal fragments bearing telomeres

    Energy Technology Data Exchange (ETDEWEB)

    Woo, Seon Rang [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Department of Biochemistry, College of Medicine, Korea University, Seoul 136-705 (Korea, Republic of); Park, Jeong-Eun; Juhn, Kyoung-Mi; Ju, Yeun-Jin; Jeong, Jaemin [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Kang, Chang-Mo; Yun, Hyun Jin [Division of Radiation Effect, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Yun, Mi Yong; Shin, Hyun-Jin; Joo, Hyun-Yoo; Park, Eun-Ran; Park, In-Chul; Hong, Sung Hee; Hwang, Sang-Gu [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Kim, Haekwon [Department of Biotechnology, Seoul Woman' s University, Seoul 139-774 (Korea, Republic of); Cho, Myung-Haing [Laboratory of Toxicology, College of Veterinary Medicine, Seoul National University, Seoul 151-742 (Korea, Republic of); Kim, Sang Hoon [Department of Biology, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Park, Gil Hong [Department of Biochemistry, College of Medicine, Korea University, Seoul 136-705 (Korea, Republic of); Lee, Kee-Ho, E-mail: khlee@kirams.re.kr [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Under conditions of telomere erosion, cells become extremely sensitive to H{sub 2}O{sub 2}. Black-Right-Pointing-Pointer Chromosomal regions adjacent to telomeres are cleaved by H{sub 2}O{sub 2} under such conditions. Black-Right-Pointing-Pointer H{sub 2}O{sub 2} thus causes multichromosomal fusions and generation of small chromosomal fragments. Black-Right-Pointing-Pointer N-acetylcysteine prevents H{sub 2}O{sub 2}-induced chromosomal aberrations. -- Abstract: During genotoxic stress, reactive oxygen species hydrogen peroxide (H{sub 2}O{sub 2}) is a prime mediator of the DNA damage response. Telomeres function both to assist in DNA damage repair and to inhibit chromosomal end-to-end fusion. Here, we show that telomere dysfunction renders cells susceptible to H{sub 2}O{sub 2}, via generation of multichromosomal fusion and chromosomal fragments. H{sub 2}O{sub 2} caused formation of multichromosomal end-to-end fusions involving more than three chromosomes, preferentially when telomeres were erosive. Interestingly, extensive chromosomal fragmentation (yielding small-sized fragments) occurred only in cells exhibiting such multichromosomal fusions. Telomeres were absent from fusion points, being rather present in the small fragments, indicating that H{sub 2}O{sub 2} cleaves chromosomal regions adjacent to telomeres. Restoration of telomere function or addition of the antioxidant N-acetylcysteine prevented development of chromosomal aberrations and rescued the observed hypersensitivity to H{sub 2}O{sub 2}. Thus, chromosomal regions adjacent to telomeres become sensitive to reactive oxygen species hydrogen peroxide when telomeres are dysfunctional, and are cleaved to produce multichromosomal fusions and small chromosomal fragments bearing the telomeres.

  20. Radioactivity and fusion energy

    International Nuclear Information System (INIS)

    Kudo, H.

    1995-01-01

    Nuclear fusion is expected to give an ultimate solution to energy problems over the long term. From recent progress in developing technology for fusion reactors, we can anticipate a prototype fusion reactor by 2030. This review article describes the present status of nuclear fusion research, including muon catalyzed fusion (μCF) which attracts quite new physical interest. Tritium is an essential component of fusion reactors, because the first-stage fusion reactors will utilize a mixture of deuterium and tritium as their fuel. The knowledge about tritium as well as the fusion-neutron induced radioactivity is summarized in terms of nuclear fusion research. (orig.)

  1. Identification of target genes of synovial sarcoma-associated fusion oncoprotein using human pluripotent stem cells.

    Science.gov (United States)

    Hayakawa, Kazuo; Ikeya, Makoto; Fukuta, Makoto; Woltjen, Knut; Tamaki, Sakura; Takahara, Naoko; Kato, Tomohisa; Sato, Shingo; Otsuka, Takanobu; Toguchida, Junya

    2013-03-22

    Synovial sarcoma (SS) is a malignant soft tissue tumor harboring chromosomal translocation t(X; 18)(p11.2; q11.2), which produces SS-specific fusion gene, SYT-SSX. Although precise function of SYT-SSX remains to be investigated, accumulating evidences suggest its role in gene regulation via epigenetic mechanisms, and the product of SYT-SSX target genes may serve as biomarkers of SS. Lack of knowledge about the cell-of-origin of SS, however, has placed obstacle in the way of target identification. Here we report a novel approach to identify SYT-SSX2 target genes using human pluripotent stem cells (hPSCs) containing a doxycycline-inducible SYT-SSX2 gene. SYT-SSX2 was efficiently induced both at mRNA and protein levels within three hours after doxycycline administration, while no morphological change of hPSCs was observed until 24h. Serial microarray analyses identified genes of which the expression level changed more than twofold within 24h. Surprisingly, the majority (297/312, 95.2%) were up-regulated genes and a result inconsistent with the current concept of SYT-SSX as a transcriptional repressor. Comparing these genes with SS-related genes which were selected by a series of in silico analyses, 49 and 2 genes were finally identified as candidates of up- and down-regulated target of SYT-SSX, respectively. Association of these genes with SYT-SSX in SS cells was confirmed by knockdown experiments. Expression profiles of SS-related genes in hPSCs and human mesenchymal stem cells (hMSCs) were strikingly different in response to the induction of SYT-SSX, and more than half of SYT-SSX target genes in hPSCs were not induced in hMSCs. These results suggest the importance of cellular context for correct understanding of SYT-SSX function, and demonstrated how our new system will help to overcome this issue. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Accumulation of specific sterol precursors targets a MAP kinase cascade mediating cell–cell recognition and fusion

    Science.gov (United States)

    Weichert, Martin; Lichius, Alexander; Priegnitz, Bert-Ewald; Brandt, Ulrike; Gottschalk, Johannes; Nawrath, Thorben; Groenhagen, Ulrike; Read, Nick D.; Schulz, Stefan; Fleißner, André

    2016-01-01

    Sterols are vital components of eukaryotic cell membranes. Defects in sterol biosynthesis, which result in the accumulation of precursor molecules, are commonly associated with cellular disorders and disease. However, the effects of these sterol precursors on the metabolism, signaling, and behavior of cells are only poorly understood. In this study, we show that the accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain specifically disrupts cell–cell communication and fusion in the fungus Neurospora crassa. Genetically identical germinating spores of this fungus undergo cell–cell fusion, thereby forming a highly interconnected supracellular network during colony initiation. Before fusion, the cells use an unusual signaling mechanism that involves the coordinated and alternating switching between signal sending and receiving states of the two fusion partners. Accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain disrupts this coordinated cell–cell communication and suppresses cell fusion. These specific sterol precursors target a single ERK-like mitogen-activated protein (MAP) kinase (MAK-1)-signaling cascade, whereas a second MAP kinase pathway (MAK-2), which is also involved in cell fusion, is unaffected. These observations indicate that a minor specific change in sterol structure can exert a strong detrimental effect on a key signaling pathway of the cell, resulting in the absence of cell fusion. PMID:27708165

  3. Detection of E2A-PBX1 fusion transcripts in human non-small-cell lung cancer

    OpenAIRE

    Mo, Min-Li; Chen, Zhao; Zhou, Hai-Meng; Li, Hui; Hirata, Tomomi; Jablons, David M; He, Biao

    2013-01-01

    Background E2A-PBX1 fusion gene caused by t(1;19)(q23;p13), has been well characterized in acute lymphoid leukemia (ALL). There is no report on E2A-PBX1 fusion transcripts in non-small-cell lung cancer (NSCLC). Methods We used polymerase chain reaction (PCR) to detect E2A-PBX1 fusion transcripts in human NSCLC tissue specimens and cell lines. We analyzed correlation of E2A-PBX1 fusion transcripts with clinical outcomes in 76 patients with adenocarcinoma in situ (AIS) and other subgroups. We c...

  4. Discovery of a fusion kinase in EOL-1 cells and idiopathic hypereosinophilic syndrome.

    Science.gov (United States)

    Griffin, John H; Leung, Joey; Bruner, Rebecca J; Caligiuri, Michael A; Briesewitz, Roger

    2003-06-24

    Idiopathic hypereosinophilic syndrome (HES) is a myeloproliferative disease of unknown etiology. Recently, it has been reported that imatinib mesylate (Gleevec), an inhibitor of Bcr-Abl kinase useful in the treatment of chronic myeloid leukemia, is also effective in treating HES; however, the molecular target of imatinib in HES is unknown. This report identifies a genetic rearrangement in the eosinophilic cell line EOL-1 that results in the expression of a fusion protein comprising an N-terminal region encoded by a gene of unknown function with the GenBank accession number NM_030917 and a C-terminal region derived from the intracellular domain of the platelet-derived growth factor receptor alpha (PDGFRalpha). The fusion gene was also detected in blood cells from two patients with HES. We propose naming NM_030917 Rhe for Rearranged in hypereosinophilia. Rhe-PDGFRalpha fusions result from an apparent interstitial deletion that links Rhe to exon 12 of PDGFRalpha on chromosome 4q12. The fusion kinase Rhe-PDGFRalpha is constitutively phosphorylated and supports IL-3-independent growth when expressed in BaF3 cells. Proliferation and viability of EOL-1 and BaF3 cells expressing Rhe-PDGFRalpha are ablated by the PDGFRalpha inhibitors imatinib, vatalanib, and THRX-165724.

  5. A Unique Opportunity to Test Whether Cell Fusion is a Mechanism of Breast Cancer Metastasis

    Science.gov (United States)

    2013-07-01

    We and others13, 74, 141 argue that a “ sexual ” method of reproduction – such as cell fusion – that allows for genetic recombination offers a more...2007;282(20):14906-15. 8. Wu M, Jung L, Cooper AB, Fleet C, Chen L, Breault L, Clark K, Cai Z, Vincent S, Bottega S. Dissecting genetic

  6. Engineering spinal fusion: evaluating ceramic materials for cell based tissue engineered approaches

    NARCIS (Netherlands)

    Wilson, C.E.

    2011-01-01

    The principal aim of this thesis was to advance the development of tissue engineered posterolateral spinal fusion by investigating the potential of calcium phosphate ceramic materials to support cell based tissue engineered bone formation. This was accomplished by developing several novel model

  7. Adenovirus-Mediated Expression of the p14 Fusion-Associated Small Transmembrane Protein Promotes Cancer Cell Fusion and Apoptosis In Vitro but Does Not Provide Therapeutic Efficacy in a Xenograft Mouse Model of Cancer.

    Directory of Open Access Journals (Sweden)

    Carmen M Wong

    Full Text Available Adenoviruses (Ads are used in numerous preclinical and clinical studies for delivery of anti-cancer therapeutic genes. Unfortunately, Ad has a poor ability to distribute throughout a tumor mass after intratumoral injection, and infects cells primarily within the immediate area of the injection tract. Thus, Ad-encoded transgene expression is typically limited to only a small percentage of cells within the tumor. One method to increase the proportion of the tumor impacted by Ad is through expression of fusogenic proteins. Infection of a single cell with an Ad vector encoding a fusogenic protein should lead to syncytium formation with adjacent cells, effectively spreading the effect of Ad and Ad-encoded therapeutic transgenes to a greater percentage of the tumor mass. Moreover, syncytium formation can be cytotoxic, suggesting that such proteins may be effective sole therapeutics. We show that an early region 1 (E1-deleted Ad expressing reptilian reovirus p14 fusion-associated small transmembrane (FAST protein caused extensive cell fusion in the replication-permissive 293 cell line and at high multiplicity of infection in non-permissive human lung adenocarcinoma A549 cells in vitro. FAST protein expression in the A549 cancer cell line led to a loss of cellular metabolic activity and membrane integrity, which correlated with induction of apoptosis. However, in an A549 xenograft CD-1 nude mouse cancer model, Ad-mediated FAST gene delivery did not induce detectable cell fusion, reduce tumor burden nor enhance mouse survival compared to controls. Taken together, our results show that, although AdFAST can enhance cancer cell killing in vitro, it is not effective as a sole therapeutic in the A549 tumor model in vivo.

  8. Exceptionally potent anti-tumor bystander activity of an scFv : sTRAIL fusion protein with specificity for EGP2 toward target antigen-negative tumor cells

    NARCIS (Netherlands)

    Bremer, E; Samplonius, D; Kroesen, BJ; van Genne, L; de Leij, L; Helfrich, W

    2004-01-01

    Previously, we reported on the target cell-restricted fratricide apoptotic activity of scFvC54:sTRAIL, a fusion protein comprising human-soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) genetically linked to the antibody fragment scFvC54 specific for the cell surface target

  9. Fusions of Breast Carcinoma and Dendritic Cells as a Vaccine for the Treatment of Metastatic Breast Cancer

    Science.gov (United States)

    2005-07-01

    AD Award Number: DAMD17-03-1-0487 TITLE: Fusions of Breast Carcinoma and Dendritic Cells as a Vaccine for the Treatment of Metastatic Breast Cancer...4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Fusions of Breast Carcinoma and Dendritic Cells as a Vaccine for the Treatment of Metastatic Breast Cancer... Borras -Cuesta, F., and Lasarte, J. J. CD4+/CD25+ regulatory cells inhibit activation of tumor-primed CD4+ T cells with IFN- gamma-dependent

  10. Research Progress in Non-small Cell Lung Cancer 
with Concomitant EML4-ALK Fusion Gene and EGFR Gene Mutation

    Directory of Open Access Journals (Sweden)

    Zhu ZENG

    2011-11-01

    Full Text Available Lung cancer is one of the most prevalence malignances, of which non-small cell lung cancer (NSCLC account for 80%-85% of lung cancer. Molecular target therapy is one of the most popular and promising field of NSCLC treatment, and its hotspots includes EGFR (epidermal growth factor receptor, EML4-ALK (echinoderm microtubule associated protein like4-anaplastic lymphoma kinase, etc. Former researches indicated that EML4-ALK fusion and EGFR mutation were excluded mutually. However, cases of patients harbored concomitant EML4-ALK fusion gene and EGFR mutation have been reported continuously at recent. This review aims to summarize the incidence and molecular structure of EML4-ALK fusion gene and EGFR mutation, as well as clinical features of patients with the concomitant genes induced NSCLC.

  11. A New Target in Non-small Cell Lung Cancer: EML4-ALK Fusion Gene

    Directory of Open Access Journals (Sweden)

    Huijuan WANG

    2011-06-01

    Full Text Available It was only 3 years ago that the fusion gene between echinoderm microtubule-associated protein-like4 (EML4 and anaplastic lymphoma kinase (ALK has been identified in a subset of non-small cell lung cancer (NSCLC. EML4-ALK is most often detected in never smokers with lung adenocarcinoma and has unique pathologic features. EML4-ALK fusion gene is oncogenic, which could be suppressed by ALK-inhibitor through blocking the downstream signaling passway of EML4-ALK. This review will focus on the molecular structure, function, biology, detection method and the diagnostic and therapeutic meaning of EML4-ALK of lung cancer.

  12. ChimPipe: accurate detection of fusion genes and transcription-induced chimeras from RNA-seq data.

    Science.gov (United States)

    Rodríguez-Martín, Bernardo; Palumbo, Emilio; Marco-Sola, Santiago; Griebel, Thasso; Ribeca, Paolo; Alonso, Graciela; Rastrojo, Alberto; Aguado, Begoña; Guigó, Roderic; Djebali, Sarah

    2017-01-03

    Chimeric transcripts are commonly defined as transcripts linking two or more different genes in the genome, and can be explained by various biological mechanisms such as genomic rearrangement, read-through or trans-splicing, but also by technical or biological artefacts. Several studies have shown their importance in cancer, cell pluripotency and motility. Many programs have recently been developed to identify chimeras from Illumina RNA-seq data (mostly fusion genes in cancer). However outputs of different programs on the same dataset can be widely inconsistent, and tend to include many false positives. Other issues relate to simulated datasets restricted to fusion genes, real datasets with limited numbers of validated cases, result inconsistencies between simulated and real datasets, and gene rather than junction level assessment. Here we present ChimPipe, a modular and easy-to-use method to reliably identify fusion genes and transcription-induced chimeras from paired-end Illumina RNA-seq data. We have also produced realistic simulated datasets for three different read lengths, and enhanced two gold-standard cancer datasets by associating exact junction points to validated gene fusions. Benchmarking ChimPipe together with four other state-of-the-art tools on this data showed ChimPipe to be the top program at identifying exact junction coordinates for both kinds of datasets, and the one showing the best trade-off between sensitivity and precision. Applied to 106 ENCODE human RNA-seq datasets, ChimPipe identified 137 high confidence chimeras connecting the protein coding sequence of their parent genes. In subsequent experiments, three out of four predicted chimeras, two of which recurrently expressed in a large majority of the samples, could be validated. Cloning and sequencing of the three cases revealed several new chimeric transcript structures, 3 of which with the potential to encode a chimeric protein for which we hypothesized a new role. Applying ChimPipe to

  13. Expression of a preproinsulin-beta-galactosidase gene fusion in mammalian cells.

    OpenAIRE

    Nielsen, D A; Chou, J; MacKrell, A J; Casadaban, M J; Steiner, D F

    1983-01-01

    As an approach to the study of mammalian gene expression, the promoters and translation initiation regions of the rat preproinsulin II and the simian virus 40 early genes were fused to the structural gene of Escherichia coli beta-galactosidase, a sensitive probe for gene expression. These fusions were introduced into COS-7 cells, a simian virus 40 large tumor-antigen-producing monkey kidney cell line, where they directed the synthesis of enzymatically active hybrid beta-galactosidase proteins...

  14. Autoprocessing of human immunodeficiency virus type 1 protease miniprecursor fusions in mammalian cells

    Directory of Open Access Journals (Sweden)

    Chen Chaoping

    2010-07-01

    Full Text Available Abstract Background HIV protease (PR is a virus-encoded aspartic protease that is essential for viral replication and infectivity. The fully active and mature dimeric protease is released from the Gag-Pol polyprotein as a result of precursor autoprocessing. Results We here describe a simple model system to directly examine HIV protease autoprocessing in transfected mammalian cells. A fusion precursor was engineered encoding GST fused to a well-characterized miniprecursor, consisting of the mature protease along with its upstream transframe region (TFR, and small peptide epitopes to facilitate detection of the precursor substrate and autoprocessing products. In HEK 293T cells, the resulting chimeric precursor undergoes effective autoprocessing, producing mature protease that is rapidly degraded likely via autoproteolysis. The known protease inhibitors Darunavir and Indinavir suppressed both precursor autoprocessing and autoproteolysis in a dose-dependent manner. Protease mutations that inhibit Gag processing as characterized using proviruses also reduced autoprocessing efficiency when they were introduced to the fusion precursor. Interestingly, autoprocessing of the fusion precursor requires neither the full proteolytic activity nor the majority of the N-terminal TFR region. Conclusions We suggest that the fusion precursors provide a useful system to study protease autoprocessing in mammalian cells, and may be further developed for screening of new drugs targeting HIV protease autoprocessing.

  15. Augmentation of antitumor immunity by fusions of ethanol-treated tumor cells and dendritic cells stimulated via dual TLRs through TGF-β1 blockade and IL-12p70 production.

    Directory of Open Access Journals (Sweden)

    Shigeo Koido

    Full Text Available The therapeutic efficacy of fusion cell (FC-based cancer vaccine generated with whole tumor cells and dendritic cells (DCs requires the improved immunogenicity of both cells. Treatment of whole tumor cells with ethanol resulted in blockade of immune-suppressive soluble factors such as transforming growth factor (TGF-β1, vascular endothelial growth factor, and IL-10 without decreased expression of major histocompatibility complex (MHC class I and the MUC1 tumor-associated antigen. Moreover, the ethanol-treated tumor cells expressed "eat-me" signals such as calreticulin (CRT on the cell surface and released immunostimulatory factors such as heat shock protein (HSP90α and high-mobility group box 1 (HMGB1. A dual stimulation of protein-bound polysaccharides isolated from Coriolus versicolor (TLR2 agonist and penicillin-inactivated Streptococcus pyogenes (TLR4 agonist led human monocyte-derived DCs to produce HSP90α and multiple cytokines such as IL-12p70 and IL-10. Interestingly, incorporating ethanol-treated tumor cells and TLRs-stimulated DCs during the fusion process promoted fusion efficiency and up-regulated MHC class II molecules on a per fusion basis. Moreover, fusions of ethanol-treated tumor cells and dual TLRs-stimulated DCs (E-tumor/FCs inhibited the production of multiple immune-suppressive soluble factors including TGF-β1 and up-regulated the production of IL-12p70 and HSP90α. Most importantly, E-tumor/FCs activated T cells capable of producing high levels of IFN-γ, resulting in augmented MUC1-specific CTL induction. Collectively, our results illustrate the synergy between ethanol-treated whole tumor cells and dual TLRs-stimulated DCs in inducing augmented CTL responses in vitro by FC preparations. The alternative system is simple and may provide a platform for adoptive immunotherapy.

  16. Tritium production in He-3 gas cells immersed in the Tokamak Fusion Test Reactor neutron field

    Energy Technology Data Exchange (ETDEWEB)

    Jassby, D.L.; Gentile, C.A.; Ascione, G.; Kugel, H.W.; Roquemore, A.L. [Princeton Plasma Physics Laboratory, Princeton, New Jersey 08543 (United States); Kumar, A. [University of California, Los Angeles, California 90024 (United States)

    1999-01-01

    Tritium generated in an external cell by the reaction {sup 3}He(n,p)T can be used as a gauge of long-term fusion neutron production, because of the 12-year half-life of T and the relative ease of measuring the T content either by sampling or from the saturation current of the cell when operated as an ionization chamber. Two high-pressure {sup 3}He gas cells enclosed in polyethylene neutron moderators were exposed to Tokamak Fusion Test Reactor (TFTR) neutrons during high-power D{endash}T operation. The tritium produced in the cells was assayed by the Princeton Differential Atmospheric Tritium Sampler. The measured tritium generated per 10{sup 19} fusion neutrons was 510 pCi/cc at 2.3 m from the TFTR vessel and 1.3 m below the midplane, and 2020 pCi/cc at 1.0 m from the TFTR vessel in the midplane. Combining these results with previous measurements at a third location, we found 0.11 to 0.23 triton produced per neutron incident on the projected cell cross section, with an asymptotic local tritium breeding ratio of 0.32. {copyright} {ital 1999 American Institute of Physics.}

  17. Nuclear localization and transactivating capacities of the papillary renal cell carcinoma-associated TFE3 and PRCC (fusion) proteins

    NARCIS (Netherlands)

    Weterman, M. A. J.; van Groningen, J. J.; Jansen, A.; van Kessel, A. G.

    2000-01-01

    The papillary renal cell carcinoma-associated t(X;1)(p11;q21) leads to fusion of the transcription factor TFE3 gene on the X-chromosome to a novel gene, PRCC, on chromosome 1. As a result, two putative fusion proteins are formed: PRCCTFE3, which contains all known domains for DNA binding,

  18. Molecular alterations in tumorigenic human bronchial and breast epithelial cells induced by high let radiation

    Science.gov (United States)

    Hei, T. K.; Zhao, Y. L.; Roy, D.; Piao, C. Q.; Calaf, G.; Hall, E. J.

    Carcinogenesis is a multi-stage process with sequence of genetic events governing the phenotypic expression of a series of transformation steps leading to the development of metastatic cancer. In the present study, immortalized human bronchial (BEP2D) and breast (MCF-10F) cells were irradiated with graded doses of either 150 keV/μm alpha particles or 1 GeV/nucleon 56Fe ions. Transformed cells developed through a series of successive steps before becoming tumorigenic in nude mice. Cell fusion studies indicated that radiation-induced tumorigenic phenotype in BEP2D cells could be completely suppressed by fusion with non-tumorigenic BEP2D cells. The differential expressions of known genes between tumorigenic bronchial and breast cells induced by alpha particles and their respective control cultures were compared using cDNA expression array. Among the 11 genes identified to be differentially expressed in BEP2D cells, three ( DCC, DNA-PK and p21 CIPI) were shown to be consistently down-regulated by 2 to 4 fold in all the 5 tumor cell lines examined. In contrast, their expressions in the fusion cell lines were comparable to control BEP2D cells. Similarly, expression levels of a series of genes were found to be altered in a step-wise manner among tumorigenic MCF-10F cells. The results are highly suggestive that functional alterations of these genes may be causally related to the carcinogenic process.

  19. Tipping the balance: robustness of tip cell selection, migration and fusion in angiogenesis.

    Directory of Open Access Journals (Sweden)

    Katie Bentley

    2009-10-01

    Full Text Available Vascular abnormalities contribute to many diseases such as cancer and diabetic retinopathy. In angiogenesis new blood vessels, headed by a migrating tip cell, sprout from pre-existing vessels in response to signals, e.g., vascular endothelial growth factor (VEGF. Tip cells meet and fuse (anastomosis to form blood-flow supporting loops. Tip cell selection is achieved by Dll4-Notch mediated lateral inhibition resulting, under normal conditions, in an interleaved arrangement of tip and non-migrating stalk cells. Previously, we showed that the increased VEGF levels found in many diseases can cause the delayed negative feedback of lateral inhibition to produce abnormal oscillations of tip/stalk cell fates. Here we describe the development and implementation of a novel physics-based hierarchical agent model, tightly coupled to in vivo data, to explore the system dynamics as perpetual lateral inhibition combines with tip cell migration and fusion. We explore the tipping point between normal and abnormal sprouting as VEGF increases. A novel filopodia-adhesion driven migration mechanism is presented and validated against in vivo data. Due to the unique feature of ongoing lateral inhibition, 'stabilised' tip/stalk cell patterns show sensitivity to the formation of new cell-cell junctions during fusion: we predict cell fates can reverse. The fusing tip cells become inhibited and neighbouring stalk cells flip fate, recursively providing new tip cells. Junction size emerges as a key factor in establishing a stable tip/stalk pattern. Cell-cell junctions elongate as tip cells migrate, which is shown to provide positive feedback to lateral inhibition, causing it to be more susceptible to pathological oscillations. Importantly, down-regulation of the migratory pathway alone is shown to be sufficient to rescue the sprouting system from oscillation and restore stability. Thus we suggest the use of migration inhibitors as therapeutic agents for vascular

  20. Osteoclast Fusion

    DEFF Research Database (Denmark)

    Marie Julie Møller, Anaïs; Delaissé, Jean-Marie; Søe, Kent

    2017-01-01

    Investigations addressing the molecular keys of osteoclast fusion are primarily based on end-point analyses. No matter if investigations are performed in vivo or in vitro the impact of a given factor is predominantly analyzed by counting the number of multi-nucleated cells, the number of nuclei per...... multinucleated cell or TRAcP activity. But end-point analyses do not show how the fusion came about. This would not be a problem if fusion of osteoclasts was a random process and occurred by the same molecular mechanism from beginning to end. However, we and others have in the recent period published data...... suggesting that fusion partners may specifically select each other and that heterogeneity between the partners seems to play a role. Therefore, we set out to directly test the hypothesis that fusion factors have a heterogenic involvement at different stages of nuclearity. Therefore, we have analyzed...

  1. Discovery of CTCF-sensitive Cis-spliced fusion RNAs between adjacent genes in human prostate cells.

    Science.gov (United States)

    Qin, Fujun; Song, Zhenguo; Babiceanu, Mihaela; Song, Yansu; Facemire, Loryn; Singh, Ritambhara; Adli, Mazhar; Li, Hui

    2015-02-01

    Genes or their encoded products are not expected to mingle with each other unless in some disease situations. In cancer, a frequent mechanism that can produce gene fusions is chromosomal rearrangement. However, recent discoveries of RNA trans-splicing and cis-splicing between adjacent genes (cis-SAGe) support for other mechanisms in generating fusion RNAs. In our transcriptome analyses of 28 prostate normal and cancer samples, 30% fusion RNAs on average are the transcripts that contain exons belonging to same-strand neighboring genes. These fusion RNAs may be the products of cis-SAGe, which was previously thought to be rare. To validate this finding and to better understand the phenomenon, we used LNCaP, a prostate cell line as a model, and identified 16 additional cis-SAGe events by silencing transcription factor CTCF and paired-end RNA sequencing. About half of the fusions are expressed at a significant level compared to their parental genes. Silencing one of the in-frame fusions resulted in reduced cell motility. Most out-of-frame fusions are likely to function as non-coding RNAs. The majority of the 16 fusions are also detected in other prostate cell lines, as well as in the 14 clinical prostate normal and cancer pairs. By studying the features associated with these fusions, we developed a set of rules: 1) the parental genes are same-strand-neighboring genes; 2) the distance between the genes is within 30kb; 3) the 5' genes are actively transcribing; and 4) the chimeras tend to have the second-to-last exon in the 5' genes joined to the second exon in the 3' genes. We then randomly selected 20 neighboring genes in the genome, and detected four fusion events using these rules in prostate cancer and non-cancerous cells. These results suggest that splicing between neighboring gene transcripts is a rather frequent phenomenon, and it is not a feature unique to cancer cells.

  2. Chromatin-prebound Crm1 recruits Nup98-HoxA9 fusion to induce aberrant expression of Hox cluster genes.

    Science.gov (United States)

    Oka, Masahiro; Mura, Sonoko; Yamada, Kohji; Sangel, Percival; Hirata, Saki; Maehara, Kazumitsu; Kawakami, Koichi; Tachibana, Taro; Ohkawa, Yasuyuki; Kimura, Hiroshi; Yoneda, Yoshihiro

    2016-01-07

    The nucleoporin Nup98 is frequently rearranged to form leukemogenic Nup98-fusion proteins with various partners. However, their function remains largely elusive. Here, we show that Nup98-HoxA9, a fusion between Nup98 and the homeobox transcription factor HoxA9, forms nuclear aggregates that frequently associate with facultative heterochromatin. We demonstrate that stable expression of Nup98-HoxA9 in mouse embryonic stem cells selectively induces the expression of Hox cluster genes. Genome-wide binding site analysis revealed that Nup98-HoxA9 is preferentially targeted and accumulated at Hox cluster regions where the export factor Crm1 is originally prebound. In addition, leptomycin B, an inhibitor of Crm1, disassembled nuclear Nup98-HoxA9 dots, resulting in the loss of chromatin binding of Nup98-HoxA9 and Nup98-HoxA9-mediated activation of Hox genes. Collectively, our results indicate that highly selective targeting of Nup98-fusion proteins to Hox cluster regions via prebound Crm1 induces the formation of higher order chromatin structures that causes aberrant Hox gene regulation.

  3. NMR structure and localization of a large fragment of the SARS-CoV fusion protein: Implications in viral cell fusion.

    Science.gov (United States)

    Mahajan, Mukesh; Chatterjee, Deepak; Bhuvaneswari, Kannaian; Pillay, Shubhadra; Bhattacharjya, Surajit

    2018-02-01

    The lethal Coronaviruses (CoVs), Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV) and most recently Middle East Respiratory Syndrome Coronavirus, (MERS-CoV) are serious human health hazard. A successful viral infection requires fusion between virus and host cells carried out by the surface spike glycoprotein or S protein of CoV. Current models propose that the S2 subunit of S protein assembled into a hexameric helical bundle exposing hydrophobic fusogenic peptides or fusion peptides (FPs) for membrane insertion. The N-terminus of S2 subunit of SARS-CoV reported to be active in cell fusion whereby FPs have been identified. Atomic-resolution structure of FPs derived either in model membranes or in membrane mimic environment would glean insights toward viral cell fusion mechanism. Here, we have solved 3D structure, dynamics and micelle localization of a 64-residue long fusion peptide or LFP in DPC detergent micelles by NMR methods. Micelle bound structure of LFP is elucidated by the presence of discretely folded helical and intervening loops. The C-terminus region, residues F42-Y62, displays a long hydrophobic helix, whereas the N-terminus is defined by a short amphipathic helix, residues R4-Q12. The intervening residues of LFP assume stretches of loops and helical turns. The N-terminal helix is sustained by close aromatic and aliphatic sidechain packing interactions at the non-polar face. 15 N{ 1 H}NOE studies indicated dynamical motion, at ps-ns timescale, of the helices of LFP in DPC micelles. PRE NMR showed that insertion of several regions of LFP into DPC micelle core. Together, the current study provides insights toward fusion mechanism of SARS-CoV. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Survival of the fittest: positive selection of CD4+ T cells expressing a membrane-bound fusion inhibitor following HIV-1 infection.

    Science.gov (United States)

    Kimpel, Janine; Braun, Stephen E; Qiu, Gang; Wong, Fay Eng; Conolle, Michelle; Schmitz, Jörn E; Brendel, Christian; Humeau, Laurent M; Dropulic, Boro; Rossi, John J; Berger, Annemarie; von Laer, Dorothee; Johnson, R Paul

    2010-08-23

    Although a variety of genetic strategies have been developed to inhibit HIV replication, few direct comparisons of the efficacy of these inhibitors have been carried out. Moreover, most studies have not examined whether genetic inhibitors are able to induce a survival advantage that results in an expansion of genetically-modified cells following HIV infection. We evaluated the efficacy of three leading genetic strategies to inhibit HIV replication: 1) an HIV-1 tat/rev-specific small hairpin (sh) RNA; 2) an RNA antisense gene specific for the HIV-1 envelope; and 3) a viral entry inhibitor, maC46. In stably transduced cell lines selected such that >95% of cells expressed the genetic inhibitor, the RNA antisense envelope and viral entry inhibitor maC46 provided the strongest inhibition of HIV-1 replication. However, when mixed populations of transduced and untransduced cells were challenged with HIV-1, the maC46 fusion inhibitor resulted in highly efficient positive selection of transduced cells, an effect that was evident even in mixed populations containing as few as 1% maC46-expressing cells. The selective advantage of the maC46 fusion inhibitor was also observed in HIV-1-infected cultures of primary T lymphocytes as well as in HIV-1-infected humanized mice. These results demonstrate robust inhibition of HIV replication with the fusion inhibitor maC46 and the antisense Env inhibitor, and importantly, a survival advantage of cells expressing the maC46 fusion inhibitor both in vitro and in vivo. Evaluation of the ability of genetic inhibitors of HIV-1 replication to confer a survival advantage on genetically-modified cells provides unique information not provided by standard techniques that may be important in the in vivo efficacy of these genes.

  5. VAMP2-NRG1 Fusion Gene is a Novel Oncogenic Driver of Non-Small-Cell Lung Adenocarcinoma.

    Science.gov (United States)

    Jung, Yeonjoo; Yong, Seunghui; Kim, Pora; Lee, Hee-Young; Jung, Yeonhwa; Keum, Juhee; Lee, Sanghyuk; Kim, Jhingook; Kim, Jaesang

    2015-07-01

    Neuregulin 1 (NRG1) has been discovered as the tail moiety of fusion genes with several distinct partner head genes in lung cancers. These fusion genes activate ERBB2/ERBB3 receptor-mediated cell signaling and thereby function as oncogenic drivers. We have carried out whole-transcriptome sequencing of 100 non-small-cell lung carcinoma tumors and isolated a novel fusion gene consisting of vesicle-associated membrane protein 2 (VAMP2) and NRG1. Reverse transcription-polymerase chain reaction and genomic DNA analysis were used to demonstrate interchromosomal translocation. Immunoblotting and soft agar assays were used to examine stimulating activity of the fusion gene through ERBB2/ERBB3 signaling pathway. The most highly expressed splice variant of VAMP2-NRG1 fusion gene was shown to be membrane bound and display EGF-like domain of NRG1 extracellularly. VAMP2-NRG1 promotes anchorage-independent colony formation of H1568 lung adenocarcinoma cells. Ectopic expression of the fusion gene stimulates phosphorylation of ERBB2 and ERBB3 as well as downstream targets, AKT and ERK, confirming activation of the signaling pathway. VAMP2-NRG1 is a novel oncogenic fusion gene representing a new addition to the list of NRG1 fusion genes, which together may form an important diagnostic and clinical category of lung adenocarcinoma cases.

  6. CD4 RAT X RAT AND MOUSE X RAT T-CELL HYBRIDOMAS PRODUCED BY FUSION OF ESTABLISHED T-CELL LINES AND CLONES TO W/FU (C58NT)D

    NARCIS (Netherlands)

    BOOTS, AMH; VANLIEROP, MJ; WAUBEN, MHM; VANKOOTEN, PJS; HENSEN, EJ; VANEDEN, W; Boots, Annemieke

    1991-01-01

    Previously, fusion of established T cell lines or clones has been claimed to be difficult. We now report our experiences in the fusion of both long term cultures of rat T cell clones and mouse T cell lines to rat W/Fu (C58NT)D. Upon fusion of rat T cell clones the hybrids obtained expressed antigen

  7. of Hypoxia-Inducible Factor-1α Activity by the Fusion of High-Resolution SPECT and Morphological Imaging Tests

    Directory of Open Access Journals (Sweden)

    Hirofumi Fujii

    2012-01-01

    Full Text Available Purpose. We aimed to clearly visualize heterogeneous distribution of hypoxia-inducible factor 1α (HIF activity in tumor tissues in vivo. Methods. We synthesized of 125I-IPOS, a 125I labeled chimeric protein probe, that would visualize HIF activity. The biodistribution of 125I-IPOS in FM3A tumor-bearing mice was evaluated. Then, the intratumoral localization of this probe was observed by autoradiography, and it was compared with histopathological findings. The distribution of 125I-IPOS in tumors was imaged by a small animal SPECT/CT scanner. The obtained in vivo SPECT-CT fusion images were compared with ex vivo images of excised tumors. Fusion imaging with MRI was also examined. Results. 125I-IPOS well accumulated in FM3A tumors. The intratumoral distribution of 125I-IPOS by autoradiography was quite heterogeneous, and it partially overlapped with that of pimonidazole. High-resolution SPECT-CT fusion images successfully demonstrated the heterogeneity of 125I-IPOS distribution inside tumors. SPECT-MRI fusion images could give more detailed information about the intratumoral distribution of 125I-IPOS. Conclusion. High-resolution SPECT images successfully demonstrated heterogeneous intratumoral distribution of 125I-IPOS. SPECT-CT fusion images, more favorably SPECT-MRI fusion images, would be useful to understand the features of heterogeneous intratumoral expression of HIF activity in vivo.

  8. TRAIL death receptor 4 signaling via lysosome fusion and membrane raft clustering in coronary arterial endothelial cells: evidence from ASM knockout mice.

    Science.gov (United States)

    Li, Xiang; Han, Wei-Qing; Boini, Krishna M; Xia, Min; Zhang, Yang; Li, Pin-Lan

    2013-01-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor, death receptor 4 (DR4), have been implicated in the development of endothelial dysfunction and atherosclerosis. However, the signaling mechanism mediating DR4 activation leading to endothelial injury remains unclear. We recently demonstrated that ceramide production via hydrolysis of membrane sphingomyelin by acid sphingomyelinase (ASM) results in membrane raft (MR) clustering and the formation of important redox signaling platforms, which play a crucial role in amplifying redox signaling in endothelial cells leading to endothelial dysfunction. The present study aims to investigate whether TRAIL triggers MR clustering via lysosome fusion and ASM activation, thereby conducting transmembrane redox signaling and changing endothelial function. Using confocal microscopy, we found that TRAIL induced MR clustering and co-localized with DR4 in coronary arterial endothelial cells (CAECs) isolated from wild-type (Smpd1 (+/+)) mice. Furthermore, TRAIL triggered ASM translocation, ceramide production, and NADPH oxidase aggregation in MR clusters in Smpd1 ( +/+ ) CAECs, whereas these observations were not found in Smpd1 (-/-) CAECs. Moreover, ASM deficiency reduced TRAIL-induced O(2) (-[Symbol: see text]) production in CAECs and abolished TRAIL-induced impairment on endothelium-dependent vasodilation in small resistance arteries. By measuring fluorescence resonance energy transfer, we found that Lamp-1 (lysosome membrane marker protein) and ganglioside G(M1) (MR marker) were trafficking together in Smpd1 (+/+) CAECs, which was absent in Smpd1 (-/-) CAECs. Consistently, fluorescence imaging of living cells with specific lysosome probes demonstrated that TRAIL-induced lysosome fusion with membrane was also absent in Smpd1 (-/-) CAECs. Taken together, these results suggest that ASM is essential for TRAIL-induced lysosomal trafficking, membrane fusion and formation of MR redox signaling platforms

  9. Discovery of a fusion kinase in EOL-1 cells and idiopathic hypereosinophilic syndrome

    OpenAIRE

    Griffin, John H.; Leung, Joey; Bruner, Rebecca J.; Caligiuri, Michael A.; Briesewitz, Roger

    2003-01-01

    Idiopathic hypereosinophilic syndrome (HES) is a myeloproliferative disease of unknown etiology. Recently, it has been reported that imatinib mesylate (Gleevec), an inhibitor of Bcr-Abl kinase useful in the treatment of chronic myeloid leukemia, is also effective in treating HES; however, the molecular target of imatinib in HES is unknown. This report identifies a genetic rearrangement in the eosinophilic cell line EOL-1 that results in the expression of a fusion protein comprising an N...

  10. FOXO1 is a direct target of EWS-Fli1 oncogenic fusion protein in Ewing's sarcoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Liu, E-mail: lyang@u.washington.edu [Department of Orthopedics, University of Washington, Seattle, WA 98195 (United States); Medical Research Service, VA Puget Sound Health Care System, Seattle, WA 98108 (United States); Hu, Hsien-Ming; Zielinska-Kwiatkowska, Anna; Chansky, Howard A. [Department of Orthopedics, University of Washington, Seattle, WA 98195 (United States); Medical Research Service, VA Puget Sound Health Care System, Seattle, WA 98108 (United States)

    2010-11-05

    Research highlights: {yields} Inducible and reversible siRNA knockdown of an oncogenic fusion protein such as EWS-Fli1 is feasible and more advantageous than other siRNA methods. {yields} The tumor suppressor gene FOXO1 is a new EWS-Fli1 target. {yields} While trans-activators are known for the FOXO1 gene, there has been no report on negative regulators of FOXO1 transcription. {yields} This study provides first evidence that the EWS-Fli1 oncogenic fusion protein can function as a transcriptional repressor of the FOXO1 gene. -- Abstract: Ewing's family tumors are characterized by a specific t(11;22) chromosomal translocation that results in the formation of EWS-Fli1 oncogenic fusion protein. To investigate the effects of EWS-Fli1 on gene expression, we carried out DNA microarray analysis after specific knockdown of EWS-Fli1 through transfection of synthetic siRNAs. EWS-Fli1 knockdown increased expression of genes such as DKK1 and p57 that are known to be repressed by EWS-Fli1 fusion protein. Among other potential EWS-Fli1 targets identified by our microarray analysis, we have focused on the FOXO1 gene since it encodes a potential tumor suppressor and has not been previously reported in Ewing's cells. To better understand how EWS-Fli1 affects FOXO1 expression, we have established a doxycycline-inducible siRNA system to achieve stable and reversible knockdown of EWS-Fli1 in Ewing's sarcoma cells. Here we show that FOXO1 expression in Ewing's cells has an inverse relationship with EWS-Fli1 protein level, and FOXO1 promoter activity is increased after doxycycline-induced EWS-Fli1 knockdown. In addition, we have found that direct binding of EWS-Fli1 to FOXO1 promoter is attenuated after doxycycline-induced siRNA knockdown of the fusion protein. Together, these results suggest that suppression of FOXO1 function by EWS-Fli1 fusion protein may contribute to cellular transformation in Ewing's family tumors.

  11. PARP inhibition sensitizes to low dose-rate radiation TMPRSS2-ERG fusion gene-expressing and PTEN-deficient prostate cancer cells.

    Directory of Open Access Journals (Sweden)

    Payel Chatterjee

    Full Text Available Exposure to genotoxic agents, such as irradiation produces DNA damage, the toxicity of which is augmented when the DNA repair is impaired. Poly (ADP-ribose polymerase (PARP inhibitors were found to be "synthetic lethal" in cells deficient in BRCA1 and BRCA2 that impair homologous recombination. However, since many tumors, including prostate cancer (PCa rarely have on such mutations, there is considerable interest in finding alternative determinants of PARP inhibitor sensitivity. We evaluated the effectiveness of radiation in combination with the PARP inhibitor, rucaparib in PCa cells. The combination index for clonogenic survival following radiation and rucaparib treatments revealed synergistic interactions in a panel of PCa cell lines, being strongest for LNCaP and VCaP cells that express ETS gene fusion proteins. These findings correlated with synergistic interactions for senescence activation, as indicated by β--galactosidase staining. Absence of PTEN and presence of ETS gene fusion thus facilitated activation of senescence, which contributed to decreased clonogenic survival. Increased radiosensitivity in the presence of rucaparib was associated with persistent DNA breaks, as determined by χ-H2AX, p53BP1, and Rad51 foci. VCaP cells, which harbor the TMPRSS2-ERG gene fusion and PC3 cells that stably express a similar construct (fusion III showed enhanced sensitivity towards rucaparib, which, in turn, increased the radiation response to a similar extent as the DNA-PKcs inhibitor NU7441. Rucaparib radiosensitized PCa cells, with a clear benefit of low dose-rate radiation (LDR administered over a longer period of time that caused enhanced DNA damage. LDR mimicking brachytherapy, which is used successfully in the clinic, was most effective when combined with rucaparib by inducing persistent DNA damage and senescence, leading to decreased clonogenic survival. This combination was most effective in the presence of the TMPRSS2-ERG and in the

  12. Deciphering the Role of microRNAs in BRD4-NUT Fusion Gene Induced NUT Midline Carcinoma.

    Science.gov (United States)

    Pathak, Ekta; Bhavya; Mishra, Divya; Atri, Neelam; Mishra, Rajeev

    2017-01-01

    NUT midline carcinoma (NMC) is a very aggressive and lethal type of squamous epithelial cell cancer caused due to fusion of BRD4 and NUT genes. The gene fusion results into a new fusion protein that promotes oncogenesis. The detailed molecular mechanisms underlying the NMC are still not clear and new findings are urgently required to complement the current efforts. Abnormal microRNAs (miRNA) expression promotes tumour formation by modulating the functional expression of critical genes other than the parent genes involved in tumour cell proliferation or survival. Here, using Insilco methods, miRNA targeting the transcripts of parent genes (BRD4 and NUT) and the BRD4-NUT fusion gene were predicted. We investigated a situation, wherein abnormal miRNA expression in malignant cells could arise due to deletion and fusion of genomic regions encompassing the target site of miRNA genes. A set of 48 dysregulated miRNAs targeting the critical genes other than the parent genes (BRD4 and NUT) was identified. Functional enrichment analysis of KEGG pathways of target genes of these Ex-miRNAs implicates their role in cancer pathways. Amplification in the expression level of these miRNAs can be used for NMC diagnosis and prognosis.

  13. Using Fluorescent Protein Fusions to Study Protein Subcellular Localization and Dynamics in Plant Cells.

    Science.gov (United States)

    Cui, Yong; Gao, Caiji; Zhao, Qiong; Jiang, Liwen

    2016-01-01

    Studies of protein subcellular localization and dynamics are helpful in understanding the cellular functions of proteins in an organism. In the past decade, the use of green fluorescent protein (GFP) as a fusion tag has dramatically extended our knowledge in this field. Transient expression and stable transformation of GFP-tagged proteins have been wildly used to study protein localization in vivo in different systems. Although GFP-based tags provide a fast and convenient way to characterize protein properties in living cells, several reports have demonstrated that GFP fusions might not accurately reflect the localization of the native protein as GFP tags may alter the protein properties. To facilitate proper usage of GFP tags in plant cell biology study, we describe detailed protocols to identify possible inhibitory effects of fluorescent tags on protein subcellular localization and to determine if a fluorescently tagged protein is localized to the correct subcellular compartment. Using Arabidopsis Endomembrane protein 12 (EMP12) as an example, we first show the possible inhibitory effect of GFP tags on proper protein localization and then describe the immunofluorescence labeling method to verify the correct localization of GFP fusion proteins. Next, a method is presented using the ImageJ program with the Pearson-Spearman correlation (PSC) colocalization plug-in for statistical quantification of colocalization ratios of two fluorophores. Finally we provide a detailed method for protein dynamics studies using spinning disk confocal microscopy in Arabidopsis cells.

  14. Dynamic in vivo imaging and cell tracking using a histone fluorescent protein fusion in mice

    Directory of Open Access Journals (Sweden)

    Papaioannou Virginia E

    2004-12-01

    Full Text Available Abstract Background Advances in optical imaging modalities and the continued evolution of genetically-encoded fluorescent proteins are coming together to facilitate the study of cell behavior at high resolution in living organisms. As a result, imaging using autofluorescent protein reporters is gaining popularity in mouse transgenic and targeted mutagenesis applications. Results We have used embryonic stem cell-mediated transgenesis to label cells at sub-cellular resolution in vivo, and to evaluate fusion of a human histone protein to green fluorescent protein for ubiquitous fluorescent labeling of nucleosomes in mice. To this end we have generated embryonic stem cells and a corresponding strain of mice that is viable and fertile and exhibits widespread chromatin-localized reporter expression. High levels of transgene expression are maintained in a constitutive manner. Viability and fertility of homozygous transgenic animals demonstrates that this reporter is developmentally neutral and does not interfere with mitosis or meiosis. Conclusions Using various optical imaging modalities including wide-field, spinning disc confocal, and laser scanning confocal and multiphoton excitation microscopy, we can identify cells in various stages of the cell cycle. We can identify cells in interphase, cells undergoing mitosis or cell death. We demonstrate that this histone fusion reporter allows the direct visualization of active chromatin in situ. Since this reporter segments three-dimensional space, it permits the visualization of individual cells within a population, and so facilitates tracking cell position over time. It is therefore attractive for use in multidimensional studies of in vivo cell behavior and cell fate.

  15. HIV-1-Induced Small T Cell Syncytia Can Transfer Virus Particles to Target Cells through Transient Contacts.

    Science.gov (United States)

    Symeonides, Menelaos; Murooka, Thomas T; Bellfy, Lauren N; Roy, Nathan H; Mempel, Thorsten R; Thali, Markus

    2015-12-12

    HIV-1 Env mediates fusion of viral and target cell membranes, but it can also mediate fusion of infected (producer) and target cells, thus triggering the formation of multinucleated cells, so-called syncytia. Large, round, immobile syncytia are readily observable in cultures of HIV-1-infected T cells, but these fast growing "fusion sinks" are largely regarded as cell culture artifacts. In contrast, small HIV-1-induced syncytia were seen in the paracortex of peripheral lymph nodes and other secondary lymphoid tissue of HIV-1-positive individuals. Further, recent intravital imaging of lymph nodes in humanized mice early after their infection with HIV-1 demonstrated that a significant fraction of infected cells were highly mobile, small syncytia, suggesting that these entities contribute to virus dissemination. Here, we report that the formation of small, migratory syncytia, for which we provide further quantification in humanized mice, can be recapitulated in vitro if HIV-1-infected T cells are placed into 3D extracellular matrix (ECM) hydrogels rather than being kept in traditional suspension culture systems. Intriguingly, live-cell imaging in hydrogels revealed that these syncytia, similar to individual infected cells, can transiently interact with uninfected cells, leading to rapid virus transfer without cell-cell fusion. Infected cells were also observed to deposit large amounts of viral particles into the extracellular space. Altogether, these observations suggest the need to further evaluate the biological significance of small, T cell-based syncytia and to consider the possibility that these entities do indeed contribute to virus spread and pathogenesis.

  16. The human leukocyte antigen G promotes trophoblast fusion and β-hCG production through the Erk1/2 pathway in human choriocarcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Ji-meng [School of Medicine, Nankai University, Tianjin 300071 (China); State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China); Zhao, Hong-xi [Department of Obstetrics and Gynecology, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038 (China); Wang, Li [Department of Obstetrics and Gynecology, General Hospital of Chinese People’s Liberation Army, Beijing 100853 (China); Gao, Zhi-ying, E-mail: gaozy301@yahoo.com.cn [Department of Obstetrics and Gynecology, General Hospital of Chinese People’s Liberation Army, Beijing 100853 (China); Yao, Yuan-qing, E-mail: yqyao@126.com [Department of Obstetrics and Gynecology, General Hospital of Chinese People’s Liberation Army, Beijing 100853 (China)

    2013-05-10

    Highlights: •HLA-G expression promotes BeWo cells fusion and fusogenic gene expression. •HLA-G is capable of inducing β-hCG production in human choriocarcinoma cell lines. •Up-regulation of β-hCG production by HLA-G is mediated via the Erk1/2 pathway. -- Abstract: The human leukocyte antigen G (HLA-G) is expressed on the fetal–maternal interface and plays a role in protecting fetal-derived trophoblasts from the maternal immune response, allowing trophoblasts to invade the uterus. However, HLA-G also possesses immune suppressing-independent functions. We found that HLA-G expressing BeWo choriocarcinoma cells increased cell–cell fusion compared to control BeWo cells under forskolin treatment. Regardless of forskolin treatment, the expression of fusogenic gene mRNAs, including syncytin-1, the transcription factor glial cell missing 1 (Gcm1), and beta human chorionic gonadotropin (β-hCG) were elevated. HLA-G up-regulates β-hCG production in human choriocarcinoma cells because HLA-G knockdown in JEG-3 cells induces a dramatic decrease in β-hCG compared with control cells. The defect in β-hCG production in HLA-G knocked-down cells could not be completely overcome by stimulating hCG production through increasing intracellular cAMP levels. HLA-G expressing cells have increased phosphorylation levels for extracellular signal-regulated kinase1/2 (Erk1/2) in BeWo cells. The Erk1/2 pathway is inactivated after the inhibition of HLA-G expression in JEG-3 cells. Finally, Erk1/2 inhibition was able to suppress the increased hCG production induced by HLA-G expression. Together, these data suggest novel roles for HLA-G in regulating β-hCG production via the modulation of the Erk1/2 pathway and by inducing trophoblast cell fusion.

  17. The human leukocyte antigen G promotes trophoblast fusion and β-hCG production through the Erk1/2 pathway in human choriocarcinoma cell lines

    International Nuclear Information System (INIS)

    Wang, Ji-meng; Zhao, Hong-xi; Wang, Li; Gao, Zhi-ying; Yao, Yuan-qing

    2013-01-01

    Highlights: •HLA-G expression promotes BeWo cells fusion and fusogenic gene expression. •HLA-G is capable of inducing β-hCG production in human choriocarcinoma cell lines. •Up-regulation of β-hCG production by HLA-G is mediated via the Erk1/2 pathway. -- Abstract: The human leukocyte antigen G (HLA-G) is expressed on the fetal–maternal interface and plays a role in protecting fetal-derived trophoblasts from the maternal immune response, allowing trophoblasts to invade the uterus. However, HLA-G also possesses immune suppressing-independent functions. We found that HLA-G expressing BeWo choriocarcinoma cells increased cell–cell fusion compared to control BeWo cells under forskolin treatment. Regardless of forskolin treatment, the expression of fusogenic gene mRNAs, including syncytin-1, the transcription factor glial cell missing 1 (Gcm1), and beta human chorionic gonadotropin (β-hCG) were elevated. HLA-G up-regulates β-hCG production in human choriocarcinoma cells because HLA-G knockdown in JEG-3 cells induces a dramatic decrease in β-hCG compared with control cells. The defect in β-hCG production in HLA-G knocked-down cells could not be completely overcome by stimulating hCG production through increasing intracellular cAMP levels. HLA-G expressing cells have increased phosphorylation levels for extracellular signal-regulated kinase1/2 (Erk1/2) in BeWo cells. The Erk1/2 pathway is inactivated after the inhibition of HLA-G expression in JEG-3 cells. Finally, Erk1/2 inhibition was able to suppress the increased hCG production induced by HLA-G expression. Together, these data suggest novel roles for HLA-G in regulating β-hCG production via the modulation of the Erk1/2 pathway and by inducing trophoblast cell fusion

  18. Identification of Alternative Splicing and Fusion Transcripts in Non-Small Cell Lung Cancer by RNA Sequencing.

    Science.gov (United States)

    Hong, Yoonki; Kim, Woo Jin; Bang, Chi Young; Lee, Jae Cheol; Oh, Yeon-Mok

    2016-04-01

    Lung cancer is the most common cause of cancer related death. Alterations in gene sequence, structure, and expression have an important role in the pathogenesis of lung cancer. Fusion genes and alternative splicing of cancer-related genes have the potential to be oncogenic. In the current study, we performed RNA-sequencing (RNA-seq) to investigate potential fusion genes and alternative splicing in non-small cell lung cancer. RNA was isolated from lung tissues obtained from 86 subjects with lung cancer. The RNA samples from lung cancer and normal tissues were processed with RNA-seq using the HiSeq 2000 system. Fusion genes were evaluated using Defuse and ChimeraScan. Candidate fusion transcripts were validated by Sanger sequencing. Alternative splicing was analyzed using multivariate analysis of transcript sequencing and validated using quantitative real time polymerase chain reaction. RNA-seq data identified oncogenic fusion genes EML4-ALK and SLC34A2-ROS1 in three of 86 normal-cancer paired samples. Nine distinct fusion transcripts were selected using DeFuse and ChimeraScan; of which, four fusion transcripts were validated by Sanger sequencing. In 33 squamous cell carcinoma, 29 tumor specific skipped exon events and six mutually exclusive exon events were identified. ITGB4 and PYCR1 were top genes that showed significant tumor specific splice variants. In conclusion, RNA-seq data identified novel potential fusion transcripts and splice variants. Further evaluation of their functional significance in the pathogenesis of lung cancer is required.

  19. Fusion toxin BLyS-gelonin inhibits growth of malignant human B cell lines in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Troy A Luster

    Full Text Available B lymphocyte stimulator (BLyS is a member of the TNF superfamily of cytokines. The biological activity of BLyS is mediated by three cell surface receptors: BR3/BAFF-R, TACI and BCMA. The expression of these receptors is highly restricted to B cells, both normal and malignant. A BLyS-gelonin fusion toxin (BLyS-gel was generated consisting of the recombinant plant-derived toxin gelonin fused to the N-terminus of BLyS and tested against a large and diverse panel of B-NHL cell lines. Interestingly, B-NHL subtypes mantle cell lymphoma (MCL, diffuse large B cell lymphoma (DLBCL and B cell precursor-acute lymphocytic leukemia (BCP-ALL were preferentially sensitive to BLyS-gel mediated cytotoxicity, with low picomolar EC(50 values. BLyS receptor expression did not guarantee sensitivity to BLyS-gel, even though the construct was internalized by both sensitive and resistant cells. Resistance to BLyS-gel could be overcome by treatment with the endosomotropic drug chloroquine, suggesting BLyS-gel may become trapped within endosomal/lysosomal compartments in resistant cells. BLyS-gel induced cell death was caspase-independent and shown to be at least partially mediated by the "ribotoxic stress response." This response involves activation of p38 MAPK and JNK/SAPK, and BLyS-gel mediated cytotoxicity was inhibited by the p38/JNK inhibitor SB203580. Finally, BLyS-gel treatment was shown to localize to sites of disease, rapidly reduce tumor burden, and significantly prolong survival in xenograft mouse models of disseminated BCP-ALL, DLBCL, and MCL. Together, these findings suggest BLyS has significant potential as a targeting ligand for the delivery of cytotoxic "payloads" to malignant B cells.

  20. Membrane cholesterol regulates lysosome-plasma membrane fusion events and modulates Trypanosoma cruzi invasion of host cells.

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    Bárbara Hissa

    Full Text Available BACKGROUND: Trypomastigotes of Trypanosoma cruzi are able to invade several types of non-phagocytic cells through a lysosomal dependent mechanism. It has been shown that, during invasion, parasites trigger host cell lysosome exocytosis, which initially occurs at the parasite-host contact site. Acid sphingomyelinase released from lysosomes then induces endocytosis and parasite internalization. Lysosomes continue to fuse with the newly formed parasitophorous vacuole until the parasite is completely enclosed by lysosomal membrane, a process indispensable for a stable infection. Previous work has shown that host membrane cholesterol is also important for the T. cruzi invasion process in both professional (macrophages and non-professional (epithelial phagocytic cells. However, the mechanism by which cholesterol-enriched microdomains participate in this process has remained unclear. METHODOLOGY/PRINCIPAL FINDING: In the present work we show that cardiomyocytes treated with MβCD, a drug able to sequester cholesterol from cell membranes, leads to a 50% reduction in invasion by T. cruzi trypomastigotes, as well as a decrease in the number of recently internalized parasites co-localizing with lysosomal markers. Cholesterol depletion from host membranes was accompanied by a decrease in the labeling of host membrane lipid rafts, as well as excessive lysosome exocytic events during the earlier stages of treatment. Precocious lysosomal exocytosis in MβCD treated cells led to a change in lysosomal distribution, with a reduction in the number of these organelles at the cell periphery, and probably compromises the intracellular pool of lysosomes necessary for T. cruzi invasion. CONCLUSION/SIGNIFICANCE: Based on these results, we propose that cholesterol depletion leads to unregulated exocytic events, reducing lysosome availability at the cell cortex and consequently compromise T. cruzi entry into host cells. The results also suggest that two different pools of

  1. Analysis of Induced Gamma Activation by D-T Neutrons in Selected Fusion Reactor Relevant Materials with EAF-2010

    Directory of Open Access Journals (Sweden)

    Klix Axel

    2016-01-01

    Full Text Available Samples of lanthanum, erbium and titanium which are constituents of structural materials, insulating coatings and tritium breeder for blankets of fusion reactor designs have been irradiated in a fusion peak neutron field. The induced gamma activities were measured and the results were used to check calculations with the European activation system EASY-2010. Good agreement for the prediction of major contributors to the contact dose rate of the materials was found, but for minor contributors the calculation deviated up to 50%.

  2. A Particle-in-Cell Simulation for the Traveling Wave Direct Energy Converter (TWDEC) for Fusion Propulsion

    Science.gov (United States)

    Chap, Andrew; Tarditi, Alfonso G.; Scott, John H.

    2013-01-01

    A Particle-in-cell simulation model has been developed to study the physics of the Traveling Wave Direct Energy Converter (TWDEC) applied to the conversion of charged fusion products into electricity. In this model the availability of a beam of collimated fusion products is assumed; the simulation is focused on the conversion of the beam kinetic energy into alternating current (AC) electric power. The model is electrostatic, as the electro-dynamics of the relatively slow ions can be treated in the quasistatic approximation. A two-dimensional, axisymmetric (radial-axial coordinates) geometry is considered. Ion beam particles are injected on one end and travel along the axis through ring-shaped electrodes with externally applied time-varying voltages, thus modulating the beam by forming a sinusoidal pattern in the beam density. Further downstream, the modulated beam passes through another set of ring electrodes, now electrically oating. The modulated beam induces a time alternating potential di erence between adjacent electrodes. Power can be drawn from the electrodes by connecting a resistive load. As energy is dissipated in the load, a corresponding drop in beam energy is measured. The simulation encapsulates the TWDEC process by reproducing the time-dependent transfer of energy and the particle deceleration due to the electric eld phase time variations.

  3. HIV transcription is induced in dying cells

    Energy Technology Data Exchange (ETDEWEB)

    Woloschak, G.E.; Chang-Liu, Chin-Mei [Argonne National Lab., IL (United States); Schreck, S. [Argonne National Lab., IL (United States)]|[Univ. of South Carolina, Columbia, SC (United States). Dept. of Chemistry; Panozzo, J. [Loyola Univ. Medical Center, Maywood, IL (United States); Libertin, C.R. [Loyola Univ. Medical Center, Maywood, IL (United States)

    1996-02-01

    Using HeLa cells stably transfected with an HIV-LTR-CAT construct, we demonstrated a peak in CAT induction that occurs in viable (but not necessarily cell-division-competent) cells 24 h following exposure to some cell-killing agents. {gamma} rays were the only cell-killing agent which did not induce HIV transcription; this can be attributed to the fact that {gamma}-ray-induced apoptotic death requires functional p53, which is not present in HeLa cells. For all other agents, HIV-LTR induction was dose-dependent and correlated with the amount of cell killing that occurred in the culture. Doses which caused over 99% cell killing induced HIV-LTR transcription maximally, demonstrating that cells that will go on to die by 14 days are the cells expressing HIV-LTR-CAT.

  4. Combined virus-like particle and fusion protein-encoding DNA vaccination of cotton rats induces protection against respiratory syncytial virus without causing vaccine-enhanced disease

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Hye Suk; Lee, Young-Tae; Kim, Ki-Hye; Park, Soojin; Kwon, Young-Man; Lee, Youri; Ko, Eun-Ju; Jung, Yu-Jin [Center for Inflammation, Immunity & Infection, Institute for Biomedical Sciences and Department of Biology, Georgia State University, Atlanta, GA (United States); Lee, Jong Seok [Center for Inflammation, Immunity & Infection, Institute for Biomedical Sciences and Department of Biology, Georgia State University, Atlanta, GA (United States); National Institute of Biological Resources, Incheon (Korea, Republic of); Kim, Yu-Jin [Center for Inflammation, Immunity & Infection, Institute for Biomedical Sciences and Department of Biology, Georgia State University, Atlanta, GA (United States); Lee, Yu-Na; Kim, Min-Chul [Center for Inflammation, Immunity & Infection, Institute for Biomedical Sciences and Department of Biology, Georgia State University, Atlanta, GA (United States); Animal and Plant Quarantine Agency, Gyeonggi-do, Gimcheon, Gyeongsangbukdo (Korea, Republic of); Cho, Minkyoung [Center for Inflammation, Immunity & Infection, Institute for Biomedical Sciences and Department of Biology, Georgia State University, Atlanta, GA (United States); Kang, Sang-Moo, E-mail: skang24@gsu.edu [Center for Inflammation, Immunity & Infection, Institute for Biomedical Sciences and Department of Biology, Georgia State University, Atlanta, GA (United States)

    2016-07-15

    A safe and effective vaccine against respiratory syncytial virus (RSV) should confer protection without causing vaccine-enhanced disease. Here, using a cotton rat model, we investigated the protective efficacy and safety of an RSV combination vaccine composed of F-encoding plasmid DNA and virus-like particles containing RSV fusion (F) and attachment (G) glycoproteins (FFG-VLP). Cotton rats with FFG-VLP vaccination controlled lung viral replication below the detection limit, and effectively induced neutralizing activity and antibody-secreting cell responses. In comparison with formalin inactivated RSV (FI-RSV) causing severe RSV disease after challenge, FFG-VLP vaccination did not cause weight loss, airway hyper-responsiveness, IL-4 cytokines, histopathology, and infiltrates of proinflammatory cells such as eosinophils. FFG-VLP was even more effective in preventing RSV-induced pulmonary inflammation than live RSV infections. This study provides evidence that FFG-VLP can be developed into a safe and effective RSV vaccine candidate. - Highlights: • Combined RSV FFG VLP vaccine is effective in inducing F specific responses. • FFG VLP vaccine confers RSV neutralizing activity and viral control in cotton rats. • Cotton rats with RSV FFG VLP vaccination do not show vaccine-enhanced disease. • Cotton rats with FFG VLP vaccine induce F specific antibody secreting cell responses. • Cotton rats with FFG VLP do not induce lung cellular infiltrates and Th2 cytokine.

  5. Combined virus-like particle and fusion protein-encoding DNA vaccination of cotton rats induces protection against respiratory syncytial virus without causing vaccine-enhanced disease

    International Nuclear Information System (INIS)

    Hwang, Hye Suk; Lee, Young-Tae; Kim, Ki-Hye; Park, Soojin; Kwon, Young-Man; Lee, Youri; Ko, Eun-Ju; Jung, Yu-Jin; Lee, Jong Seok; Kim, Yu-Jin; Lee, Yu-Na; Kim, Min-Chul; Cho, Minkyoung; Kang, Sang-Moo

    2016-01-01

    A safe and effective vaccine against respiratory syncytial virus (RSV) should confer protection without causing vaccine-enhanced disease. Here, using a cotton rat model, we investigated the protective efficacy and safety of an RSV combination vaccine composed of F-encoding plasmid DNA and virus-like particles containing RSV fusion (F) and attachment (G) glycoproteins (FFG-VLP). Cotton rats with FFG-VLP vaccination controlled lung viral replication below the detection limit, and effectively induced neutralizing activity and antibody-secreting cell responses. In comparison with formalin inactivated RSV (FI-RSV) causing severe RSV disease after challenge, FFG-VLP vaccination did not cause weight loss, airway hyper-responsiveness, IL-4 cytokines, histopathology, and infiltrates of proinflammatory cells such as eosinophils. FFG-VLP was even more effective in preventing RSV-induced pulmonary inflammation than live RSV infections. This study provides evidence that FFG-VLP can be developed into a safe and effective RSV vaccine candidate. - Highlights: • Combined RSV FFG VLP vaccine is effective in inducing F specific responses. • FFG VLP vaccine confers RSV neutralizing activity and viral control in cotton rats. • Cotton rats with RSV FFG VLP vaccination do not show vaccine-enhanced disease. • Cotton rats with FFG VLP vaccine induce F specific antibody secreting cell responses. • Cotton rats with FFG VLP do not induce lung cellular infiltrates and Th2 cytokine.

  6. Lipid droplets fusion in adipocyte differentiated 3T3-L1 cells: A Monte Carlo simulation

    International Nuclear Information System (INIS)

    Boschi, Federico; Rizzatti, Vanni; Zamboni, Mauro; Sbarbati, Andrea

    2014-01-01

    Several human worldwide diseases like obesity, type 2 diabetes, hepatic steatosis, atherosclerosis and other metabolic pathologies are related to the excessive accumulation of lipids in cells. Lipids accumulate in spherical cellular inclusions called lipid droplets (LDs) whose sizes range from fraction to one hundred of micrometers in adipocytes. It has been suggested that LDs can grow in size due to a fusion process by which a larger LD is obtained with spherical shape and volume equal to the sum of the progenitors’ ones. In this study, the size distribution of two populations of LDs was analyzed in immature and mature (5-days differentiated) 3T3-L1 adipocytes (first and second populations, respectively) after Oil Red O staining. A Monte Carlo simulation of interaction between LDs has been developed in order to quantify the size distribution and the number of fusion events needed to obtain the distribution of the second population size starting from the first one. Four models are presented here based on different kinds of interaction: a surface weighted interaction (R2 Model), a volume weighted interaction (R3 Model), a random interaction (Random model) and an interaction related to the place where the LDs are born (Nearest Model). The last two models mimic quite well the behavior found in the experimental data. This work represents a first step in developing numerical simulations of the LDs growth process. Due to the complex phenomena involving LDs (absorption, growth through additional neutral lipid deposition in existing droplets, de novo formation and catabolism) the study focuses on the fusion process. The results suggest that, to obtain the observed size distribution, a number of fusion events comparable with the number of LDs themselves is needed. Moreover the MC approach results a powerful tool for investigating the LDs growth process. Highlights: • We evaluated the role of the fusion process in the synthesis of the lipid droplets. • We compared the

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

    International Nuclear Information System (INIS)

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

    2008-01-01

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

  8. The anti-cell death FNK protein protects cells from death induced by freezing and thawing

    International Nuclear Information System (INIS)

    Sudo, Kentaro; Asoh, Sadamitsu; Ohsawa, Ikuroh; Ozaki, Daiya; Yamagata, Kumi; Ito, Hiromoto; Ohta, Shigeo

    2005-01-01

    The FNK protein, constructed from anti-apoptotic Bcl-x L with enhanced activity, was fused with the protein transduction domain (PTD) of the HIV/Tat protein to mediate the delivery of FNK into cells. The fusion protein PTD-FNK was introduced into chondrocytes in isolated articular cartilage-bone sections, cultured neurons, and isolated bone marrow mononuclear cells to evaluate its ability to prevent cell death induced by freezing and thawing. PTD-FNK protected the cells from freeze-thaw damage in a concentration-dependent manner. Addition of PTD-FNK with conventional cryoprotectants (dimethyl sulfoxide and hydroxyethyl starch) increased surviving cell numbers around 2-fold compared with controls treated only with the cryoprotectants. Notably, PTD-FNK allowed CD34 + cells among bone marrow mononuclear cells to survive more efficiently (12-fold more than the control cells) from two successive freeze-thaw cycles. Thus, PTD-FNK prevented cell death induced by freezing and thawing, suggesting that it provides for the successful cryopreservation of biological materials

  9. JAK inhibitors suppress t(8;21) fusion protein-induced leukemia

    Science.gov (United States)

    Lo, Miao-Chia; Peterson, Luke F.; Yan, Ming; Cong, Xiuli; Hickman, Justin H.; DeKelver, Russel C.; Niewerth, Denise; Zhang, Dong-Er

    2014-01-01

    Oncogenic mutations in components of the JAK/STAT pathway, including those in cytokine receptors and JAKs, lead to increased activity of downstream signaling and are frequently found in leukemia and other hematological disorders. Thus, small-molecule inhibitors of this pathway have been the focus of targeted therapy in these hematological diseases. We previously showed that t(8;21) fusion protein AML1-ETO and its alternatively spliced variant AML1-ETO9a (AE9a) enhance the JAK/STAT pathway via down-regulation of CD45, a negative regulator of this pathway. To investigate the therapeutic potential of targeting JAK/STAT in t(8;21) leukemia, we examined the effects of a JAK2-selective inhibitor TG101209 and a JAK1/2-selective inhibitor INCB18424 on t(8;21) leukemia cells. TG101209 and INCB18424 inhibited proliferation and promoted apoptosis of these cells. Furthermore, TG101209 treatment in AE9a leukemia mice reduced tumor burden and significantly prolonged survival. TG101209 also significantly impaired the leukemia-initiating potential of AE9a leukemia cells in secondary recipient mice. These results demonstrate the potential therapeutic efficacy of JAK inhibitors in treating t(8;21) AML. PMID:23812420

  10. Cell fusion in osteoclasts plays a critical role in controlling bone mass and osteoblastic activity

    International Nuclear Information System (INIS)

    Iwasaki, Ryotaro; Ninomiya, Ken; Miyamoto, Kana; Suzuki, Toru; Sato, Yuiko

    2008-01-01

    The balance between osteoclast and osteoblast activity is central for maintaining the integrity of bone homeostasis. Here we show that mice lacking dendritic cell specific transmembrane protein (DC-STAMP), an essential molecule for osteoclast cell-cell fusion, exhibited impaired bone resorption and upregulation of bone formation by osteoblasts, which do not express DC-STAMP, which led to increased bone mass. On the contrary, DC-STAMP over-expressing transgenic (DC-STAMP-Tg) mice under the control of an actin promoter showed significantly accelerated cell-cell fusion of osteoclasts and bone resorption, with decreased osteoblastic activity and bone mass. Bone resorption and formation are known to be regulated in a coupled manner, whereas DC-STAMP regulates bone homeostasis in an un-coupled manner. Thus our results indicate that inhibition of a single molecule provides both decreased osteoclast activity and increased bone formation by osteoblasts, thereby increasing bone mass in an un-coupled and a tissue specific manner.

  11. Newcastle Disease Virus Establishes Persistent Infection in Tumor CellsIn Vitro: Contribution of the Cleavage Site of Fusion Protein and Second Sialic Acid Binding Site of Hemagglutinin-Neuraminidase.

    Science.gov (United States)

    Rangaswamy, Udaya S; Wang, Weijia; Cheng, Xing; McTamney, Patrick; Carroll, Danielle; Jin, Hong

    2017-08-15

    Newcastle disease virus (NDV) is an oncolytic virus being developed for the treatment of cancer. Following infection of a human ovarian cancer cell line (OVCAR3) with a recombinant low-pathogenic NDV, persistent infection was established in a subset of tumor cells. Persistently infected (PI) cells exhibited resistance to superinfection with NDV and established an antiviral state, as demonstrated by upregulation of interferon and interferon-induced genes such as myxoma resistance gene 1 (Mx1) and retinoic acid-inducing gene-I (RIG-I). Viruses released from PI cells induced higher cell-to-cell fusion than the parental virus following infection in two tumor cell lines tested, HT1080 and HeLa, and remained attenuated in chickens. Two mutations, one in the fusion (F) protein cleavage site, F117S (F 117S ), and another in hemagglutinin-neuraminidase (HN), G169R (HN 169R ), located in the second sialic acid binding region, were responsible for the hyperfusogenic phenotype. F 117S improves F protein cleavage efficiency, facilitating cell-to-cell fusion, while HN 169R possesses a multifaceted role in contributing to higher fusion, reduced receptor binding, and lower neuraminidase activity, which together result in increased fusion and reduced viral replication. Thus, establishment of persistent infection in vitro involves viral genetic changes that facilitate efficient viral spread from cell to cell as a potential mechanism to escape host antiviral responses. The results of our study also demonstrate a critical role in the viral life cycle for the second receptor binding region of the HN protein, which is conserved in several paramyxoviruses. IMPORTANCE Oncolytic Newcastle disease virus (NDV) could establish persistent infection in a tumor cell line, resulting in a steady antiviral state reflected by constitutively expressed interferon. Viruses isolated from persistently infected cells are highly fusogenic, and this phenotype has been mapped to two mutations, one each in

  12. Multiple vertebral fractures associated with glucocorticoid-induced osteoporosis treated with teriparatide followed by kyphosis correction fusion: a case report.

    Science.gov (United States)

    Uei, Hiroshi; Tokuhashi, Yasuaki; Maseda, Masafumi; Nakahashi, Masahiro; Nakayama, Enshi

    2018-02-23

    Surgical treatment of multiple vertebral fractures in patients with glucocorticoid-induced osteoporosis is difficult because of a high rate of secondary fracture postoperatively. A case is described in which initial treatment with teriparatide to improve osteoporosis followed by treatment of kyphosis with correction fusion achieved a favorable outcome. Secondary fracture frequently occurs after treatment of vertebral fracture with vertebroplasty and balloon kyphoplasty in patients with glucocorticoid-induced osteoporosis, but effective treatment of multiple vertebral fractures has rarely been reported. Thus, a treatment of kyphosis following multiple vertebral fractures associated with glucocorticoid-induced osteoporosis is required. The patient was a 24-year-old woman diagnosed with glucocorticoid-induced osteoporosis who was under treatment with oral alendronate, vitamin D, and elcatonin injection. Secondary multiple vertebral fractures occurred despite these treatments and low back pain gradually aggravated. Vertebroplasty or balloon kyphoplasty was not performed in the early phase. Instead, treatment with teriparatide was used for initial improvement of osteoporosis. Kyphosis in the center of the residual thoracolumbar junction was then treated with posterior correction fusion. At 2 years after surgery, the corrected position has been maintained and no new fracture has occurred. There is no established method for treatment of multiple vertebral fractures caused by glucocorticoid-induced osteoporosis. Initial treatment with teriparatide to improve osteoporosis followed by treatment of kyphosis with correction fusion may result in a more favorable outcome.

  13. Dual microRNA Screens Reveal That the Immune-Responsive miR-181 Promotes Henipavirus Entry and Cell-Cell Fusion

    Science.gov (United States)

    Foo, Chwan Hong; Rootes, Christina L.; Marsh, Glenn A.; Gould, Cathryn M.; Klein, Reuben; Riddell, Sarah J.; Middleton, Deborah; Simpson, Kaylene J.; Bean, Andrew G. D.; Stewart, Cameron R.

    2016-01-01

    Hendra and Nipah viruses (family Paramyxoviridae, genus Henipavirus) are bat-borne viruses that cause fatal disease in humans and a range of other mammalian species. Gaining a deeper understanding of host pathways exploited by henipaviruses for infection may identify targets for new anti-viral therapies. Here we have performed genome-wide high-throughput agonist and antagonist screens at biosafety level 4 to identify host-encoded microRNAs (miRNAs) impacting henipavirus infection in human cells. Members of the miR-181 and miR-17~93 families strongly promoted Hendra virus infection. miR-181 also promoted Nipah virus infection, but did not affect infection by paramyxoviruses from other genera, indicating specificity in the virus-host interaction. Infection promotion was primarily mediated via the ability of miR-181 to significantly enhance henipavirus-induced membrane fusion. Cell signalling receptors of ephrins, namely EphA5 and EphA7, were identified as novel negative regulators of henipavirus fusion. The expression of these receptors, as well as EphB4, were suppressed by miR-181 overexpression, suggesting that simultaneous inhibition of several Ephs by the miRNA contributes to enhanced infection and fusion. Immune-responsive miR-181 levels was also up-regulated in the biofluids of ferrets and horses infected with Hendra virus, suggesting that the host innate immune response may promote henipavirus spread and exacerbate disease severity. This study is the first genome-wide screen of miRNAs influencing infection by a clinically significant mononegavirus and nominates select miRNAs as targets for future anti-viral therapy development. PMID:27783670

  14. Dual microRNA Screens Reveal That the Immune-Responsive miR-181 Promotes Henipavirus Entry and Cell-Cell Fusion.

    Directory of Open Access Journals (Sweden)

    Chwan Hong Foo

    2016-10-01

    Full Text Available Hendra and Nipah viruses (family Paramyxoviridae, genus Henipavirus are bat-borne viruses that cause fatal disease in humans and a range of other mammalian species. Gaining a deeper understanding of host pathways exploited by henipaviruses for infection may identify targets for new anti-viral therapies. Here we have performed genome-wide high-throughput agonist and antagonist screens at biosafety level 4 to identify host-encoded microRNAs (miRNAs impacting henipavirus infection in human cells. Members of the miR-181 and miR-17~93 families strongly promoted Hendra virus infection. miR-181 also promoted Nipah virus infection, but did not affect infection by paramyxoviruses from other genera, indicating specificity in the virus-host interaction. Infection promotion was primarily mediated via the ability of miR-181 to significantly enhance henipavirus-induced membrane fusion. Cell signalling receptors of ephrins, namely EphA5 and EphA7, were identified as novel negative regulators of henipavirus fusion. The expression of these receptors, as well as EphB4, were suppressed by miR-181 overexpression, suggesting that simultaneous inhibition of several Ephs by the miRNA contributes to enhanced infection and fusion. Immune-responsive miR-181 levels was also up-regulated in the biofluids of ferrets and horses infected with Hendra virus, suggesting that the host innate immune response may promote henipavirus spread and exacerbate disease severity. This study is the first genome-wide screen of miRNAs influencing infection by a clinically significant mononegavirus and nominates select miRNAs as targets for future anti-viral therapy development.

  15. High-throughput deterministic single-cell encapsulation and droplet pairing, fusion, and shrinkage in a single microfluidic device

    NARCIS (Netherlands)

    Schoeman, R.M.; Kemna, Evelien; Wolbers, F.; van den Berg, Albert

    In this article, we present a microfluidic device capable of successive high-yield single-cell encapsulation in droplets, with additional droplet pairing, fusion, and shrinkage. Deterministic single-cell encapsulation is realized using Dean-coupled inertial ordering of cells in a Yin-Yang-shaped

  16. Autophagy regulation revealed by SapM-induced block of autophagosome-lysosome fusion via binding RAB7

    International Nuclear Information System (INIS)

    Hu, Dong; Wu, Jing; Wang, Wan; Mu, Min; Zhao, Runpeng; Xu, Xuewei; Chen, Zhaoquan; Xiao, Jian; Hu, Fengyu; Yang, Yabo; Zhang, Rongbo

    2015-01-01

    The mechanism underlying autophagy alteration by mycobacterium tuberculosis remains unclear. Our previous study shows LpqH, a lipoprotein of mycobacterium tuberculosis, can cause autophagosomes accumulation in murine macrophages. It is well known that SapM, another virulence factor, plays an important role in blocking phagosome-endosome fusion. However, the mechanism that SapM interferes with autophagy remains poorly defined. In this study, we report that SapM suppresses the autophagy flux by blocking autophagosome fusion with lysosome. Exposure to SapM results in accumulations of autophagosomes and decreased co-localization of autophagosome with lysosome. Molecularly, Rab7, a small GTPase, is blocked by SapM through its CT domain and is prevented from involvement of autophagosome-lysosome fusion. In conclusion, our study reveals that SapM takes Rab7 as a previously unknown target to govern a distinct molecular mechanism underlying autophagosome-lysosome fusion, which may bring light to a new thought about developing potential drugs or vaccines against tuberculosis. - Highlights: • A mechanism for disrupting autophagosome-lysosome fusion induced by SapM. • Rab7 is involved in SapM-inhibited autophagy. • SapM interacts with Rab7 by CT-domain. • CT-domain is indispensable to SapM-inhibited autophagy

  17. Autophagy regulation revealed by SapM-induced block of autophagosome-lysosome fusion via binding RAB7

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Dong, E-mail: austhudong@126.com [Institute of Infection and Immunology, Department of Medical Immunology, Medical School, Anhui University of Science and Technology, Huainan (China); Wu, Jing, E-mail: wujing8008@126.com [Institute of Infection and Immunology, Department of Medical Immunology, Medical School, Anhui University of Science and Technology, Huainan (China); Wang, Wan; Mu, Min; Zhao, Runpeng; Xu, Xuewei; Chen, Zhaoquan [Institute of Infection and Immunology, Department of Medical Immunology, Medical School, Anhui University of Science and Technology, Huainan (China); Xiao, Jian [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Hu, Fengyu; Yang, Yabo [Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou (China); Zhang, Rongbo, E-mail: lory456@126.com [Institute of Infection and Immunology, Department of Medical Immunology, Medical School, Anhui University of Science and Technology, Huainan (China)

    2015-05-29

    The mechanism underlying autophagy alteration by mycobacterium tuberculosis remains unclear. Our previous study shows LpqH, a lipoprotein of mycobacterium tuberculosis, can cause autophagosomes accumulation in murine macrophages. It is well known that SapM, another virulence factor, plays an important role in blocking phagosome-endosome fusion. However, the mechanism that SapM interferes with autophagy remains poorly defined. In this study, we report that SapM suppresses the autophagy flux by blocking autophagosome fusion with lysosome. Exposure to SapM results in accumulations of autophagosomes and decreased co-localization of autophagosome with lysosome. Molecularly, Rab7, a small GTPase, is blocked by SapM through its CT domain and is prevented from involvement of autophagosome-lysosome fusion. In conclusion, our study reveals that SapM takes Rab7 as a previously unknown target to govern a distinct molecular mechanism underlying autophagosome-lysosome fusion, which may bring light to a new thought about developing potential drugs or vaccines against tuberculosis. - Highlights: • A mechanism for disrupting autophagosome-lysosome fusion induced by SapM. • Rab7 is involved in SapM-inhibited autophagy. • SapM interacts with Rab7 by CT-domain. • CT-domain is indispensable to SapM-inhibited autophagy.

  18. Generation of the Fluorescent HMGB1-GFP Fusion Protein in Insect Cells and Evaluation of its Immunogenicity in Two Mice Models.

    Science.gov (United States)

    Anvar, Ali; Vahabpour, Rouhollah; Salahshourifar, Iman; Bolhassani, Azam

    2017-01-01

    High mobility group box 1 (HMGB1) is a highly conserved protein present in the nuclei and cytoplasm of cells which has an important role as a mediator of inflammation in the extracellular environment. HMGB1 was identified as an innate adjuvant that induces immune responses against soluble antigens in vivo. Our goal is the generation of recombinant HMGB1-GFP fusion protein in insect cells for evaluation of immune responses in mouse model. In the current study, we used a baculovirus expression system for insect cells that was based on expression of HMGB1 with target gene (GFP), and purified the recombinant HMGB1- GFP fusion protein. We then demonstrated whether immunogenicity of GFP changes in the presence or absence of recombinant HMGB1 acting as an adjuvant in C57BL/6 and BALB/c mice. Our data showed that HMGB1 had a major influence on antibody immune responses induced by GFP in both animal models. The groups receiving HMGB1-GFP fusion protein showed total IgG and IgG2a responses significantly higher than IgG1 in BALB/c mice. Indeed, a mixed IgG1/IgG2a response was observed with high intensity toward IgG2a. In contrast, C57BL/6 mice immunized by HMGB1-GFP protein elicited the same levels of IgG1 and IgG2a. However, the levels of IgG2a and total IgG against the recombinant GFP (rGFP) in C57BL/6 mice were lower than those in BALB/c mice. We concluded that fusion of HMGB1 with GFP was immunologically more effective than GFP alone. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  19. A CRE/AP-1-like motif is essential for induced syncytin-2 expression and fusion in human trophoblast-like model.

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    Chirine Toufaily

    Full Text Available Syncytin-2 is encoded by the envelope gene of Endogenous Retrovirus-FRD (ERVFRD-1 and plays a critical role in fusion of placental trophoblasts leading to the formation of the multinucleated syncytiotrophoblast. Its expression is consequently regulated in a strict manner. In the present study, we have identified a forskolin-responsive region located between positions -300 to -150 in the Syncytin-2 promoter region. This 150 bp region in the context of a minimal promoter mediated an 80-fold induction of promoter activity following forskolin stimulation. EMSA analyses with competition experiments with nuclear extracts from forskolin-stimulated BeWo cells demonstrated that the -211 to -177 region specifically bound two forskolin-induced complexes, one of them containing a CRE/AP-1-like motif. Site-directed mutagenesis of the CRE/AP-1 binding site in the context of the Syncytin-2 promoter or a heterologous promoter showed that this motif was mostly essential for forskolin-induced promoter activity. Transfection experiments with dominant negative mutants and constitutively activated CREB expression vectors in addition to Chromatin Immunoprecipitation suggested that a CREB family member, CREB2 was binding and acting through the CRE/AP-1 motif. We further demonstrated the binding of JunD to this same motif. Similar to forskolin and soluble cAMP, CREB2 and JunD overexpression induced Syncytin-2 promoter activity in a CRE/AP-1-dependent manner and Syncytin-2 expression. In addition, BeWo cell fusion was induced by both CREB2 and JunD overexpression, while being repressed following silencing of either gene. These results thereby demonstrate that induced expression of Syncytin-2 is highly dependent on the interaction of bZIP-containing transcription factors to a CRE/AP-1 motif and that this element is important for the regulation of Syncytin-2 expression, which results in the formation of the peripheral syncytiotrophoblast layer.

  20. WASP-Arp2/3-dependent actin polymerization influences fusogen localization during cell-cell fusion in Caenorhabditis elegans embryos

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    Yan Zhang

    2017-09-01

    Full Text Available Cell-cell fusion is essential for development and physiology. Actin polymerization was implicated in the Caenorhabditis elegans fusogen EFF-1 engagement in a reconstituted Drosophila cell culture system, and the actin-binding protein spectraplakin links EFF-1 to the actin cytoskeleton and promotes cell-cell fusions in C. elegans larvae. However, it remains unclear whether and how fusogens and the actin cytoskeleton are coordinated in C. elegans embryos. Here, we used live imaging analysis of GFP knock-in and RNAi embryos to study the embryonic cell-cell fusions in C. elegans. Our results show that the inhibition of WASP-Arp2/3-dependent actin polymerization delays cell-cell fusions. EFF-1 is primarily distributed in intracellular vesicles in embryonic fusing cells, and we find that the perturbation of actin polymerization reduces the number of EFF-1-postive vesicles. Thus, the actin cytoskeleton differently promotes cell-cell fusion by regulating fusogen localization to the fusing plasma membrane in larvae or to intracellular vesicles in embryos.

  1. Effective enrichment strategy for EML4-ALK fusion gene screening in patients with non-small cell lung cancer.

    Science.gov (United States)

    Kobayashi, Makoto; Sakakibara, Tomohiro; Inoue, Akira; Fukuhara, Tatsuro; Sasano, Hironobu; Ichinose, Masakazu; Nukiwa, Toshihiro

    2014-01-01

    A novel fusion gene that comprises the echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) genes was recently identified in non-small cell lung cancer (NSCLC), particularly in adenocarcinoma. A specific ALK inhibitor has been shown to exert anti-tumor effects in NSCLC with the EML4-ALK fusion gene. Previous reports suggested an EML4-ALK incidence of approximately 5% in a pan-NSCLC population, with an increased frequency in younger patients, but an appropriate strategy for further selecting patients with the EML4-ALK fusion gene remains unknown. Patients, 55 years of age or younger, who were diagnosed with NSCLC without typical squamous cell carcinoma features at our institute were retrospectively evaluated. The tumor specimens were examined by immunohistochemistry for the EML4-ALK fusion gene and by polymerase chain reaction for epidermal growth factor receptor (EGFR) mutations. Between January 2004 and September 2011, the EML4-ALK fusion gene was detected in 19.6% (9/46) of patients. The fusion gene incidence increased to 31% (9/29) when patients with EGFR mutations were excluded. The EML4-ALK fusion gene was further detected in 2 cases of undifferentiated cell carcinoma. EML4-ALK fusion gene examinations could be more effectively performed by selecting young NSCLC patients without EGFR mutations, whereas selection on the basis of a non-smoking or adenocarcinoma history, as reported in previous studies, may not correctly identify the patient groups with potential EML4-ALK fusion gene. Copyright © 2013 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.

  2. Early Events in Chikungunya Virus Infection—From Virus CellBinding to Membrane Fusion

    Directory of Open Access Journals (Sweden)

    Mareike K. S. van Duijl-Richter

    2015-07-01

    Full Text Available Chikungunya virus (CHIKV is a rapidly emerging mosquito-borne alphavirus causing millions of infections in the tropical and subtropical regions of the world. CHIKV infection often leads to an acute self-limited febrile illness with debilitating myalgia and arthralgia. A potential long-term complication of CHIKV infection is severe joint pain, which can last for months to years. There are no vaccines or specific therapeutics available to prevent or treat infection. This review describes the critical steps in CHIKV cell entry. We summarize the latest studies on the virus-cell tropism, virus-receptor binding, internalization, membrane fusion and review the molecules and compounds that have been described to interfere with virus cell entry. The aim of the review is to give the reader a state-of-the-art overview on CHIKV cell entry and to provide an outlook on potential new avenues in CHIKV research.

  3. Coating Nanoparticles with Plant-Produced Transferrin-Hydrophobin Fusion Protein Enhances Their Uptake in Cancer Cells.

    Science.gov (United States)

    Reuter, Lauri J; Shahbazi, Mohammad-Ali; Mäkilä, Ermei M; Salonen, Jarno J; Saberianfar, Reza; Menassa, Rima; Santos, Hélder A; Joensuu, Jussi J; Ritala, Anneli

    2017-06-21

    The encapsulation of drugs to nanoparticles may offer a solution for targeted delivery. Here, we set out to engineer a self-assembling targeting ligand by combining the functional properties of human transferrin and fungal hydrophobins in a single fusion protein. We showed that human transferrin can be expressed in Nicotiana benthamiana plants as a fusion with Trichoderma reesei hydrophobins HFBI, HFBII, or HFBIV. Transferrin-HFBIV was further expressed in tobacco BY-2 suspension cells. Both partners of the fusion protein retained their functionality; the hydrophobin moiety enabled migration to a surfactant phase in an aqueous two-phase system, and the transferrin moiety was able to reversibly bind iron. Coating porous silicon nanoparticles with the fusion protein resulted in uptake of the nanoparticles in human cancer cells. This study provides a proof-of-concept for the functionalization of hydrophobin coatings with transferrin as a targeting ligand.

  4. Big Animal Cloning Using Transgenic Induced Pluripotent Stem Cells: A Case Study of Goat Transgenic Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Song, Hui; Li, Hui; Huang, Mingrui; Xu, Dan; Wang, Ziyu; Wang, Feng

    2016-02-01

    Using of embryonic stem cells (ESCs) could improve production traits and disease resistance by improving the efficiency of somatic cell nuclear transfer (SCNT) technology. However, robust ESCs have not been established from domestic ungulates. In the present study, we generated goat induced pluripotent stem cells (giPSCs) and transgenic cloned dairy goat induced pluripotent stem cells (tgiPSCs) from dairy goat fibroblasts (gFs) and transgenic cloned dairy goat fibroblasts (tgFs), respectively, using lentiviruses that contained hOCT4, hSOX2, hMYC, and hKLF4 without chemical compounds. The giPSCs and tgiPSCs expressed endogenous pluripotent markers, including OCT4, SOX2, MYC, KLF4, and NANOG. Moreover, they were able to maintain a normal karyotype and differentiate into derivatives from all three germ layers in vitro and in vivo. Using SCNT, tgFs and tgiPSCs were used as donor cells to produce embryos, which were named tgF-Embryos and tgiPSC-Embryos. The fusion rates and cleavage rates had no significant differences between tgF-Embryos and tgiPSC-Embryos. However, the expression of IGF-2, which is an important gene associated with embryonic development, was significantly lower in tgiPSC-Embryos than in tgF-Embryos and was not significantly different from vivo-Embryos.

  5. A fusion protein of interleukin-4 and interleukin-10 protects against blood-induced cartilage damage in vitro and in vivo

    NARCIS (Netherlands)

    van Vulpen, L F D; Popov-Celeketic, J; van Meegeren, M. E R; Coeleveld, K; van Laar, J M; Hack, C E; Schutgens, R E G; Mastbergen, S C; Lafeber, F P J G

    Essentials Targeted treatment for hemophilic arthropathy, still causing significant morbidity, is lacking. This study evaluates the efficacy of a fusion of protein of interleukin(IL)-4 and IL-10. In vitro the fusion protein prevents blood-induced cartilage damage in a dose-dependent manner. In

  6. E-cadherin cytoplasmic domain inhibits cell surface localization of endogenous cadherins and fusion of C2C12 myoblasts

    Directory of Open Access Journals (Sweden)

    Masayuki Ozawa

    2015-11-01

    Full Text Available Myoblast fusion is a highly regulated process that is essential for skeletal muscle formation during muscle development and regeneration in mammals. Much remains to be elucidated about the molecular mechanism of myoblast fusion although cadherins, which are Ca2+-dependent cell–cell adhesion molecules, are thought to play a critical role in this process. Mouse myoblasts lacking either N-cadherin or M-cadherin can still fuse to form myotubes, indicating that they have no specific function in this process and may be functionally replaced by either M-cadherin or N-cadherin, respectively. In this study, we show that expressing the E-cadherin cytoplasmic domain ectopically in C2C12 myoblasts inhibits cell surface localization of endogenous M-cadherin and N-cadherin, as well as cell–cell fusion. This domain, however, does not inhibit myoblast differentiation according to microarray-based gene expression analysis. In contrast, expressing a dominant-negative β-catenin mutant ectopically, which suppresses Wnt/β-catenin signaling, did not inhibit cell–cell fusion. Therefore, the E-cadherin cytoplasmic domain inhibits cell–cell fusion by inhibiting cell surface localization of endogenous cadherins and not by inhibiting Wnt/β-catenin signaling.

  7. UVC-induced stress granules in mammalian cells.

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    Mohamed Taha Moutaoufik

    Full Text Available Stress granules (SGs are well characterized cytoplasmic RNA bodies that form under various stress conditions. We have observed that exposure of mammalian cells in culture to low doses of UVC induces the formation of discrete cytoplasmic RNA granules that were detected by immunofluorescence staining using antibodies to RNA-binding proteins. UVC-induced cytoplasmic granules are not Processing Bodies (P-bodies and are bone fide SGs as they contain TIA-1, TIA-1/R, Caprin1, FMRP, G3BP1, PABP1, well known markers, and mRNA. Concomitant with the accumulation of the granules in the cytoplasm, cells enter a quiescent state, as they are arrested in G1 phase of the cell cycle in order to repair DNA damages induced by UVC irradiation. This blockage persists as long as the granules are present. A tight correlation between their decay and re-entry into S-phase was observed. However the kinetics of their formation, their low number per cell, their absence of fusion into larger granules, their persistence over 48 hours and their slow decay, all differ from classical SGs induced by arsenite or heat treatment. The induction of these SGs does not correlate with major translation inhibition nor with phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α. We propose that a restricted subset of mRNAs coding for proteins implicated in cell cycling are removed from the translational apparatus and are sequestered in a repressed form in SGs.

  8. Identification of gene fusion transcripts by transcriptome sequencing in BRCA1-mutated breast cancers and cell lines.

    Science.gov (United States)

    Ha, Kevin C H; Lalonde, Emilie; Li, Lili; Cavallone, Luca; Natrajan, Rachael; Lambros, Maryou B; Mitsopoulos, Costas; Hakas, Jarle; Kozarewa, Iwanka; Fenwick, Kerry; Lord, Chris J; Ashworth, Alan; Vincent-Salomon, Anne; Basik, Mark; Reis-Filho, Jorge S; Majewski, Jacek; Foulkes, William D

    2011-10-27

    Gene fusions arising from chromosomal translocations have been implicated in cancer. However, the role of gene fusions in BRCA1-related breast cancers is not well understood. Mutations in BRCA1 are associated with an increased risk for breast cancer (up to 80% lifetime risk) and ovarian cancer (up to 50%). We sought to identify putative gene fusions in the transcriptomes of these cancers using high-throughput RNA sequencing (RNA-Seq). We used Illumina sequencing technology to sequence the transcriptomes of five BRCA1-mutated breast cancer cell lines, three BRCA1-mutated primary tumors, two secretory breast cancer primary tumors and one non-tumorigenic breast epithelial cell line. Using a bioinformatics approach, our initial attempt at discovering putative gene fusions relied on analyzing single-end reads and identifying reads that aligned across exons of two different genes. Subsequently, latter samples were sequenced with paired-end reads and at longer cycles (producing longer reads). We then refined our approach by identifying misaligned paired reads, which may flank a putative gene fusion junction. As a proof of concept, we were able to identify two previously characterized gene fusions in our samples using both single-end and paired-end approaches. In addition, we identified three novel in-frame fusions, but none were recurrent. Two of the candidates, WWC1-ADRBK2 in HCC3153 cell line and ADNP-C20orf132 in a primary tumor, were confirmed by Sanger sequencing and RT-PCR. RNA-Seq expression profiling of these two fusions showed a distinct overexpression of the 3' partner genes, suggesting that its expression may be under the control of the 5' partner gene's regulatory elements. In this study, we used both single-end and paired-end sequencing strategies to discover gene fusions in breast cancer transcriptomes with BRCA1 mutations. We found that the use of paired-end reads is an effective tool for transcriptome profiling of gene fusions. Our findings suggest that

  9. Identification of gene fusion transcripts by transcriptome sequencing in BRCA1-mutated breast cancers and cell lines

    Directory of Open Access Journals (Sweden)

    Ha Kevin CH

    2011-10-01

    Full Text Available Abstract Background Gene fusions arising from chromosomal translocations have been implicated in cancer. However, the role of gene fusions in BRCA1-related breast cancers is not well understood. Mutations in BRCA1 are associated with an increased risk for breast cancer (up to 80% lifetime risk and ovarian cancer (up to 50%. We sought to identify putative gene fusions in the transcriptomes of these cancers using high-throughput RNA sequencing (RNA-Seq. Methods We used Illumina sequencing technology to sequence the transcriptomes of five BRCA1-mutated breast cancer cell lines, three BRCA1-mutated primary tumors, two secretory breast cancer primary tumors and one non-tumorigenic breast epithelial cell line. Using a bioinformatics approach, our initial attempt at discovering putative gene fusions relied on analyzing single-end reads and identifying reads that aligned across exons of two different genes. Subsequently, latter samples were sequenced with paired-end reads and at longer cycles (producing longer reads. We then refined our approach by identifying misaligned paired reads, which may flank a putative gene fusion junction. Results As a proof of concept, we were able to identify two previously characterized gene fusions in our samples using both single-end and paired-end approaches. In addition, we identified three novel in-frame fusions, but none were recurrent. Two of the candidates, WWC1-ADRBK2 in HCC3153 cell line and ADNP-C20orf132 in a primary tumor, were confirmed by Sanger sequencing and RT-PCR. RNA-Seq expression profiling of these two fusions showed a distinct overexpression of the 3' partner genes, suggesting that its expression may be under the control of the 5' partner gene's regulatory elements. Conclusions In this study, we used both single-end and paired-end sequencing strategies to discover gene fusions in breast cancer transcriptomes with BRCA1 mutations. We found that the use of paired-end reads is an effective tool for

  10. Zipping into fusion

    NARCIS (Netherlands)

    Zheng, Tingting

    2014-01-01

    Fusion of lipid bilayers in cells facilitates the active transport of chemicals. Non-viral membrane fusion is regulated by a cascade of proteins as the process is highly regulated both in space and time. In eukaryotic cells, the so-called SNARE protein complex is at the heart of fusion. However,

  11. Targeted delivery of siRNA into breast cancer cells via phage fusion proteins.

    Science.gov (United States)

    Bedi, Deepa; Gillespie, James W; Petrenko, Vasily A; Ebner, Andreas; Leitner, Michael; Hinterdorfer, Peter; Petrenko, Valery A

    2013-02-04

    Nucleic acids, including antisense oligonucleotides, small interfering RNA (siRNA), aptamers, and rybozymes, emerged as versatile therapeutics due to their ability to interfere in a well-planned manner with the flow of genetic information from DNA to protein. However, a systemic use of NAs is hindered by their instability in physiological liquids and inability of intracellular accumulation in the site of action. We first evaluated the potential of cancer specific phage fusion proteins as targeting ligands that provide encapsulation, protection, and navigation of siRNA to the target cell. The tumor-specific proteins were isolated from phages that were affinity selected from a landscape phage library against target breast cancer cells. It was found that fusion phage coat protein fpVIII displaying cancer-targeting peptides can effectively encapsulate siRNAs and deliver them into the cells leading to specific silencing of the model gene GAPDH. Complexes of siRNA and phage protein form nanoparticles (nanophages), which were characterized by atomic force microscopy and ELISA, and their stability was demonstrated by resistance of encapsulated siRNA to degradation by serum nucleases. The phage protein/siRNA complexes can make a new type of highly selective, stable, active, and physiologically acceptable cancer nanomedicine.

  12. Recombinant GDNF: Tetanus toxin fragment C fusion protein produced from insect cells

    International Nuclear Information System (INIS)

    Li, Jianhong; Chian, Ru-Ju; Ay, Ilknur; Celia, Samuel A.; Kashi, Brenda B.; Tamrazian, Eric; Matthews, Jonathan C.; Remington, Mary P.; Pepinsky, R. Blake; Fishman, Paul S.; Brown, Robert H.; Francis, Jonathan W.

    2009-01-01

    Glial cell line-derived neurotrophic factor (GDNF) has potent survival-promoting effects on CNS motor neurons in experimental animals. Its therapeutic efficacy in humans, however, may have been limited by poor bioavailability to the brain and spinal cord. With a view toward improving delivery of GDNF to CNS motor neurons in vivo, we generated a recombinant fusion protein comprised of rat GDNF linked to the non-toxic, neuron-binding fragment of tetanus toxin. Recombinant GDNF:TTC produced from insect cells was a soluble homodimer like wild-type GDNF and was bi-functional with respect to GDNF and TTC activity. Like recombinant rat GDNF, the fusion protein increased levels of immunoreactive phosphoAkt in treated NB41A3-hGFRα-1 neuroblastoma cells. Like TTC, GDNF:TTC bound to immobilized ganglioside GT1b in vitro with high affinity and selectivity. These results support further testing of recombinant GDNF:TTC as a non-viral vector to improve delivery of GDNF to brain and spinal cord in vivo.

  13. Recombinant GDNF: Tetanus toxin fragment C fusion protein produced from insect cells

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jianhong; Chian, Ru-Ju; Ay, Ilknur; Celia, Samuel A.; Kashi, Brenda B.; Tamrazian, Eric; Matthews, Jonathan C. [Cecil B. Day Laboratory for Neuromuscular Research, Department of Neurology, Massachusetts General Hospital, Charlestown, MA 02129 (United States); Remington, Mary P. [Research Service, Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201 (United States); Pepinsky, R. Blake [BiogenIdec, Inc., 14 Cambridge Center, Cambridge, MA 02142 (United States); Fishman, Paul S. [Research Service, Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201 (United States); Department of Neurology, University of Maryland School of Medicine, Baltimore, MD 21201 (United States); Brown, Robert H. [Cecil B. Day Laboratory for Neuromuscular Research, Department of Neurology, Massachusetts General Hospital, Charlestown, MA 02129 (United States); Francis, Jonathan W., E-mail: jwfrancisby@gmail.com [Cecil B. Day Laboratory for Neuromuscular Research, Department of Neurology, Massachusetts General Hospital, Charlestown, MA 02129 (United States)

    2009-07-31

    Glial cell line-derived neurotrophic factor (GDNF) has potent survival-promoting effects on CNS motor neurons in experimental animals. Its therapeutic efficacy in humans, however, may have been limited by poor bioavailability to the brain and spinal cord. With a view toward improving delivery of GDNF to CNS motor neurons in vivo, we generated a recombinant fusion protein comprised of rat GDNF linked to the non-toxic, neuron-binding fragment of tetanus toxin. Recombinant GDNF:TTC produced from insect cells was a soluble homodimer like wild-type GDNF and was bi-functional with respect to GDNF and TTC activity. Like recombinant rat GDNF, the fusion protein increased levels of immunoreactive phosphoAkt in treated NB41A3-hGFR{alpha}-1 neuroblastoma cells. Like TTC, GDNF:TTC bound to immobilized ganglioside GT1b in vitro with high affinity and selectivity. These results support further testing of recombinant GDNF:TTC as a non-viral vector to improve delivery of GDNF to brain and spinal cord in vivo.

  14. Fusion of bone marrow-derived cells with renal tubules contributes to renal dysfunction in diabetic nephropathy.

    Science.gov (United States)

    Yamashita, Tomohisa; Fujimiya, Mineko; Nagaishi, Kanna; Ataka, Koji; Tanaka, Marenao; Yoshida, Hideaki; Tsuchihashi, Kazufumi; Shimamoto, Kazuaki; Miura, Tetsuji

    2012-04-01

    Although diabetic nephropathy (DN) is a major cause of end-stage renal disease, the mechanism of dysfunction has not yet been clarified. We previously reported that in diabetes proinsulin-producing bone marrow-derived cells (BMDCs) fuse with hepatocytes and neurons. Fusion cells are polyploidy and produce tumor necrosis factor (TNF)-α, ultimately causing diabetic complications. In this study, we assessed whether the same mechanism is involved in DN. We performed bone marrow transplantation from male GFP-Tg mice to female C57BL/6J mice and produced diabetes by streptozotocin (STZ) or a high-fat diet. In diabetic kidneys, massive infiltration of BMDCs and tubulointerstitial injury were prominent. BMDCs and damaged tubular epithelial cells were positively stained with proinsulin and TNF-α. Cell fusion between BMDCs and renal tubules was confirmed by the presence of Y chromosome. Of tubular epithelial cells, 15.4% contain Y chromosomes in STZ-diabetic mice, 8.6% in HFD-diabetic mice, but only 1.5% in nondiabetic mice. Fusion cells primarily expressed TNF-α and caspase-3 in diabetic kidney. These in vivo findings were confirmed by in vitro coculture experiments between isolated renal tubular cells and BMDCs. It was concluded that cell fusion between BMDCs and renal tubular epithelial cells plays a crucial role in DN.

  15. Microdroplet fusion mass spectrometry: accelerated kinetics of acid-induced chlorophyll demetallation.

    Science.gov (United States)

    Lee, Jae Kyoo; Nam, Hong Gil; Zare, Richard N

    2017-01-01

    Kinetics of acid-induced chlorophyll demetallation was recorded in microdroplets by fusing a stream of microdroplets containing 40 µM chlorophyll a or b dissolved in methanol with a stream of aqueous microdroplets containing 35 mM hydrochloric acid (pH = 1·46). The kinetics of the demetallation of chlorophyll in the fused microdroplets (14 ± 6 µm diameter; 84 ± 18 m s-1 velocity) was recorded by controlling the traveling distance of the fused microdroplets between the fusion region and the inlet of a mass spectrometer. The rate of acid-induced chlorophyll demetallation was about 960 ± 120 times faster in the charged microdroplets compared with that reported in bulk solution. If no voltage was applied to the sprayed microdroplets, then the acceleration factor was about 580 ± 90, suggesting that the applied voltage is not a major factor determining the acceleration. Chlorophyll a was more rapidly demetallated than chlorophyll b by a factor of ~26 in bulk solution and ~5 in charged microdroplets. The demetallation kinetics was second order in the H+ concentration, but the acceleration factor of microdroplets compared with bulk solution appeared to be unchanged in going from pH = 1·3 to 7·0. The water:methanol ratio of the fused microdroplets was varied from 7:3 to 3:7 causing an increase in the reaction rate of chlorophyll a demetallation by 20%. This observation demonstrates that the solvent composition, which has different evaporation rates, does not significantly affect the acceleration. We believe that a major portion of the acceleration can be attributed to confinement effects involving surface reactions rather than either to evaporation of solvents or to the introduction of charges to the microdroplets.

  16. Transfer of an expression YAC into goat fetal fibroblasts by cell fusion for mammary gland bioreactor

    International Nuclear Information System (INIS)

    Zhang Xufeng; Wu Guoxiang; Chen, Jian-Quan; Zhang Aimin; Liu Siguo; Jiao Binghua; Cheng Guoxiang

    2005-01-01

    Yeast artificial chromosomes (YACs) as transgenes in transgenic animals are likely to ensure optimal expression levels. Microinjection of YACs is the exclusive technique used to produce YACs transgenic livestock so far. However, low efficiency and high cost are its critical restrictive factors. In this study, we presented a novel procedure to produce YACs transgenic livestock as mammary gland bioreactor. A targeting vector, containing the gene of interest-a human serum albumin minigene (intron 1, 2), yeast selectable marker (G418R), and mammalian cell resistance marker (neo r ), replaced the α-lactalbumin gene in a 210 kb human α-lactalbumin YAC by homogeneous recombination in yeasts. The chimeric YAC was introduced into goat fetal fibroblasts using polyethylene glycol-mediated spheroplast fusion. PCR and Southern analysis showed that intact YAC was integrated in the genome of resistant cells. Perhaps, it may offer a cell-based route by nuclear transfer to produce YACs transgenic livestock

  17. A Polymorphism within the Internal Fusion Loop of the Ebola Virus Glycoprotein Modulates Host Cell Entry.

    Science.gov (United States)

    Hoffmann, Markus; Crone, Lisa; Dietzel, Erik; Paijo, Jennifer; González-Hernández, Mariana; Nehlmeier, Inga; Kalinke, Ulrich; Becker, Stephan; Pöhlmann, Stefan

    2017-05-01

    The large scale of the Ebola virus disease (EVD) outbreak in West Africa in 2013-2016 raised the question whether the host cell interactions of the responsible Ebola virus (EBOV) strain differed from those of other ebolaviruses. We previously reported that the glycoprotein (GP) of the virus circulating in West Africa in 2014 (EBOV2014) exhibited reduced ability to mediate entry into two nonhuman primate (NHP)-derived cell lines relative to the GP of EBOV1976. Here, we investigated the molecular determinants underlying the differential entry efficiency. We found that EBOV2014-GP-driven entry into diverse NHP-derived cell lines, as well as human monocyte-derived macrophages and dendritic cells, was reduced compared to EBOV1976-GP, although entry into most human- and all bat-derived cell lines tested was comparable. Moreover, EBOV2014 replication in NHP but not human cells was diminished relative to EBOV1976, suggesting that reduced cell entry translated into reduced viral spread. Mutagenic analysis of EBOV2014-GP and EBOV1976-GP revealed that an amino acid polymorphism in the receptor-binding domain, A82V, modulated entry efficiency in a cell line-independent manner and did not account for the reduced EBOV2014-GP-driven entry into NHP cells. In contrast, polymorphism T544I, located in the internal fusion loop in the GP2 subunit, was found to be responsible for the entry phenotype. These results suggest that position 544 is an important determinant of EBOV infectivity for both NHP and certain human target cells. IMPORTANCE The Ebola virus disease outbreak in West Africa in 2013 entailed more than 10,000 deaths. The scale of the outbreak and its dramatic impact on human health raised the question whether the responsible virus was particularly adept at infecting human cells. Our study shows that an amino acid exchange, A82V, that was acquired during the epidemic and that was not observed in previously circulating viruses, increases viral entry into diverse target cells

  18. CRISPR/Cas9 Engineering of Adult Mouse Liver Demonstrates That the Dnajb1-Prkaca Gene Fusion Is Sufficient to Induce Tumors Resembling Fibrolamellar Hepatocellular Carcinoma.

    Science.gov (United States)

    Engelholm, Lars H; Riaz, Anjum; Serra, Denise; Dagnæs-Hansen, Frederik; Johansen, Jens V; Santoni-Rugiu, Eric; Hansen, Steen H; Niola, Francesco; Frödin, Morten

    2017-12-01

    Fibrolamellar hepatocellular carcinoma (FL-HCC) is a primary liver cancer that predominantly affects children and young adults with no underlying liver disease. A somatic, 400 Kb deletion on chromosome 19 that fuses part of the DnaJ heat shock protein family (Hsp40) member B1 gene (DNAJB1) to the protein kinase cAMP-activated catalytic subunit alpha gene (PRKACA) has been repeatedly identified in patients with FL-HCC. However, the DNAJB1-PRKACA gene fusion has not been shown to induce liver tumorigenesis. We used the CRISPR/Cas9 technique to delete in mice the syntenic region on chromosome 8 to create a Dnajb1-Prkaca fusion and monitored the mice for liver tumor development. We delivered CRISPR/Cas9 vectors designed to juxtapose exon 1 of Dnajb1 with exon 2 of Prkaca to create the Dnajb1-Prkaca gene fusion associated with FL-HCC, or control Cas9 vector, via hydrodynamic tail vein injection to livers of 8-week-old female FVB/N mice. These mice did not have any other engineered genetic alterations and were not exposed to liver toxins or carcinogens. Liver tissues were collected 14 months after delivery; genomic DNA was analyzed by PCR to detect the Dnajb1-Prkaca fusion, and tissues were characterized by histology, immunohistochemistry, RNA sequencing, and whole-exome sequencing. Livers from 12 of the 15 mice given the vectors to induce the Dnajb1-Prkaca gene fusion, but none of the 11 mice given the control vector, developed neoplasms. The tumors contained the Dnajb1-Prkaca gene fusion and had histologic and cytologic features of human FL-HCCs: large polygonal cells with granular, eosinophilic, and mitochondria-rich cytoplasm, prominent nucleoli, and markers of hepatocytes and cholangiocytes. In comparing expression levels of genes between the mouse tumor and non-tumor liver cells, we identified changes similar to those detected in human FL-HCC, which included genes that affect cell cycle and mitosis regulation. Genomic analysis of mouse neoplasms induced by

  19. Inducible expression of a fusion gene encoding two proteinase inhibitors leads to insect and pathogen resistance in transgenic rice.

    Science.gov (United States)

    Quilis, Jordi; López-García, Belén; Meynard, Donaldo; Guiderdoni, Emmanuel; San Segundo, Blanca

    2014-04-01

    Plant proteinase inhibitors (PIs) are considered as candidates for increased insect resistance in transgenic plants. Insect adaptation to PI ingestion might, however, compromise the benefits received by transgenic expression of PIs. In this study, the maize proteinase inhibitor (MPI), an inhibitor of insect serine proteinases, and the potato carboxypeptidase inhibitor (PCI) were fused into a single open reading frame and introduced into rice plants. The two PIs were linked using either the processing site of the Bacillus thuringiensis Cry1B precursor protein or the 2A sequence from the foot-and-mouth disease virus (FMDV). Expression of each fusion gene was driven by the wound- and pathogen-inducible mpi promoter. The mpi-pci fusion gene was stably inherited for at least three generations with no penalty on plant phenotype. An important reduction in larval weight of Chilo suppressalis fed on mpi-pci rice, compared with larvae fed on wild-type plants, was observed. Expression of the mpi-pci fusion gene confers resistance to C. suppressalis (striped stem borer), one of the most important insect pest of rice. The mpi-pci expression systems described may represent a suitable strategy for insect pest control, better than strategies based on the use of single PI genes, by preventing insect adaptive responses. The rice plants expressing the mpi-pci fusion gene also showed enhanced resistance to infection by the fungus Magnaporthe oryzae, the causal agent of the rice blast disease. Our results illustrate the usefulness of the inducible expression of the mpi-pci fusion gene for dual resistance against insects and pathogens in rice plants. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  20. Fluorescence fluctuation analysis of BACE1-GFP fusion protein in cultured HEK293 cells

    Science.gov (United States)

    Gardeen, Spencer; Johnson, Joseph L.; Heikal, Ahmed A.

    2016-10-01

    Beta-site APP cleaving enzyme 1 (BACE1) is a type I transmembrane aspartyl protease. In the amyloidogenic pathway, BACE1 provides β-secretase activity that cleaves the amyloid precursor protein (APP) that leads to amyloid beta (Aβ) peptides. The aggregation of these Aβ will ultimately results in amyloid plaque formation, a hallmark of Alzheimer's disease (AD). Amyloid aggregation leads to progressive memory impairment and neural loss. Recent detergent protein extraction studies suggest that the untreated BACE1 protein forms a dimer that has significantly higher catalytic activity than its monomeric counterpart. Here, we examine the dimerization hypothesis of BACE1 in cultured HEK293 cells using fluorescence correlation spectroscopy (FCS). Cells were transfected with a BACE1-EGFP fusion protein construct and imaged using confocal and DIC microscopy to monitor labeled BACE1 localization and distribution within the cell. Our one-photon fluorescence fluctuation autocorrelation of BACE1- EGFP on the plasma membrane of HEK cells is modeled using two diffusing species on the plasma membrane with estimated diffusion coefficients of 1.39 x 10-7 cm2/sec and 2.8 x 10-8 cm2/sec under resting conditions and STA-200 inhibition, respectively. Anomalous diffusion model also provided adequate description of the observed autocorrelation function of BACE1- EGFP on the plasma membrane with an estimate exponent (α) of 0.8 and 0.5 for resting and STA-200 treated cells, respectively. The corresponding hydrodynamic radius of this transmembrane fusion protein was estimated using the measured diffusion coefficients assuming both Stokes-Einstein and Saffman-Delbruck models. Our results suggest a complex diffusion pattern of BACE1-EGFP on the plasma membrane of HEK cells with the possibility for dimer formation, especially under STA-200 inhibition.

  1. NUP98-PHF23 fusion is recurrent in acute myeloid leukemia and shares gene expression signature of leukemic stem cells.

    Science.gov (United States)

    Ho, Hao; Skaist, Alyza M; Pallavajjala, Aparna; Yonescu, Raluca; Batista, Denise; Wheelan, Sarah J; Ning, Yi

    2016-06-01

    Chromosome translocations involving nucleoporin 98 gene (NUP98) have been identified in a wide array of hematologic malignancies, and the resulting NUP98-associated fusions are known to play a critical role in leukemogensis through dysregulation of gene expression. Although NUP98-associated fusions were initially thought to be rare, application of molecular technologies has revealed that cryptic translocations involving NUP98 are more frequent than previously appreciated. We report an additional case of t(11;17)(p15;p13) resulting in the fusion of NUP98 and plant homeodomain finger 23 (PHF23) in a pediatric patient with acute myeloid leukemia (AML). Using RNA sequencing, we determined in-frame fusion points and also analyzed the gene expression profile of NUP98-PHF23 positive AML. Gene set enrichment analysis (GSEA) demonstrates that NUP98-PHF23 fusion shares gene expression signature of NUP98-HOXA9 fusion, the prototype of the NUP98-associated fusions, as well as the signature of leukemic stem cells. To our knowledge this is the first transcriptome analysis of human samples with NUP98-PHF23 positive AML. Our findings are in support of the gene expression study of NUP98-PHF23 mouse model and validate the usefulness of the mouse model in developing therapeutic strategies for the treatment of subsets of AML. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Molecular Process Producing Oncogene Fusion in Lung Cancer Cells by Illegitimate Repair of DNA Double-Strand Breaks

    Directory of Open Access Journals (Sweden)

    Yoshitaka Seki

    2015-09-01

    Full Text Available Constitutive activation of oncogenes by fusion to partner genes, caused by chromosome translocation and inversion, is a critical genetic event driving lung carcinogenesis. Fusions of the tyrosine kinase genes ALK (anaplastic lymphoma kinase, ROS1 (c-ros oncogene 1, or RET (rearranged during transfection occur in 1%–5% of lung adenocarcinomas (LADCs and their products constitute therapeutic targets for kinase inhibitory drugs. Interestingly, ALK, RET, and ROS1 fusions occur preferentially in LADCs of never- and light-smokers, suggesting that the molecular mechanisms that cause these rearrangements are smoking-independent. In this study, using previously reported next generation LADC genome sequencing data of the breakpoint junction structures of chromosome rearrangements that cause oncogenic fusions in human cancer cells, we employed the structures of breakpoint junctions of ALK, RET, and ROS1 fusions in 41 LADC cases as “traces” to deduce the molecular processes of chromosome rearrangements caused by DNA double-strand breaks (DSBs and illegitimate joining. We found that gene fusion was produced by illegitimate repair of DSBs at unspecified sites in genomic regions of a few kb through DNA synthesis-dependent or -independent end-joining pathways, according to DSB type. This information will assist in the understanding of how oncogene fusions are generated and which etiological factors trigger them.

  3. Protective effects of a bacterially expressed NIF-KGF fusion protein against bleomycin-induced acute lung injury in mice.

    Science.gov (United States)

    Li, Xinping; Li, Shengli; Zhang, Miaotao; Li, Xiukun; Zhang, Xiaoming; Zhang, Wenlong; Li, Chuanghong

    2010-08-01

    Current evidence suggests that the keratinocyte growth factor (KGF) and the polymorphonuclear leukocyte may play key roles in the development of lung fibrosis. Here we describe the construction, expression, purification, and identification of a novel NIF (neutrophil inhibitory factor)-KGF mutant fusion protein (NKM). The fusion gene was ligated via a flexible octapeptide hinge and expressed as an insoluble protein in Escherichia coli BL21 (DE3). The fusion protein retained the activities of KGF and NIF, as it inhibited both fibroblast proliferation and leukocyte adhesion. Next, the effects of NKM on bleomycin-induced lung fibrosis in mice were examined. The mice were divided into the following four groups: (i) saline group; (ii) bleomycin group (instilled with 5 mg/kg bleomycin intratracheally); (iii) bleomycin plus dexamethasone (Dex) group (Dex was given intraperitoneally (i.p.) at 1 mg/kg/day 2 days prior to bleomycin instillation and daily after bleomycin instillation until the end of the treatment); and (iv) bleomycin plus NKM group (NKM was given i.p. at 2 mg/kg/day using the same protocol as the Dex group). NKM significantly improved the survival rates of mice exposed to bleomycin. The marked morphological changes and increased hydroxyproline levels resulted from the instillation of bleomycin (on Day 17) in the lungs were significantly inhibited by NKM. These results revealed that NKM can attenuate bleomycin-induced lung fibrosis, suggesting that NKM could be used to prevent bleomycin-induced lung damage or other interstitial pulmonary fibrosis.

  4. Lipopolysaccharide induces multinuclear cell from RAW264.7 line with increased phagocytosis activity

    Energy Technology Data Exchange (ETDEWEB)

    Nakanishi-Matsui, Mayumi, E-mail: nakanim@iwate-med.ac.jp [Department of Biochemistry, Faculty of Pharmaceutical Sciences, Iwate Medical University, Futai Special Laboratory, Yahaba, Iwate 028-3694 (Japan); Yano, Shio; Matsumoto, Naomi; Futai, Masamitsu [Department of Biochemistry, Faculty of Pharmaceutical Sciences, Iwate Medical University, Futai Special Laboratory, Yahaba, Iwate 028-3694 (Japan)

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer LPS induces multinuclear cells from murine macrophage-derived RAW264.7 cells. Black-Right-Pointing-Pointer The multinuclear cells are formed through cell-cell fusion in the presence of Ca{sup 2+}. Black-Right-Pointing-Pointer The multinuclear cells do not express osteoclast-specific enzymes. Black-Right-Pointing-Pointer They internalized more and larger beads than mononuclear cells and osteoclasts. -- Abstract: Lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria, induces strong proinflammatory responses, including the release of cytokines and nitric oxide from macrophage. In this study, we found that a murine macrophage-derived line, RAW264.7, became multinuclear through cell-cell fusion after incubation with highly purified LPS or synthetic lipid A in the presence of Ca{sup 2+}. The same cell line is known to differentiate into multinuclear osteoclast, which expresses a specific proton pumping ATPase together with osteoclast markers on stimulation by the extracellular domain of receptor activator of nuclear factor {kappa}B ligand (Toyomura, T., Murata, Y., Yamamoto, A., Oka, T., Sun-Wada, G.-H., Wada, Y. and Futai, M., 2003). The LPS-induced multinuclear cells did not express osteoclast-specific enzymes including tartrate-resistant acid phosphatase and cathepsin K. During multinuclear cell formation, the cells internalized more and larger polystyrene beads (diameter 6-15 {mu}m) than mononuclear cells and osteoclasts. The internalized beads were located in lysosome-marker positive organelles, which were probably phagolysosomes. The LPS-induced multinuclear cell could be a good model system to study phagocytosis of large foreign bodies.

  5. Fusion Machinery

    DEFF Research Database (Denmark)

    Sørensen, Jakob Balslev; Milosevic, Ira

    2015-01-01

    SNARE proteins constitute the minimal machinery needed for membrane fusion. SNAREs operate by forming a complex, which pulls the lipid bilayers into close contact and provides the mechanical force needed for lipid bilayer fusion. At the chemical synapse, SNARE-complex formation between...... the vesicular SNARE VAMP2/synaptobrevin-2 and the target (plasma membrane) SNAREs SNAP25 and syntaxin-1 results in fusion and release of neurotransmitter, synchronized to the electrical activity of the cell by calcium influx and binding to synaptotagmin. Formation of the SNARE complex is tightly regulated...... and appears to start with syntaxin-1 bound to an SM (Sec1/Munc18-like) protein. Proteins of the Munc13-family are responsible for opening up syntaxin and allowing sequential binding of SNAP-25 and VAMP2/synaptobrevin-2. N- to C-terminal “zippering” of the SNARE domains leads to membrane fusion...

  6. Maintainability considerations for the central cell in WITAMIR-I, a conceptual design of a tandem mirror fusion power reactor

    International Nuclear Information System (INIS)

    Sviatoslavsky, I.N.

    1980-10-01

    The concepts for maintaining the central cell reactor components for WITAMIR-I are described. WITAMIR-I is a conceptual tandem mirror fusion power reactor utilizing thermal barriers designed by the University of Wisconsin-Madison. Unique solutions to the difficult problems of routine blanket replacement and maintenance are proposed. Solutions are also proposed for maintaining the central cell coils and the shield

  7. The natural product peiminine represses colorectal carcinoma tumor growth by inducing autophagic cell death

    Energy Technology Data Exchange (ETDEWEB)

    Lyu, Qing [School of Life Sciences, Tsinghua University, Beijing, 100084 (China); Key Lab in Healthy Science and Technology, Division of Life Science, Graduate School at Shenzhen, Tsinghua University, Shenzhen, 518055 (China); Tou, Fangfang [Jiangxi Provincial Key Lab of Oncology Translation Medicine, Jiangxi Cancer Hospital, Nanchang, 330029 (China); Su, Hong; Wu, Xiaoyong [First Affiliated Hospital, Guiyang College of Traditional Chinese Medicine, Guiyang, 550002 (China); Chen, Xinyi [Department of Hematology and Oncology, Beijing University of Chinese Medicine, Beijing, 100029 (China); Zheng, Zhi, E-mail: zheng_sheva@hotmail.com [Jiangxi Provincial Key Lab of Oncology Translation Medicine, Jiangxi Cancer Hospital, Nanchang, 330029 (China)

    2015-06-19

    Autophagy is evolutionarily conservative in eukaryotic cells that engulf cellular long-lived proteins and organelles, and it degrades the contents through fusion with lysosomes, via which the cell acquires recycled building blocks for the synthesis of new molecules. In this study, we revealed that peiminine induces cell death and enhances autophagic flux in colorectal carcinoma HCT-116 cells. We determined that peiminine enhances the autophagic flux by repressing the phosphorylation of mTOR through inhibiting upstream signals. Knocking down ATG5 greatly reduced the peiminine-induced cell death in wild-type HCT-116 cells, while treating Bax/Bak-deficient cells with peiminine resulted in significant cell death. In summary, our discoveries demonstrated that peiminine represses colorectal carcinoma cell proliferation and cell growth by inducing autophagic cell death. - Highlights: • Peiminine induces autophagy and upregulates autophagic flux. • Peiminine represses colorectal carcinoma tumor growth. • Peiminine induces autophagic cell death. • Peiminine represses mTOR phosphorylation by influencing PI3K/Akt and AMPK pathway.

  8. The natural product peiminine represses colorectal carcinoma tumor growth by inducing autophagic cell death

    International Nuclear Information System (INIS)

    Lyu, Qing; Tou, Fangfang; Su, Hong; Wu, Xiaoyong; Chen, Xinyi; Zheng, Zhi

    2015-01-01

    Autophagy is evolutionarily conservative in eukaryotic cells that engulf cellular long-lived proteins and organelles, and it degrades the contents through fusion with lysosomes, via which the cell acquires recycled building blocks for the synthesis of new molecules. In this study, we revealed that peiminine induces cell death and enhances autophagic flux in colorectal carcinoma HCT-116 cells. We determined that peiminine enhances the autophagic flux by repressing the phosphorylation of mTOR through inhibiting upstream signals. Knocking down ATG5 greatly reduced the peiminine-induced cell death in wild-type HCT-116 cells, while treating Bax/Bak-deficient cells with peiminine resulted in significant cell death. In summary, our discoveries demonstrated that peiminine represses colorectal carcinoma cell proliferation and cell growth by inducing autophagic cell death. - Highlights: • Peiminine induces autophagy and upregulates autophagic flux. • Peiminine represses colorectal carcinoma tumor growth. • Peiminine induces autophagic cell death. • Peiminine represses mTOR phosphorylation by influencing PI3K/Akt and AMPK pathway

  9. Effective myotube formation in human adipose tissue-derived stem cells expressing dystrophin and myosin heavy chain by cellular fusion with mouse C2C12 myoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Eom, Young Woo [Cell Therapy and Tissue Engineering Center, Wonju College of Medicine, Yonsei Univ., Wonju (Korea, Republic of); Biomedical Research Institute, Lifeliver Co., Ltd., Suwon (Korea, Republic of); Lee, Jong Eun; Yang, Mal Sook; Jang, In Keun; Kim, Hyo Eun; Lee, Doo Hoon; Kim, Young Jin [Biomedical Research Institute, Lifeliver Co., Ltd., Suwon (Korea, Republic of); Park, Won Jin [Dr. Park' s Aesthetic Clinic, Seoul (Korea, Republic of); Kong, Jee Hyun; Shim, Kwang Yong [Department of Hematology-Oncology, Wonju College of Medicine, Yonsei Univ., Wonju (Korea, Republic of); Lee, Jong In, E-mail: oncochem@yonsei.ac.kr [Department of Hematology-Oncology, Wonju College of Medicine, Yonsei Univ., Wonju (Korea, Republic of); Kim, Hyun Soo, E-mail: khsmd@unitel.co.kr [Department of Hematology-Oncology, Wonju College of Medicine, Yonsei Univ., Wonju (Korea, Republic of)

    2011-04-29

    Highlights: {yields} hASCs were differentiated into skeletal muscle cells by treatment with 5-azacytidine, FGF-2, and the supernatant of cultured hASCs. {yields} Dystrophin and MyHC were expressed in late differentiation step by treatment with the supernatant of cultured hASCs. {yields} hASCs expressing dystrophin and MyHC contributed to myotube formation during co-culture with mouse myoblast C2C12 cells. -- Abstract: Stem cell therapy for muscular dystrophies requires stem cells that are able to participate in the formation of new muscle fibers. However, the differentiation steps that are the most critical for this process are not clear. We investigated the myogenic phases of human adipose tissue-derived stem cells (hASCs) step by step and the capability of myotube formation according to the differentiation phase by cellular fusion with mouse myoblast C2C12 cells. In hASCs treated with 5-azacytidine and fibroblast growth factor-2 (FGF-2) for 1 day, the early differentiation step to express MyoD and myogenin was induced by FGF-2 treatment for 6 days. Dystrophin and myosin heavy chain (MyHC) expression was induced by hASC conditioned medium in the late differentiation step. Myotubes were observed only in hASCs undergoing the late differentiation step by cellular fusion with C2C12 cells. In contrast, hASCs that were normal or in the early stage were not involved in myotube formation. Our results indicate that stem cells expressing dystrophin and MyHC are more suitable for myotube formation by co-culture with myoblasts than normal or early differentiated stem cells expressing MyoD and myogenin.

  10. Systematic mapping of occluded genes by cell fusion reveals prevalence and stability of cis-mediated silencing in somatic cells

    Science.gov (United States)

    Looney, Timothy J.; Zhang, Li; Chen, Chih-Hsin; Lee, Jae Hyun; Chari, Sheila; Mao, Frank Fuxiang; Pelizzola, Mattia; Zhang, Lu; Lister, Ryan; Baker, Samuel W.; Fernandes, Croydon J.; Gaetz, Jedidiah; Foshay, Kara M.; Clift, Kayla L.; Zhang, Zhenyu; Li, Wei-Qiang; Vallender, Eric J.; Wagner, Ulrich; Qin, Jane Yuxia; Michelini, Katelyn J.; Bugarija, Branimir; Park, Donghyun; Aryee, Emmanuel; Stricker, Thomas; Zhou, Jie; White, Kevin P.; Ren, Bing; Schroth, Gary P.; Ecker, Joseph R.; Xiang, Andy Peng; Lahn, Bruce T.

    2014-01-01

    Both diffusible factors acting in trans and chromatin components acting in cis are implicated in gene regulation, but the extent to which either process causally determines a cell's transcriptional identity is unclear. We recently used cell fusion to define a class of silent genes termed “cis-silenced” (or “occluded”) genes, which remain silent even in the presence of trans-acting transcriptional activators. We further showed that occlusion of lineage-inappropriate genes plays a critical role in maintaining the transcriptional identities of somatic cells. Here, we present, for the first time, a comprehensive map of occluded genes in somatic cells. Specifically, we mapped occluded genes in mouse fibroblasts via fusion to a dozen different rat cell types followed by whole-transcriptome profiling. We found that occluded genes are highly prevalent and stable in somatic cells, representing a sizeable fraction of silent genes. Occluded genes are also highly enriched for important developmental regulators of alternative lineages, consistent with the role of occlusion in safeguarding cell identities. Alongside this map, we also present whole-genome maps of DNA methylation and eight other chromatin marks. These maps uncover a complex relationship between chromatin state and occlusion. Furthermore, we found that DNA methylation functions as the memory of occlusion in a subset of occluded genes, while histone deacetylation contributes to the implementation but not memory of occlusion. Our data suggest that the identities of individual cell types are defined largely by the occlusion status of their genomes. The comprehensive reference maps reported here provide the foundation for future studies aimed at understanding the role of occlusion in development and disease. PMID:24310002

  11. Aloin induces apoptosis in Jurkat cells.

    Science.gov (United States)

    Buenz, Eric J

    2008-03-01

    Aloe is widely used as a dietary supplement. However, there are continuing concerns over the toxicity and the purity of aloe-based products. The primary class of compounds responsible for aloe-induced toxicity are anthraquinones. One of these, aloe-emodin, has been extensively investigated for apoptosis inducing effects. Conversely, the precursor to aloe-emodin, aloin, has been subjected to only minimal investigation of any cytotoxic effects. Jurkat T cells, an established model for the study of compound toxicity, were used to evaluate the effect of aloin on cell viability. Cells were analyzed using flow cytometry and microscopy for cell size and granularity, cell membrane integrity, mitochondrial membrane potential, and cell cycle profile. Treatment with aloin resulted in a reduction in cell size, compromised membrane integrity, and loss of mitochondrial membrane potential in a dose-dependent manner. Additionally, treatment with aloin resulted in alteration of the cell cycle, specifically a block at G2/M phase. Importantly, the loss of cell membrane integrity was preceded by a loss of mitochondrial membrane potential, suggesting a mitochondrial-dependent pathway for aloin-induced apoptosis. These observations provide insight into the potential mechanisms of aloin-induced toxicity and thus, perhaps, aloe preparation-induced toxicity. Furthermore, because of the concern over the safety of aloe-based supplements, this work suggests that aloe supplements not containing aloin may be safer than aloe supplements containing aloin, and that aloin should be considered in addition to concentrations of aloe-emodin.

  12. Detection of E2A-PBX1 fusion transcripts in human non-small-cell lung cancer.

    Science.gov (United States)

    Mo, Min-Li; Chen, Zhao; Zhou, Hai-Meng; Li, Hui; Hirata, Tomomi; Jablons, David M; He, Biao

    2013-05-20

    E2A-PBX1 fusion gene caused by t(1;19)(q23;p13), has been well characterized in acute lymphoid leukemia (ALL). There is no report on E2A-PBX1 fusion transcripts in non-small-cell lung cancer (NSCLC). We used polymerase chain reaction (PCR) to detect E2A-PBX1 fusion transcripts in human NSCLC tissue specimens and cell lines. We analyzed correlation of E2A-PBX1 fusion transcripts with clinical outcomes in 76 patients with adenocarcinoma in situ (AIS) and other subgroups. We compared mutation status of k-ras, p53 and EGFR in 22 patients with E2A-PBX1 fusion transcripts. We detected E2A-PBX1 transcripts in 23 of 184 (12.5%) NSCLC tissue specimens and 3 of 13 (23.1%) NSCLC cell lines. Presence of E2A-PBX1 fusion transcripts correlated with smoking status in female patients (P=0.048), AIS histology (P=0.006) and tumor size (P=0.026). The overall survival was associated with gender among AIS patients (P=0.0378) and AIS patients without E2A-PBX1 fusion transcripts (P=0.0345), but not among AIS patients with E2A-PBX1 fusion transcripts (P=0.6401). The overall survival was also associated with status of E2A-PBX1 fusion transcripts among AIS stage IA patients (P=0.0363) and AIS stage IA female patients (P=0.0174). In addition, among the 22 patients with E2A-PBX1 fusion transcripts, 12 (54.5%) patients including all four non-smokers, showed no common mutations in k-ras, p53 and EGFR. E2A-PBX1 fusion gene caused by t(1;19)(q23;p13) may be a common genetic change in AIS and a survival determinant for female AIS patients at early stage.

  13. Transfer of herpes simplex virus thymidine kinase synthesized in bacteria by a high-expression plasmid to tissue culture cells by protoplast fusion

    International Nuclear Information System (INIS)

    Waldman, A.S.; Milman, G.

    1984-01-01

    The introduction of a protein into living tissue culture cells may permit the in vivo study of functions of the protein. The authors have previously described a high-efficiency-expression plasmid, pHETK2, containing the herpes simplex virus type 1 thymidine kinase (TK) gene which, upon temperature induction, causes TK to be synthesized as greater than 4% of the bacterial protein. In this report it is shown that enzymatically active TK was transferred to mouse Ltk- cells by polyethylene glycol-mediated fusion with protoplasts prepared from bacteria containing induced levels of TK. The presence of TK in the Ltk- cells was detected by the incorporation of [ 3 H]thymidine into cell nuclei as measured by autoradiography

  14. A toolkit for graded expression of green fluorescent protein fusion proteins in mammalian cells.

    Science.gov (United States)

    Nalaskowski, Marcus M; Ehm, Patrick; Giehler, Susanne; Mayr, Georg W

    2012-09-01

    Green fluorescent protein (GFP) and GFP-like proteins of different colors are important tools in cell biology. In many studies, the intracellular targeting of proteins has been determined by transiently expressing GFP fusion proteins and analyzing their intracellular localization by fluorescence microscopy. In most vectors, expression of GFP is driven by the enhancer/promoter cassette of the immediate early gene of human cytomegalovirus (hCMV). This cassette generates high levels of protein expression in most mammalian cell lines. Unfortunately, these nonphysiologically high protein levels have been repeatedly reported to artificially alter the intracellular targeting of proteins fused to GFP. To cope with this problem, we generated a multitude of attenuated GFP expression vectors by modifying the hCMV enhancer/promoter cassette. These modified vectors were transiently expressed, and the expression levels of enhanced green fluorescent protein (EGFP) alone and enhanced yellow fluorescent protein (EYFP) fused to another protein were determined by fluorescence microscopy and/or Western blotting. As shown in this study, we were able to (i) clearly reduce the expression of EGFP alone and (ii) reduce expression of an EYFP fusion protein down to the level of the endogenous protein, both in a graded manner. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Increased Cell Fusion in Cerebral Cortex May Contribute to Poststroke Regeneration

    Directory of Open Access Journals (Sweden)

    Alexander Paltsyn

    2013-01-01

    Full Text Available In this study, we used a model of a hemorrhagic stroke in a motor zone of the cortex in rats at the age of 3 months The report shows that cortical neurons can fuse with oligodendrocytes. In formed binuclear cells, the nucleus of an oligodendrocyte undergoes neuron specific reprogramming. It can be confirmed by changes in chromatin structure and in size of the second nucleus, by expression of specific neuronal markers and increasing total transcription rate. The nucleus of an oligodendrocyte likely transforms into a second neuronal nucleus. The number of binuclear neurons was validated with quantitative analysis. Fusion of neurons with oligodendrocytes might be a regenerative process in general and specifically following a stroke. The appearance of additional neuronal nuclei increases the functional outcome of the population of neurons. Participation of a certain number of binuclear cells in neuronal function might compensate for a functional deficit that arises from the death of a subset of neurons. After a stroke, the number of binuclear neurons increased in cortex around the lesion zone. In this case, the rate of recovery of stroke-damaged locomotor behavior also increased, which indicates the regenerative role of fusion.

  16. Fusion between M2-macrophages and cancer cells results in a subpopulation of radioresistant cells with enhanced DNA-repair capacity.

    Science.gov (United States)

    Lindström, Annelie; Midtbö, Kristine; Arnesson, Lars-Gunnar; Garvin, Stina; Shabo, Ivan

    2017-08-01

    Cell fusion is a natural biological process in normal development and tissue regeneration. Fusion between cancer cells and macrophages results in hybrids that acquire genetic and phenotypic characteristics from both maternal cells. There is a growing body of in vitro and in vivo data indicating that this process also occurs in solid tumors and may play a significant role in tumor progression. However, investigations of the response of macrophage:cancer cell hybrids to radiotherapy have been lacking. In this study, macrophage:MCF-7 hybrids were generated by spontaneous in vitro cell fusion. After irradiation, both hybrids and their maternal MCF-7 cells were treated with 0 Gy, 2.5 Gy and 5 Gy γ-radiation and examined by clonogenic survival and comet assays at three time points (0 h, 24 h, and 48 h). Compared to maternal MCF-7 cells, the hybrids showed increased survival fraction and plating efficiency (colony formation ability) after radiation. The hybrids developed less DNA-damage, expressed significantly lower residual DNA-damage, and after higher radiation dose showed less heterogeneity in DNA-damage compared to their maternal MCF-7 cells. To our knowledge this is the first study that demonstrates that macrophage:cancer cell fusion generates a subpopulation of radioresistant cells with enhanced DNA-repair capacity. These findings provide new insight into how the cell fusion process may contribute to clonal expansion and tumor heterogeneity. Furthermore, our results provide support for cell fusion as a mechanism behind the development of radioresistance and tumor recurrence.

  17. Induced pluripotent stem cell lines derived from human somatic cells.

    Science.gov (United States)

    Yu, Junying; Vodyanik, Maxim A; Smuga-Otto, Kim; Antosiewicz-Bourget, Jessica; Frane, Jennifer L; Tian, Shulan; Nie, Jeff; Jonsdottir, Gudrun A; Ruotti, Victor; Stewart, Ron; Slukvin, Igor I; Thomson, James A

    2007-12-21

    Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.

  18. High-throughput deterministic single-cell encapsulation and droplet pairing, fusion, and shrinkage in a single microfluidic device.

    Science.gov (United States)

    Schoeman, Rogier M; Kemna, Evelien W M; Wolbers, Floor; van den Berg, Albert

    2014-02-01

    In this article, we present a microfluidic device capable of successive high-yield single-cell encapsulation in droplets, with additional droplet pairing, fusion, and shrinkage. Deterministic single-cell encapsulation is realized using Dean-coupled inertial ordering of cells in a Yin-Yang-shaped curved microchannel using a double T-junction, with a frequency over 2000 Hz, followed by controlled droplet pairing with a 100% success rate. Subsequently, droplet fusion is realized using electrical actuation resulting in electro-coalescence of two droplets, each containing a single HL60 cell, with 95% efficiency. Finally, volume reduction of the fused droplet up to 75% is achieved by a triple pitchfork structure. This droplet volume reduction is necessary to obtain close cell-cell membrane contact necessary for final cell electrofusion, leading to hybridoma formation, which is the ultimate aim of this research. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. A G protein-coupled receptor (GPCR) in red: live cell imaging of the kappa opioid receptor-tdTomato fusion protein (KOPR-tdT) in neuronal cells.

    Science.gov (United States)

    Huang, Peng; Chiu, Yi-Ting; Chen, Chongguang; Wang, Yujun; Liu-Chen, Lee-Yuan

    2013-01-01

    In contrast to green fluorescent protein and variants (GFPs), red fluorescent proteins (RFPs) have rarely been employed for the generation of GPCR fusion proteins, likely because of formation of aggregates and cell toxicity of some RFPs. Among all the RFPs, tdTomato (tdT), one of the non-aggregating RFP, has the highest brightness score (about 3 times that of eGFP) and unsurpassed photostability. We fused tdT to the KOPR C-terminus. The KOPR-tdT cDNA construct was transfected into a Neuro2A mouse neuroblastoma cell line (Neuro2A cells) and rat cortical primary neurons for characterization of pharmacological properties and imaging studies on KOPR trafficking. KOPR-tdT retained KOPR properties (cell surface expression, ligand binding, agonist-induced signaling and internalization) when expressed in Neuro2A cells and rat primary cortical neurons. Live cell imaging of KOPR-tdT enables visualization of the time course of agonist-induced internalization of KOPR in real time for 60 min, without photobleaching and apparent cell toxicity. U50,488H-induced KOPR internalization occurred as early as 4min and plateaued at about 30 min. A unique pattern of internalized KOPR in processes of primary neurons was induced by U50,488H. tdT is an alternative to, or even a better tool than, GFPs for fusion to GPCR for trafficking studies, because tdT has higher brightness and thus better resolution and less photobleaching problems due to the reduced laser power used. It also has advantages associated with its longer-wavelength emission including spectral separation from autofluorescence and GFPs, reduced cell toxicity that the laser may impose, and greater tissue penetration. These advantages of tdT over GPFs may be critical for live cell imaging studies of GPCRs in vitro and for studying GPCRs in vivo because of their low abundance. © 2013.

  20. 5-Fluorocytosine combined with Fcy–hEGF fusion protein targets EGFR-expressing cancer cells

    International Nuclear Information System (INIS)

    Lan, Keng-Hsueh; Shih, Yi-Sheng; Chang, Cheng Allen; Yen, Sang-Hue; Lan, Keng-Li

    2012-01-01

    Highlights: ► EGFR-expressing epithelial cancers account for significant portion of cancer deaths. ► EGF–EGFR signaling pathway is validated as an important anticancer drug target. ► EGF and Fcy fusion protein (Fcy–hEGF) can bind to EGFR and convert 5-FC to 5-FU. ► Fcy–hEGF combined with 5-FC preferentially inhibits EGFR-expressing cells viability. -- Abstract: Human epithelial cancers account for approximately 50% of all cancer deaths. This type of cancer is characterized by excessive activation and expression of the epidermal growth factor receptor (EGFR). The EGFR pathway is critical for cancer cell proliferation, survival, metastasis and angiogenesis. The EGF–EGFR signaling pathway has been validated as an important anticancer drug target. Increasing numbers of targeted therapies against this pathway have been either approved or are currently under development. Here, we adopted a prodrug system that uses 5-fluorocytosine (5-FC) and human EGF (hEGF) fused with yeast cytosine deaminase (Fcy) to target EGFR-overexpressing cancer cells and to convert 5-FC to a significantly more toxic chemotherapeutic, 5-fluorouracil (5-FU). We cloned and purified the Fcy–hEGF fusion protein from Pichia pastoris yeast. This fusion protein specifically binds to EGFR with a similar affinity as hEGF, approximately 10 nM. Fcy–hEGF binds tightly to A431 and MDA-MB-468 cells, which overexpress EGFR, but it binds with a lower affinity to MDA-MB-231 and MCF-7, which express lower levels of EGFR. Similarly, the viability of EGFR-expressing cells was suppressed by Fcy–hEGF in the presence of increasing concentrations of 5-FC, and the IC 50 values for A431 and MDA-MB-468 were approximately 10-fold lower than those of MDA-MB-231 and MCF-7. This novel prodrug system, Fcy–hEGF/5-FC, might represent a promising addition to the available class of inhibitors that specifically target EGFR-expressing cancers.

  1. Application of optical tweezers and excimer laser to study protoplast fusion

    Science.gov (United States)

    Kantawang, Titirat; Samipak, Sompid; Limtrakul, Jumras; Chattham, Nattaporn

    2015-07-01

    Protoplast fusion is a physical phenomenon that two protoplasts come in contact and fuse together. Doing so, it is possible to combine specific genes from one protoplast to another during fusion such as drought resistance and disease resistance. There are a few possible methods to induce protoplast fusion, for example, electrofusion and chemical fusion. In this study, chemical fusion was performed with laser applied as an external force to enhance rate of fusion and observed under a microscope. Optical tweezers (1064 nm with 100X objective N.A. 1.3) and excimer laser (308 nm LMU-40X-UVB objective) were set with a Nikon Ti-U inverted microscope. Samples were prepared by soaking in hypertonic solution in order to induce cell plasmolysis. Elodea Canadensis and Allium cepa plasmolysed leaves were cut and observed under microscope. Concentration of solution was varied to induce difference turgor pressures on protoplasts pushing at cell wall. Free protoplasts in solution were trapped by optical tweezers to study the effect of Polyethylene glycol (PEG) solution. PEG was diluted by Ca+ solution during the process to induced protoplast cell contact and fusion. Possibility of protoplast fusion by excimer laser was investigated and found possible. Here we report a novel tool for plant cell fusion using excimer laser. Plant growth after cell fusion is currently conducted.

  2. Exceptionally Potent Anti-Tumor Bystander Activity of an scFv:sTRAIL Fusion Protein with Specificity for EGP2 Toward Target Antigen-Negative Tumor Cells

    Directory of Open Access Journals (Sweden)

    Edwin Bremer

    2004-09-01

    Full Text Available Previously, we reported on the target cell-restricted fratricide apoptotic activity of scFvC54:sTRAIL, a fusion protein comprising human-soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL genetically linked to the antibody fragment scFvC54 specific for the cell surface target antigen EGP2. In the present study, we report that the selective binding of scFvC54:sTRAIL to EGP2-positive target cells conveys an exceptionally potent pro-apoptotic effect toward neighboring tumor cells that are devoid of EGP2 expression (bystander cells. The anti-tumor bystander activity of scFvC54:sTRAIL was detectable at target-tobystander cell ratios as low as 1:100. Treatment in the presence of EGP2-blocking or TRAIL-neutralizing antibody strongly inhibited apoptosis in both target and bystander tumor cells. In the absence of target cells, bystander cell apoptosis induction was abrogated. The bystander apoptosis activity of scFvC54:sTRAIL did not require internalization, enzymatic conversion, diffusion, or communication (gap junctional intracellular communication between target and bystander cells. Furthermore, scFvC54:sTRAIL showed no detectable signs of innocent bystander activity toward freshly isolated blood cells. Further development of this new principle is warranted for approaches where cancer cells can escape from antibody-based therapy due to partial loss of target antigen expression.

  3. Enhanced expression of full-length human cytomegalovirus fusion protein in non-swelling baculovirus-infected cells with a minimal fed-batch strategy.

    Directory of Open Access Journals (Sweden)

    Marco Patrone

    Full Text Available Human cytomegalovirus congenital infection represents an unmet medical issue and attempts are ongoing to develop an effective vaccine. The virion fusion players of this enveloped virus are the natural targets to achieve this goal and to develop novel anti-viral therapies. The secreted ectodomain of the viral fusion factor glycoprotein B (gB has been exploited so far as an alternative to the cumbersome expression of the wild type trans-membrane protein. In the soluble form, gB showed encouraging but limited potential as antigen candidate calling for further efforts. Here, the exhaustive evaluation of the Baculovirus/insect cell expression system has been coupled to an orthogonal screening for expression additives to produce full-length gB. In detail, rapamycin was found to prolong gB intracellular accumulation while inhibiting the infection-induced cell swelling. Not obvious to predict, this inhibition did not affect Baculovirus growth, revealing that the virus-induced cell size increase is a dispensable side phenotype. In parallel, a feeding strategy for the limiting nutrient cysteine has been set up which improved gB stability. This multi-modal scheme allowed the production of full-length, mutation-free gB in the milligram scale. The recombinant full-length gB obtained was embedded into a stable mono-dispersed particle substantially larger than the protein trimer itself, according to the reported association of this protein with detergent-resistant lipid domains.

  4. Internalization and fate of silica nanoparticles in C2C12 skeletal muscle cells: evidence of a beneficial effect on myoblast fusion.

    Science.gov (United States)

    Poussard, Sylvie; Decossas, Marion; Le Bihan, Olivier; Mornet, Stéphane; Naudin, Grégoire; Lambert, Olivier

    2015-01-01

    The use of silica nanoparticles for their cellular uptake capability opens up new fields in biomedical research. Among the toxicological effects associated with their internalization, silica nanoparticles induce apoptosis that has been recently reported as a biochemical cue required for muscle regeneration. To assess whether silica nanoparticles could affect muscle regeneration, we used the C2C12 muscle cell line to study the uptake of fluorescently labeled NPs and their cellular trafficking over a long period. Using inhibitors of endocytosis, we determined that the NP uptake was an energy-dependent process mainly involving macropinocytosis and clathrin-mediated pathway. NPs were eventually clustered in lysosomal structures. Myoblasts containing NPs were capable of differentiation into myotubes, and after 7 days, electron microscopy revealed that the NPs remained primarily within lysosomes. The presence of NPs stimulated the formation of myotubes in a dose-dependent manner. NP internalization induced an increase of apoptotic myoblasts required for myoblast fusion. At noncytotoxic doses, the NP uptake by skeletal muscle cells did not prevent their differentiation into myotubes but, instead, enhanced the cell fusion.

  5. Effects of an adenoviral vector containing a suicide gene fusion on growth characteristics of breast cancer cells.

    Science.gov (United States)

    Kong, Heng; Liu, Chunli; Zhu, Ting; Huang, Zonghai; Yang, Liucheng; Li, Qiang

    2014-12-01

    The herpes simplex virus thymidine kinase/ganciclovir (HSV‑TK/GCV) and the cytosine deaminase/5‑fluorocytosine (CD/5‑FC) systems have been widely applied in suicide gene therapy for cancer. Although suicide gene therapy has been successfully used in vitro and in vivo studies, the number of studies on the effects of recombinant adenoviruses (Ads) containing suicide genes on target cancer cells is limited. The aim of this study was to examine whether recombinant Ads containing the CD/TK fusion gene affect cell proliferation of breast cancer cells in vitro. In the present study, we explored the use of a recombinant adenoviral vector to deliver the CD/TK fusion gene to the breast cancer cell line MCF‑7. We found that the recombinant adenoviral vector efficiently infected MCF‑7 cells. Western blot analysis revealed that CD and TK proteins are expressed in the infected cells. The infected breast cancer cells did not show any significant changes in morphology, ultrastructure, cell growth, and cell‑cycle distribution compared to the uninfected cells. This study revealed that the Ad‑vascular endothelial growth factor promoter (VEGFp)‑CD/TK vector is non‑toxic to MCF‑7 cells at the appropriate titer. Our results indicate that it is feasible to use a recombinant adenoviral vector containing the CD/TK fusion gene in suicide gene therapy to target breast cancer cells.

  6. Bilateral bony fusion around the supraspinatus muscle inducing muscle hypoplasia and shoulder pain.

    Science.gov (United States)

    Son, YeNa; Ryu, Kyung Nam; Jin, Wook; Park, Ji Seon; Park, So Young

    2017-03-01

    We describe the case of a 30-year-old man who developed chronic bilateral shoulder pain that relapsed and remitted over the course of 1 year. The patient was diagnosed with congenital shoulder fusion anomalies. The right shoulder showed anomalous accessory articulation between the distal third of the clavicle and the acromion along with normal articulation of the shoulder on CT. At the left shoulder, bony fusions were present between the distal portion of the clavicle, the acromion, and the coracoid process, and between the coracoid process, upper portion of the glenoid, and upper body of the scapula, which formed a bony canal and was responsible for hypoplasia of the supraspinatus muscle on CT and MRI. To our knowledge, this is the first description of such congenital shoulder anomalies with extreme bony fusion and is an illustrative example of how imaging may be used to differentiate fusion from other congenital abnormalities of the shoulder to aid diagnosis.

  7. Bilateral bony fusion around the supraspinatus muscle inducing muscle hypoplasia and shoulder pain

    Energy Technology Data Exchange (ETDEWEB)

    Son, YeNa; Jin, Wook; Park, So Young [Kyung Hee University Hospital at Gangdong, Department of Radiology, 892, Dongnam-ro, Gangdong-gu, Seoul (Korea, Republic of); Ryu, Kyung Nam; Park, Ji Seon [Kyung Hee University Hospital, Department of Radiology, 23 Kyunghee-daero, Dongdaemun-gu, Seoul (Korea, Republic of)

    2017-03-15

    We describe the case of a 30-year-old man who developed chronic bilateral shoulder pain that relapsed and remitted over the course of 1 year. The patient was diagnosed with congenital shoulder fusion anomalies. The right shoulder showed anomalous accessory articulation between the distal third of the clavicle and the acromion along with normal articulation of the shoulder on CT. At the left shoulder, bony fusions were present between the distal portion of the clavicle, the acromion, and the coracoid process, and between the coracoid process, upper portion of the glenoid, and upper body of the scapula, which formed a bony canal and was responsible for hypoplasia of the supraspinatus muscle on CT and MRI. To our knowledge, this is the first description of such congenital shoulder anomalies with extreme bony fusion and is an illustrative example of how imaging may be used to differentiate fusion from other congenital abnormalities of the shoulder to aid diagnosis. (orig.)

  8. Nuclei of non-muscle cells bind centrosome proteins upon fusion with differentiating myoblasts.

    Directory of Open Access Journals (Sweden)

    Xavier Fant

    2009-12-01

    Full Text Available In differentiating myoblasts, the microtubule network is reorganized from a centrosome-bound, radial array into parallel fibres, aligned along the long axis of the cell. Concomitantly, proteins of the centrosome relocalize from the pericentriolar material to the outer surface of the nucleus. The mechanisms that govern this relocalization are largely unknown.In this study, we perform experiments in vitro and in cell culture indicating that microtubule nucleation at the centrosome is reduced during myoblast differentiation, while nucleation at the nuclear surface increases. We show in heterologous cell fusion experiments, between cultures of differentiating mouse myoblasts and human cells of non-muscular origin, that nuclei from non-muscle cells recruit centrosome proteins once fused with the differentiating myoblasts. This recruitment still occurs in the presence of cycloheximide and thus appears to be independent of new protein biosynthesis.Altogether, our data suggest that nuclei of undifferentiated cells have the dormant potential to bind centrosome proteins, and that this potential becomes activated during myoblast differentiation.

  9. An induced pocket for the binding of potent fusion inhibitor CL-385319 with H5N1 influenza virus hemagglutinin.

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    Runming Li

    Full Text Available The influenza glycoprotein hemagglutinin (HA plays crucial roles in the early stage of virus infection, including receptor binding and membrane fusion. Therefore, HA is a potential target for developing anti-influenza drugs. Recently, we characterized a novel inhibitor of highly pathogenic H5N1 influenza virus, CL-385319, which specifically inhibits HA-mediated viral entry. Studies presented here identified the critical binding residues for CL-385319, which clustered in the stem region of the HA trimer by site-directed mutagenesis. Extensive computational simulations, including molecular docking, molecular dynamics simulations, molecular mechanics generalized Born surface area (MM_GBSA calculations, charge density and Laplacian calculations, have been carried out to uncover the detailed molecular mechanism that underlies the binding of CL-385319 to H5N1 influenza virus HA. It was found that the recognition and binding of CL-385319 to HA proceeds by a process of "induced fit" whereby the binding pocket is formed during their interaction. Occupation of this pocket by CL-385319 stabilizes the neutral pH structure of hemagglutinin, thus inhibiting the conformational rearrangements required for membrane fusion. This "induced fit" pocket may be a target for structure-based design of more potent influenza fusion inhibitors.

  10. Imaging plant cell death: GFP-Nit1 aggregation marks an early step of wound and herbicide induced cell death

    Directory of Open Access Journals (Sweden)

    Somerville Chris R

    2005-03-01

    Full Text Available Abstract Background A great deal is known about the morphological endpoints of plant cell death, but relatively little is known about its sequence of events and / or its execution at the biochemical level. Live cell imaging using GFP-tagged markers is a powerful way to provide dynamic portraits of a cellular process that can in turn provide a descriptive foundation valuable for future biochemical and genetic investigations. Results While characterizing a collection of random GFP-protein fusion markers we discovered that mechanical wounding induces rapid aggregation of a GFP-Nitrilase 1 fusion protein in Arabidopsis cells directly abutting wound sites. Time-lapse imaging of this response shows that the aggregation occurs in cells that subsequently die 30 – 60 minutes post-wounding, indicating that GFP-Nit1 aggregation is an early marker of cell death at wound sites. Time-lapse confocal imaging was used to characterize wound-induced cell death using GFP-Nit1 and markers of the nucleus and endoplasmic reticulum. These analyses provide dynamic portraits of well-known death-associated responses such as nuclear contraction and cellular collapse and reveal novel features such as nuclear envelope separation, ER vesiculation and loss of nuclear-lumen contents. As a parallel system for imaging cell death, we developed a chemical method for rapidly triggering cell death using the herbicides bromoxynil or chloroxynil which cause rapid GFP-Nit1 aggregation, loss of nuclear contents and cellular collapse, but not nuclear contraction, separating this response from others during plant cell death. Conclusion Our observations place aggregation of Nitrilase 1 as one of the earliest events associated with wound and herbicide-induced cell death and highlight several novel cellular events that occur as plant cells die. Our data create a detailed descriptive framework for future investigations of plant cell death and provide new tools for both its cellular and

  11. Imaging plant cell death: GFP-Nit1 aggregation marks an early step of wound and herbicide induced cell death

    Science.gov (United States)

    Cutler, Sean R; Somerville, Chris R

    2005-01-01

    Background A great deal is known about the morphological endpoints of plant cell death, but relatively little is known about its sequence of events and / or its execution at the biochemical level. Live cell imaging using GFP-tagged markers is a powerful way to provide dynamic portraits of a cellular process that can in turn provide a descriptive foundation valuable for future biochemical and genetic investigations. Results While characterizing a collection of random GFP-protein fusion markers we discovered that mechanical wounding induces rapid aggregation of a GFP-Nitrilase 1 fusion protein in Arabidopsis cells directly abutting wound sites. Time-lapse imaging of this response shows that the aggregation occurs in cells that subsequently die 30 – 60 minutes post-wounding, indicating that GFP-Nit1 aggregation is an early marker of cell death at wound sites. Time-lapse confocal imaging was used to characterize wound-induced cell death using GFP-Nit1 and markers of the nucleus and endoplasmic reticulum. These analyses provide dynamic portraits of well-known death-associated responses such as nuclear contraction and cellular collapse and reveal novel features such as nuclear envelope separation, ER vesiculation and loss of nuclear-lumen contents. As a parallel system for imaging cell death, we developed a chemical method for rapidly triggering cell death using the herbicides bromoxynil or chloroxynil which cause rapid GFP-Nit1 aggregation, loss of nuclear contents and cellular collapse, but not nuclear contraction, separating this response from others during plant cell death. Conclusion Our observations place aggregation of Nitrilase 1 as one of the earliest events associated with wound and herbicide-induced cell death and highlight several novel cellular events that occur as plant cells die. Our data create a detailed descriptive framework for future investigations of plant cell death and provide new tools for both its cellular and biochemical analysis. PMID

  12. Promoting spinal fusions by biomineralized silk fibroin films seeded with bone marrow stromal cells: An in vivo animal study.

    Science.gov (United States)

    Gu, Yong; Chen, Liang; Niu, Hai-Yun; Shen, Xiao-Feng; Yang, Hui-Lin

    2016-03-01

    To prepare a biomineralized nano silk fibroin film seeded with bone marrow stromal cells (BMSCs), and to evaluate its performance in spinal fusion. The silk fibroin film was mineralized in a modified, simulated body fluid, seeded with BMSCs, and evaluated in a rat model of posterolateral lumbar fusion, compared with pure silk fibroin, silk fibroin/bone marrow stromal cells, mineralized silk fibroin, mineralized silk fibroin/bone marrow stromal cells, iliac crest bone, and no graft. After 12 weeks, all rats were sacrificed and underwent manual palpation, micro-CT scanning, biomechanical testing, and histology. The infrared spectrum, X-ray diffraction, and scanning electron microscopy demonstrated deposition of mineral layers on the silk fibroin film surface. The fusion rate, bone volume, relative strength and stiffness, and histological score of the mineralized silk fibroin/bone marrow stromal cells were slightly lower than the autograft, but without any significant difference (p > 0.05). In addition, the mineralized silk fibroin was significantly greater in most parameters than the silk fibroin/bone marrow stromal cells (p spinal fusion is enhanced when the mineralized silk fibroin is seeded with bone marrow stromal cells. © The Author(s) 2015.

  13. Prompt photon yield and elliptic flow from gluon fusion induced by magnetic fields in relativistic heavy-ion collisions

    Science.gov (United States)

    Ayala, Alejandro; Castaño-Yepes, Jorge David; Dominguez, C. A.; Hernández, L. A.; Hernández-Ortiz, Saúl; Tejeda-Yeomans, María Elena

    2017-07-01

    We compute photon production at early times in semicentral relativistic heavy-ion collisions from nonequilibrium gluon fusion induced by a magnetic field. The calculation accounts for the main features of the collision at these early times, namely, the intense magnetic field and the high gluon occupation number. The gluon fusion channel is made possible by the magnetic field and would otherwise be forbidden due to charge conjugation invariance. Thus, the photon yield from this process is an excess over calculations without magnetic field effects. We compare this excess to the difference between PHENIX data and recent hydrodynamic calculations for the photon transverse momentum distribution and elliptic flow coefficient v2 . We show that with reasonable values for the saturation scale and magnetic field strength, the calculation helps us better describe the experimental results obtained at RHIC energies for the lowest part of the transverse photon momentum.

  14. Expression and cytosolic assembly of the S-layer fusion protein mSbsC-EGFP in eukaryotic cells

    Directory of Open Access Journals (Sweden)

    Veenhuis Marten

    2005-10-01

    Full Text Available Abstract Background Native as well as recombinant bacterial cell surface layer (S-layer protein of Geobacillus (G. stearothermophilus ATCC 12980 assembles to supramolecular structures with an oblique symmetry. Upon expression in E. coli, S-layer self assembly products are formed in the cytosol. We tested the expression and assembly of a fusion protein, consisting of the mature part (aa 31–1099 of the S-layer protein and EGFP (enhanced green fluorescent protein, in eukaryotic host cells, the yeast Saccharomyces cerevisiae and human HeLa cells. Results Upon expression in E. coli the recombinant mSbsC-EGFP fusion protein was recovered from the insoluble fraction. After denaturation by Guanidine (Gua-HCl treatment and subsequent dialysis the fusion protein assembled in solution and yielded green fluorescent cylindric structures with regular symmetry comparable to that of the authentic SbsC. For expression in the eukaryotic host Saccharomyces (S. cerevisiae mSbsC-EGFP was cloned in a multi-copy expression vector bearing the strong constitutive GPD1 (glyceraldehyde-3-phosophate-dehydrogenase promoter. The respective yeast transfomants were only slightly impaired in growth and exhibited a needle-like green fluorescent pattern. Transmission electron microscopy (TEM studies revealed the presence of closely packed cylindrical structures in the cytosol with regular symmetry comparable to those obtained after in vitro recrystallization. Similar structures are observed in HeLa cells expressing mSbsC-EGFP from the Cytomegalovirus (CMV IE promoter. Conclusion The mSbsC-EGFP fusion protein is stably expressed both in the yeast, Saccharomyces cerevisiae, and in HeLa cells. Recombinant mSbsC-EGFP combines properties of both fusion partners: it assembles both in vitro and in vivo to cylindrical structures that show an intensive green fluorescence. Fusion of proteins to S-layer proteins may be a useful tool for high level expression in yeast and HeLa cells of

  15. Long-Term Endurance Exercise in Humans Stimulates Cell Fusion of Myoblasts along with Fusogenic Endogenous Retroviral Genes In Vivo.

    Science.gov (United States)

    Frese, Sebastian; Ruebner, Matthias; Suhr, Frank; Konou, Thierry M; Tappe, Kim A; Toigo, Marco; Jung, Hans H; Henke, Christine; Steigleder, Ruth; Strissel, Pamela L; Huebner, Hanna; Beckmann, Matthias W; van der Keylen, Piet; Schoser, Benedikt; Schiffer, Thorsten; Frese, Laura; Bloch, Wilhelm; Strick, Reiner

    2015-01-01

    Myogenesis is defined as growth, differentiation and repair of muscles where cell fusion of myoblasts to multinucleated myofibers is one major characteristic. Other cell fusion events in humans are found with bone resorbing osteoclasts and placental syncytiotrophoblasts. No unifying gene regulation for natural cell fusions has been found. We analyzed skeletal muscle biopsies of competitive cyclists for muscle-specific attributes and expression of human endogenous retrovirus (ERV) envelope genes due to their involvement in cell fusion of osteoclasts and syncytiotrophoblasts. Comparing muscle biopsies from post- with the pre-competitive seasons a significant 2.25-fold increase of myonuclei/mm fiber, a 2.38-fold decrease of fiber area/nucleus and a 3.1-fold decrease of satellite cells (SCs) occurred. We propose that during the pre-competitive season SC proliferation occurred following with increased cell fusion during the competitive season. Expression of twenty-two envelope genes of muscle biopsies demonstrated a significant increase of putative muscle-cell fusogenic genes Syncytin-1 and Syncytin-3, but also for the non-fusogenic erv3. Immunohistochemistry analyses showed that Syncytin-1 mainly localized to the sarcolemma of myofibers positive for myosin heavy-chain isotypes. Cellular receptors SLC1A4 and SLC1A5 of Syncytin-1 showed significant decrease of expression in post-competitive muscles compared with the pre-competitive season, but only SLC1A4 protein expression localized throughout the myofiber. Erv3 protein was strongly expressed throughout the myofiber, whereas envK1-7 localized to SC nuclei and myonuclei. Syncytin-1 transcription factors, PPARγ and RXRα, showed no protein expression in the myofiber, whereas the pCREB-Ser133 activator of Syncytin-1 was enriched to SC nuclei and myonuclei. Syncytin-1, Syncytin-3, SLC1A4 and PAX7 gene regulations along with MyoD1 and myogenin were verified during proliferating or actively-fusing human primary myoblast cell

  16. Long-Term Endurance Exercise in Humans Stimulates Cell Fusion of Myoblasts along with Fusogenic Endogenous Retroviral Genes In Vivo.

    Directory of Open Access Journals (Sweden)

    Sebastian Frese

    Full Text Available Myogenesis is defined as growth, differentiation and repair of muscles where cell fusion of myoblasts to multinucleated myofibers is one major characteristic. Other cell fusion events in humans are found with bone resorbing osteoclasts and placental syncytiotrophoblasts. No unifying gene regulation for natural cell fusions has been found. We analyzed skeletal muscle biopsies of competitive cyclists for muscle-specific attributes and expression of human endogenous retrovirus (ERV envelope genes due to their involvement in cell fusion of osteoclasts and syncytiotrophoblasts. Comparing muscle biopsies from post- with the pre-competitive seasons a significant 2.25-fold increase of myonuclei/mm fiber, a 2.38-fold decrease of fiber area/nucleus and a 3.1-fold decrease of satellite cells (SCs occurred. We propose that during the pre-competitive season SC proliferation occurred following with increased cell fusion during the competitive season. Expression of twenty-two envelope genes of muscle biopsies demonstrated a significant increase of putative muscle-cell fusogenic genes Syncytin-1 and Syncytin-3, but also for the non-fusogenic erv3. Immunohistochemistry analyses showed that Syncytin-1 mainly localized to the sarcolemma of myofibers positive for myosin heavy-chain isotypes. Cellular receptors SLC1A4 and SLC1A5 of Syncytin-1 showed significant decrease of expression in post-competitive muscles compared with the pre-competitive season, but only SLC1A4 protein expression localized throughout the myofiber. Erv3 protein was strongly expressed throughout the myofiber, whereas envK1-7 localized to SC nuclei and myonuclei. Syncytin-1 transcription factors, PPARγ and RXRα, showed no protein expression in the myofiber, whereas the pCREB-Ser133 activator of Syncytin-1 was enriched to SC nuclei and myonuclei. Syncytin-1, Syncytin-3, SLC1A4 and PAX7 gene regulations along with MyoD1 and myogenin were verified during proliferating or actively-fusing human

  17. Unique amplification of BCR-ABL1 gene fusion in a case of T-cell acute lymphoblastic leukemia.

    Science.gov (United States)

    Koka, Rima; Bade, Najeebah A; Sausville, Edward A; Ning, Yi; Zou, Ying

    2017-01-01

    ABL1 gene translocations can be seen in precursor T-acute lymphoblastic leukemia (T-ALL). The typical translocation partner is the NUP214 gene. BCR-ABL translocations are relatively rare in this entity. Furthermore, while there have been unique patterns of amplification noted among the NUP214-ABL fusion genes, there have been few such reports among cases with BCR-ABL fusion genes. Here we report a unique case of a 44-year old patient with T-ALL in which the blasts demonstrated a derivative chromosome 9 involving a 9;22 translocation and a dicentric Philadelphia chromosome 22 with a homogeneously staining region at the interface of the 9;22 translocation, leading to BCR-ABL1 gene amplification. Fluorescence in-situ hybridization (FISH) showed abnormal BCR/ABL1 fusions with the BCR-ABL1 gene amplification in 48% of the interphase cells analyzed. The translocation was confirmed by SNP array. We present a novel derivative chromosome 9 that shows BCR-ABL gene fusion along with a dicentric Philadelphia chromosome 22 with BCR-ABL1 gene amplification. This is a unique pattern of BCR-ABL fusion which has never been described in T-ALL. It is significant that the patient responded to standard treatment with the CALGB 10403 protocol and supplementation with a tyrosine kinase inhibitor. Identification of additional patients with this pattern of BCR-ABL fusion will allow for enhanced risk assessment and prognostication.

  18. Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins.

    Science.gov (United States)

    Hein, Birka; Willig, Katrin I; Wurm, Christian A; Westphal, Volker; Jakobs, Stefan; Hell, Stefan W

    2010-01-06

    We show far-field fluorescence nanoscopy of different structural elements labeled with an organic dye within living mammalian cells. The diffraction barrier limiting far-field light microscopy is outperformed by using stimulated emission depletion. We used the tagging protein hAGT (SNAP-tag), which covalently binds benzylguanine-substituted organic dyes, for labeling. Tetramethylrhodamine was used to image the cytoskeleton (vimentin and microtubule-associated protein 2) as well as structures located at the cell membrane (caveolin and connexin-43) with a resolution down to 40 nm. Comparison with structures labeled with the yellow fluorescent protein Citrine validates this labeling approach. Nanoscopic movies showing the movement of connexin-43 clusters across the cell membrane evidence the capability of this technique to observe structural changes on the nanoscale over time. Pulsed or continuous-wave lasers for excitation and stimulated emission depletion yield images of similar resolution in living cells. Hence fusion proteins that bind modified organic dyes expand widely the application range of far-field fluorescence nanoscopy of living cells. Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  19. Search for cold fusion using Pd-D2O cells and Ti-D mixtures

    Science.gov (United States)

    Hill, John C.; Stassis, C.; Shinar, J.; Goldman, A. I.; Folkerts, R.; Schwellenbach, D. D.; Peterson, D. T.; Widrig, C.; Porter, M.; Benesh, C. J.; Vary, J. P.

    1990-09-01

    We have searched for cold fusion produced in an electrolytic cell with Pd cathode and Pt anode. The electrolyte was 0.1 molar LiOD in 99.8% D2O. A 2-mm rod of polycrystalline Pd and a 4-mm rod of single crystal Pd were used. No radiation was detected above background by a BF3 neutron and Ge γ-X detector. The D2 loading of the Pd was 0.8 D per Pd atom reaching saturation after 4 hours. We also attempted to duplicate the work of Scaramuzzi and co-workers on the Ti-D2 system. Both powder and pieces of Ti were used. The material was cycled several times between 1100 K and 77 K. No neutron, γ- or x-ray emission above background was observed. The results of a barrier penetration calculation for H-like atoms are presented. The high fusion rates reported for PdD x . are much larger than those expected from theoretical calculations on these systems.

  20. Productive infection of human immunodeficiency virus type 1 in dendritic cells requires fusion-mediated viral entry

    International Nuclear Information System (INIS)

    Janas, Alicia M.; Dong, Chunsheng; Wang Jianhua; Wu Li

    2008-01-01

    Human immunodeficiency virus type 1 (HIV-1) enters dendritic cells (DCs) through endocytosis and viral receptor-mediated fusion. Although endocytosis-mediated HIV-1 entry can generate productive infection in certain cell types, including human monocyte-derived macrophages, productive HIV-1 infection in DCs appears to be dependent on fusion-mediated viral entry. It remains to be defined whether endocytosed HIV-1 in DCs can initiate productive infection. Using HIV-1 infection and cellular fractionation assays to measure productive viral infection and entry, here we show that HIV-1 enters monocyte-derived DCs predominately through endocytosis; however, endocytosed HIV-1 cannot initiate productive HIV-1 infection in DCs. In contrast, productive HIV-1 infection in DCs requires fusion-mediated viral entry. Together, these results provide functional evidence in understanding HIV-1 cis-infection of DCs, suggesting that different pathways of HIV-1 entry into DCs determine the outcome of viral infection

  1. Oncogenic fusion proteins expressed in immature hematopoietic cells fail to recapitulate the transcriptional changes observed in human AML

    DEFF Research Database (Denmark)

    Rapin, N; Porse, B T

    2014-01-01

    Reciprocal chromosomal translocations are observed in one-third of acute myeloid leukemia (AML) cases. Targeting and understanding the effects of the resulting aberrant oncogenic fusion proteins may help developing drugs against specific leukemic subtypes, as demonstrated earlier by the use of ATRA....... Surprisingly, we found that the gene-expression profiles of CD34+ human HSPCs transformed with the potent oncogenic fusion proteins AML-ETO or MLL-AF9, only weakly resembled those derived from primary AML samples. Hence, our work raises concerns as to the relevance of the use of in vitro transduced cells...... in acute promyelocytic leukemia. Hematopoietic stem/progenitor (HSPCs) cells transduced with oncogenic fusion genes are regarded as promising in vitromodels of their corresponding AML subtypes. Here, we critically assessed the potential of such in vitro models using an integrative bioinformatics approach...

  2. Pre-Clinical Studies of Dendritic Cell-Tumor Cell Fusion Vaccines to Treat Breast Cancer

    National Research Council Canada - National Science Library

    Akporiaye, Emmanuel

    2002-01-01

    ...+ T-helper cells, CD8+ cytotoxic T lymphocytes (CTLs), NK and NKT cells (1,2). Because DC have the capacity to take up various types of molecules, the cells can be loaded with tumor-associated antigens (TAAs...

  3. Neutron induced displacement damage in beryllium in the blanket of a (d,t)-fusion reactor

    International Nuclear Information System (INIS)

    Hermanutz, D.

    1995-09-01

    Beryllium is a favoured candidate for a neutron multiplier in solid breeder blankets of fusion reactors. This is mainly due to its low (n, 2n)-reaction threshold and because of its good thermal and mechanical properties. Its behaviour under intense neutron irradiation, however, is a crucial issue for its use in future fusion reactors. Displacement damage in beryllium so far has been calculated both with data related and methodological deficiencies. First of all, there is a need to have accurate cross-section data in order to obtain reliable spectra of primary knock-on atoms (PKA's). Furthermore, there are principal restrictions of the NRT-model in general used to calculate secondary displacements initiated by PKA's. The underlying theory of damage-energy (part of kinetic energy of PKA transferred elastically to matrix atoms) according to Lindhard is strictly valid only for medium and heavy mass ions with moderate energies in targets of the same element. In this work improved damage cross-sections and displacement rates (dpa/s) in beryllium have been calculated based on cross-section data from ENDF/B-VI (with a significantly improved (n, 2n)-evaluation) and on an appropriate treatment of damage-energy that is suitable for fusion relevant damage of light mass materials. ''This work has been performed in the framework of the Nuclear Fusion Project of the Forschungszentrum Karlsruhe and is supported by the European Communities within the European Fusion Technology Program''. (orig.)

  4. Production of Hev b5 as a fluorescent biotin-binding tripartite fusion protein in insect cells

    International Nuclear Information System (INIS)

    Nordlund, Henri R.; Laitinen, Olli H.; Uotila, Sanna T.H.; Kulmala, Minna; Kalkkinen, Nisse; Kulomaa, Markku S.

    2005-01-01

    The presented green fluorescent protein and streptavidin core-based tripartite fusion system provides a simple and efficient way for the production of proteins fused to it in insect cells. This fusion protein forms a unique tag, which serves as a multipurpose device enabling easy optimization of production, one-step purification via streptavidin-biotin interaction, and visualization of the fusion protein during downstream processing and in applications. In the present study, we demonstrate the successful production, purification, and detection of a natural rubber latex allergen Hev b5 with this system. We also describe the production of another NRL allergen with the system, Hev b1, which formed large aggregates and gave small yields in purification. The aggregates were detected at early steps by microscopical inspection of the infected insect cells producing this protein. Therefore, this fusion system can also be utilized as a fast indicator of the solubility of the expressed fusion proteins and may therefore be extremely useful in high-throughput expression approaches

  5. Exploring incomplete fusion fraction in 6,7Li induced nuclear reactions

    Directory of Open Access Journals (Sweden)

    Parkar V. V.

    2017-01-01

    Full Text Available We have included breakup effects explicitly to simultaneously calculate the measured cross-sections of the complete fusion, incomplete fusion, and total fusion for 6,7Li projectiles on various targets using the Continuum Discretized Coupled Channels method. The breakup absorption cross-sections obtained with different choices of short range imaginary potentials are utilized to evaluate the individual α-capture and d/t-capture cross-sections and compare with the measured data. It is interesting to note, while in case of 7Li projectile the cross-sections for triton-ICF/triton-capture is far more dominant than α-ICF/α-capture at all energies, similar behavior is not observed in case of 6Li projectile for the deuteron-ICF/deuteron-capture and α-ICF/α-capture. Both these observations are also corroborated by the experimental data for all the systems studied.

  6. Exploring incomplete fusion fraction in 6,7Li induced nuclear reactions

    Science.gov (United States)

    Parkar, V. V.; Jha, V.; Kailas, S.

    2017-11-01

    We have included breakup effects explicitly to simultaneously calculate the measured cross-sections of the complete fusion, incomplete fusion, and total fusion for 6,7Li projectiles on various targets using the Continuum Discretized Coupled Channels method. The breakup absorption cross-sections obtained with different choices of short range imaginary potentials are utilized to evaluate the individual α-capture and d/t-capture cross-sections and compare with the measured data. It is interesting to note, while in case of 7Li projectile the cross-sections for triton-ICF/triton-capture is far more dominant than α-ICF/α-capture at all energies, similar behavior is not observed in case of 6Li projectile for the deuteron-ICF/deuteron-capture and α-ICF/α-capture. Both these observations are also corroborated by the experimental data for all the systems studied.

  7. NUTM2A-CIC fusion small round cell sarcoma: a genetically distinct variant of CIC-rearranged sarcoma.

    Science.gov (United States)

    Sugita, Shintaro; Arai, Yasuhito; Aoyama, Tomoyuki; Asanuma, Hiroko; Mukai, Wakako; Hama, Natsuko; Emori, Makoto; Shibata, Tatsuhiro; Hasegawa, Tadashi

    2017-07-01

    CIC-rearranged sarcoma is a new entity of undifferentiated small round cell sarcoma characterized by chimeric fusions with CIC rearrangement. We report a NUTM2A-CIC fusion sarcoma in a 43-year-old woman who died of rapidly progressive disease. Histologic analysis revealed multinodular proliferation of small round tumor cells with mild nuclear pleomorphism. The sclerotic fibrous septa separated the tumor into multiple nodules. Immunohistochemistry showed that the tumor cells were diffusely positive for vimentin, focally positive for cytokeratin, and negative for CD99 and NKX2.2. Tumor cells were also negative for ETV4, which was recently identified as a specific marker for CIC-rearranged sarcoma. High-throughput RNA sequencing of a formalin-fixed, paraffin-embedded clinical sample unveiled a novel NUTM2A-CIC fusion between NUTM2A exon 7 and CIC exon 12, and fluorescence in situ hybridization identified CIC and NUTM2A split signals. This case shared several clinicopathological findings with previously reported CIC-rearranged cases. We recognized the tumor as a genetically distinct variant of CIC-rearranged sarcomas with a novel NUTM2A-CIC fusion. Copyright © 2017. Published by Elsevier Inc.

  8. Alloy development for fast induced radioactivity decay for fusion reactor applications

    International Nuclear Information System (INIS)

    Klueh, R.L.; Bloom, E.E.

    1984-01-01

    During the operation of a fusion reactor, the structural material of the first wall and blanket structure will become highly radioactive from activation by the high-energy fusion neutrons. A difficult radioactive waste management problem will be involved in the disposal of this material after the service lifetime is complete. One way to minimize the management problem is the use of structural materials where the radioactive isotopes in the irradiated material decay to levels that allow for simplified disposal techniques. We are exploring how ferritic and austenitic steels could be developed to meet this objective

  9. The transmembrane domain and acidic lipid flip-flop regulates voltage-dependent fusion mediated by class II and III viral proteins.

    Directory of Open Access Journals (Sweden)

    Ruben M Markosyan

    Full Text Available Voltage dependence of fusion induced by class II and class III viral fusion proteins was investigated. Class II proteins from Ross River and Sindbus virus and a mutant class III protein from Epstein Barr virus were found to induce cell-cell fusion that is voltage dependent. Combined with previous studies, in all, four class II and two class III protein have now been shown to exhibit voltage-dependent fusion, demonstrating that this is probably a general phenomenon for these two classes of viral fusion proteins. In the present study, monitoring fusion of pseudovirus expressing Vesicular Stomatitis virus (VSV G within endosomes shows that here, too, fusion is voltage dependent. This supports the claim that voltage dependence of fusion is biologically relevant and that cell-cell fusion reliably models the voltage dependence. Fusion induced by class I viral proteins is independent of voltage; chimeras expressing the ectodomain of a class I fusion protein and the transmembrane domain of VSV G could therefore be used to explore the location within the protein responsible for voltage dependence. Results showed that the transmembrane domain is the region associated with voltage dependence. Experiments in which cells were enriched with acidic lipids led to the conclusion that it is the flip-flop of acidic lipids that carries the charge responsible for the observed voltage dependence of fusion. This flip-flop occurred downstream of hemifusion, in accord with previous findings that the voltage dependent steps of fusion occur at a stage subsequent to hemifusion.

  10. Neutrophils Induced Licensing of Natural Killer Cells

    Directory of Open Access Journals (Sweden)

    Keishiro Amano

    2015-01-01

    Full Text Available Natural killer (NK cells acquire effector function through a licensing process and exert anti-leukemia/tumor effect. However, there is no means to promote a licensing effect of allogeneic NK cells other than cytomegalovirus reactivation-induced licensing in allogeneic hematopoietic stem cell transplantation in human. In mice, a licensing process is mediated by Ly49 receptors which recognize self-major histocompatibility complex class I. The distribution of four Ly49 receptors showed similar pattern in congenic mice, B10, B10.BR, and B10.D2, which have B10 background. Forty Gy-irradiated 2×106 B10.D2 cells including splenocytes, peripheral blood mononuclear cells in untreated mice, or granulocyte colony-stimulating factor treated mice were injected intraperitoneally into B10 mice. We found that murine NK cells were effectively licensed by intraperitoneal injection of donor neutrophils with its corresponding NK receptor ligand in B10 mice as a recipient and B10.D2 as a donor. Mechanistic studies revealed that NK cells showed the upregulation of intracellular interferon-γ and CD107a expression as markers of NK cell activation. Moreover, enriched neutrophils enhanced licensing effect of NK cells; meanwhile, licensing effect was diminished by depletion of neutrophils. Collectively, injection of neutrophils induced NK cell licensing (activation via NK receptor ligand interaction.

  11. [Construction of Eukaryotic Expression Vector of siRNA Specific for BCR/ABL Fusion Gene and Its Effects on K562 Cells].

    Science.gov (United States)

    Li, Ming; Wang, Bao-Lin; Wang, Li-Na; Xi, Ya-Ming

    2016-12-01

    To construct eukaryotic expression vector of siRNA specific for BCR/ABL and to investigate the effect of recombinant plasmid on BCR/ABL and P210 protein expression in K562 cells. siRNA(small interfering RNA)was designed according to the Tuschl's principle of Ai-based medicine, and was converted into cDNA coding expression of shRNA(small hairpin RNAs)of siRNA for BCR/ABL fusion gene. The cDNA was synthesized and inserted into plasmid pTER. The pTER117 and pTER363 of recombinant plasmid being eukaryotic expression vector was controlled by the H1 promoter of RNA polymerase III, and identified by the restriction map and the sequence analysis. The recombinant plasmid did not only have the screening resisting antibiotics, its expression but also are induced by tetracycline (tet). After steadily transfection into K562 cells by Lipofectamine, their positive mono-cell clones being resistant to Zeocin were isolated. TaqMan real-time quantitative RT-PCR (RQ-PCR) and Western blot respectively detected expression of BCR/ABL mRNA and P210 protein. Trypaum blue dying was used to analyze the proliferation of K562 cells. Cell apoptosis was observed by flow cytometer. the recombinant plasmid was steadily transfected into K562 cells by Lipofectamine 2000, Their positive mono-cell clones being resistant to Zeocin were isolated. The proliferation of K562 cells were remarkably inhibited by the recombinant plasmid induced gene expression by tetracycline. Tetracycline induced its expression for 48 h and 72 h. pTER117, pTER363 decreased the mRNA level of BCR/ABL 90%, 82% and 91.5%, 84%, respectively, P210 protein were almost measured in K562 cells. FCM analysis showed that the recombinant plasmid induced apoptosis in K562 cells, the apoptosis rate were respectively 34.4%, 58.1% in K562 cells treated by pTER117 for 48 h and 72 h, apoptosis rate were 31.8%, 54.6% by pTER363, but the control groups did not show these effects on K562 cells. The siRNA eukaryotic expression vector against BCR

  12. Long-term Radiological and Clinical Outcomes After Using Bone Marrow Mesenchymal Stem Cells Concentrate Obtained With Selective Retention Cell Technology in Posterolateral Spinal Fusion.

    Science.gov (United States)

    Yousef, Mohamed Abdelhamid Ali; La Maida, Giovanni Andrea; Misaggi, Bernardo

    2017-12-15

    Retrospective study. The aim of this study was to evaluate the long-term clinical and radiological outcomes of the use of bone marrow mesenchymal stem cell concentrate obtained with selective cell retention technology using Cellect with a particular collagen scaffold, Healos for posterolateral spinal fusion. With the increasing rate of spinal fusion, the problem of pseudarthrosis, which contributes to recurrent pain with patient disability, is considered to be the most common cause of revision lumbar spine surgery. Intensive research is being carried out to develop an alternative source of bone grafting and improve the spinal fusion rate. A retrospective review of hospital records was performed. Identified patients were contacted to have a clinical and radiological evaluation follow-up. Clinical outcome was evaluated using visual analog scales for the back pain (VAS), Oswestry Disability Index (ODI) scores, and quality of life (EQ-5D) questionnaire. Radiological outcome was evaluated by performing dynamic flexion/extension lateral views and calculation of segmental Cobb angle. Any implant-associated complication was reported. Computed tomography (CT) scans were also performed. Twenty-one patients were included and all patients achieved successful fusion. The mean difference of the segmental Cobb angle was 0.48° (range 0.3°-0.7°). Computed tomography scans showed solid bilateral fusion with bridging bone (Grade I) in all patients, but solid unilateral fusion with bridging bone (Grade II) was detected for one patient at one level. Patients started to resume working activities within a mean period of 3.5 months. The VAS score for the residual back pain was 4.1 ± 2.1, whereas the ODI was 10.5 ± 5.6 points, and the mean disability index was 21.1%. The use of bone marrow mesenchymal stem cell concentrate obtained with selective cell retention technology could be considered as an effective means for augmenting spinal fusion. 3.

  13. [Detection of ALK, ROS1 and RET fusion genes in non-small cell lung cancer patients and its clinicopathologic correlation].

    Science.gov (United States)

    Zhong, Shan; Zhang, Haiping; Bai, Dongyu; Gao, Dehong; Zheng, Jie; Ding, Yi

    2015-09-01

    To study the prevalence of ALK, ROS1 and RET fusion genes in non-small cell lung cancer (NSCLC), and its correlation with clinicopathologic features. Formalin-fixed and paraffin-embedded tissue sections from samples of 302 patients with NSCLC were screened for ALK, ROS1, RET fusions by real-time polymerase chain reaction (PCR). All of the cases were validated by Sanger DNA sequencing. The relationship between ALK, ROS1, RET fusion genes and clinicopathologic features were analyzed. In the cohort of 302 NSCLC samples, 3.97% (12/302) were found to contain ALK fusion genes, including 3 cases with E13; A20 gene fusion, 3 cases with E6; A20 gene fusion and 3 cases with E20; A20 gene fusion. There was no statistically significant difference in patient's gender, age, smoking history and histologic type. Moreover, in the 302 NSCLC samples studied, 3.97% (12/302) were found to contain ROS1 fusion genes, with CD74-ROS1 fusion identified in 9 cases. There was no statistically significant difference in patients' gender, age, smoking history and histologic type. One non-smoking elderly female patient with pulmonary adenocarcinoma had RET gene fusion. None of the cases studied had concurrent ALK, ROS1 and RET mutations. The ALK, ROS1 and RET fusion gene mutation rates in NSCLC are low, they represent some specific molecular subtypes of NSCLC. Genetic testing has significant meaning to guide clinical targeted therapy.

  14. Sodium Valproate Induces Cell Senescence in Human Hepatocarcinoma Cells

    Directory of Open Access Journals (Sweden)

    Hong-Mei An

    2013-12-01

    Full Text Available Hepatocarcinogenesis is associated with epigenetic changes, including histone deacetylases (HDACs. Epigenetic modulation by HDAC inhibition is a potentially valuable approach for hepatocellular carcinoma treatment. In present study, we evaluated the anticancer effects of sodium valproate (SVP, a known HDAC inhibitor, in human hepatocarcinoma cells. The results showed SVP inhibited the proliferation of Bel-7402 cells in a dose-dependent manner. Low dose SVP treatment caused a large and flat morphology change, positive SA-β-gal staining, and G0/G1 phase cell cycle arrest in human hepatocarcinoma cells. Low dose SVP treatment also increased acetylation of histone H3 and H4 on p21 promoter, accompanied by up-regulation of p21 and down-regulation of RB phosphorylation. These observations suggested that a low dose of SVP could induce cell senescence in hepatocarcinoma cells, which might correlate with hyperacetylation of histone H3 and H4, up-regulation of p21, and inhibition of RB phosphorylation. Since the effective concentration inducing cell senescence in hepatocarcinoma cells is clinically available, whether a clinical dose of SVP could induce cell senescence in clinical hepatocarcinoma is worthy of further study.

  15. Intracellular delivery of cell-penetrating peptide-transcriptional factor fusion protein and its role in selective osteogenesis

    Science.gov (United States)

    Suh, Jin Sook; Lee, Jue Yeon; Choi, Yoon Jung; You, Hyung Keun; Hong, Seong-Doo; Chung, Chong Pyoung; Park, Yoon Jeong

    2014-01-01

    Protein-transduction technology has been attempted to deliver macromolecular materials, including protein, nucleic acids, and polymeric drugs, for either diagnosis or therapeutic purposes. Herein, fusion protein composed of an arginine-rich cell-penetrating peptide, termed low-molecular-weight protamine (LMWP), and a transcriptional coactivator with a PDZ-binding motif (TAZ) protein was prepared and applied in combination with biomaterials to increase bone-forming capacity. TAZ has been recently identified as a specific osteogenic stimulating transcriptional coactivator in human mesenchymal stem cell (hMSC) differentiation, while simultaneously blocking adipogenic differentiation. However, TAZ by itself cannot penetrate the cells, and thus needs a transfection tool for translocalization. The LMWP-TAZ fusion proteins were efficiently translocalized into the cytosol of hMSCs. The hMSCs treated with cell-penetrating LMWP-TAZ exhibited increased expression of osteoblastic genes and protein, producing significantly higher quantities of mineralized matrix compared to free TAZ. In contrast, adipogenic differentiation of the hMSCs was blocked by treatment of LMWP-TAZ fusion protein, as reflected by reduced marker-protein expression, adipocyte fatty acid-binding protein 2, and peroxisome proliferator-activated receptor-γ messenger ribonucleic acid levels. LMWP-TAZ was applied in alginate gel for the purpose of localization and controlled release. The LMWP-TAZ fusion protein-loaded alginate gel matrix significantly increased bone formation in rabbit calvarial defects compared with alginate gel matrix mixed with free TAZ protein. The protein transduction of TAZ fused with cell-penetrating LMWP peptide was able selectively to stimulate osteogenesis in vitro and in vivo. Taken together, this fusion protein-transduction technology for osteogenic protein can thus be applied in combination with biomaterials for tissue regeneration and controlled release for tissue

  16. Cell-cycle dependent expression of a translocation-mediated fusion oncogene mediates checkpoint adaptation in rhabdomyosarcoma.

    Directory of Open Access Journals (Sweden)

    Ken Kikuchi

    2014-01-01

    Full Text Available Rhabdomyosarcoma is the most commonly occurring soft-tissue sarcoma in childhood. Most rhabdomyosarcoma falls into one of two biologically distinct subgroups represented by alveolar or embryonal histology. The alveolar subtype harbors a translocation-mediated PAX3:FOXO1A fusion gene and has an extremely poor prognosis. However, tumor cells have heterogeneous expression for the fusion gene. Using a conditional genetic mouse model as well as human tumor cell lines, we show that that Pax3:Foxo1a expression is enriched in G2 and triggers a transcriptional program conducive to checkpoint adaptation under stress conditions such as irradiation in vitro and in vivo. Pax3:Foxo1a also tolerizes tumor cells to clinically-established chemotherapy agents and emerging molecularly-targeted agents. Thus, the surprisingly dynamic regulation of the Pax3:Foxo1a locus is a paradigm that has important implications for the way in which oncogenes are modeled in cancer cells.

  17. Development of FET-switched induction accelerator cells for heavy-ion fusion recirculators

    International Nuclear Information System (INIS)

    Newton, M.A.; Cravey, W.R.; Hawkins, S.A.; Kirbie, H.C.; Ollis, C.W.

    1993-01-01

    The ''recirculator,'' a recirculating heavy-ion induction accelerator, has been identified as a promising approach for an inertial fusion driver. One of the technical challenges to building a recirculator is the requirement for a modulator that can drive the induction accelerator cells at repetition rates ≥ 100 kHz with variable pulse width and pulse repetition rate capability. A high repetition rate modulator and cell is presently being developed for use on a proposed heavy-ion recirculator. The goal is to develop an array of field-effect transistors to switch 5 kV, 1 μs pulses onto a Metglas induction core at pulse rates exceeding 100 kHz. Each transistor in the array is driven by a fiber-optic isolated gate signal that is powered by a dc/dc converter. The circuit architecture provides for core reset between pulses and produces bursts of pulses that are variable in pulse width and prf. The transistor switching array, energy storage capacitors, reset circuit and cell core are all combined into a single compact, low-impedance package. Progress of this development work will be presented with supporting data

  18. Detection of trans-acting factors for hemoglobin switching by cell fusions

    International Nuclear Information System (INIS)

    Broyles, R.H.; Palmer, J.C.; Smith, D.J.; Ramseyer, T.H.

    1986-01-01

    The authors have devised protocols for chemically fusing erythroid cells from amphibians of different developmental stages, so that we may study the short-term effects of trans-acting factors on globin gene expression. The authors are performing both homospecifc (Rana x Rana) and heterospecific (Rana x Xenopus) fusions; and they are detecting the expression of specific globin genes with selective radioactive labeling ( 35 S-methionine is incorporated only into adult globins), polyacrylamide gel electrophoresis, monospecific antisera, and cDNA probes that are species-and developmental stage-specific. Their results indicate that: (1) there are factors in tadpole erythroblasts that can reactivate adult Hb synthesis in mature, synthetically-inactive adult RBCs; (2) there are factors in tadpole erythroblasts that can reactivate tadpole globin genes in adult RBCs; and (3) there are factors in adult erythroid cells that apparently activate adult globin genes in tadpole RBCs. These results suggests that erythroid cells from animals of different developmental stages possess different sets of globin gene-specific trans-acting factors which can be studied with a system that exhibits normal developmental Hb switching

  19. Online energy management strategy of fuel cell hybrid electric vehicles based on data fusion approach

    Science.gov (United States)

    Zhou, Daming; Al-Durra, Ahmed; Gao, Fei; Ravey, Alexandre; Matraji, Imad; Godoy Simões, Marcelo

    2017-10-01

    Energy management strategy plays a key role for Fuel Cell Hybrid Electric Vehicles (FCHEVs), it directly affects the efficiency and performance of energy storages in FCHEVs. For example, by using a suitable energy distribution controller, the fuel cell system can be maintained in a high efficiency region and thus saving hydrogen consumption. In this paper, an energy management strategy for online driving cycles is proposed based on a combination of the parameters from three offline optimized fuzzy logic controllers using data fusion approach. The fuzzy logic controllers are respectively optimized for three typical driving scenarios: highway, suburban and city in offline. To classify patterns of online driving cycles, a Probabilistic Support Vector Machine (PSVM) is used to provide probabilistic classification results. Based on the classification results of the online driving cycle, the parameters of each offline optimized fuzzy logic controllers are then fused using Dempster-Shafer (DS) evidence theory, in order to calculate the final parameters for the online fuzzy logic controller. Three experimental validations using Hardware-In-the-Loop (HIL) platform with different-sized FCHEVs have been performed. Experimental comparison results show that, the proposed PSVM-DS based online controller can achieve a relatively stable operation and a higher efficiency of fuel cell system in real driving cycles.

  20. Membrane fusion is induced by a distinct peptide sequence of the sea urchin fertilization protein bindin

    NARCIS (Netherlands)

    Ulrich, AS; Glabe, CG; Hoekstra, D

    1998-01-01

    Fertilization in the sea urchin is mediated by the membrane-associated acrosomal protein bindin, which plays a key role in the adhesion and fusion between sperm and egg. We have investigated the structure/function relationship of an 18-amino acid peptide fragment "B18," which represents the minimal

  1. EML4-ALK induces epithelial–mesenchymal transition consistent with cancer stem cell properties in H1299 non-small cell lung cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Fuchun; Liu, Xiaoke, E-mail: liuxk57@163.com; Qing, Qin, E-mail: qinqingscu@126.com; Sang, Yaxiong, E-mail: yaxiongsang@gmail.com; Feng, Chengjun, E-mail: leymj@163.com; Li, Xiaoyu, E-mail: lixiaoyu2012huaxi@163.com; Jiang, Li, E-mail: summer.jl06@foxmail.com; Su, Pei, E-mail: keyanxiaozhu@163.com; Wang, Yongsheng, E-mail: wangys@scu.edu.cn

    2015-04-10

    The echinoderm microtubule-associated protein-like 4(EML4) – anaplastic lymphoma kinase (ALK) fusion gene has been identified as a driver mutation in non-small-cell lung cancer (NSCLC). However, the role of EML4-ALK in malignant transformation is not entirely clear. Here, for the first time, we showed that H1299 NSCLC cells stably expressing EML4-ALK acquire EMT phenotype, associated with enhanced invasive migration and increased expression of EMT-inducing transcription factors. H1299-EML4-ALK cells also displayed cancer stem cell-like properties with a concomitant up-regulation of CD133 and enhanced ability of mammospheres formation. Moreover, we found that inhibition of ERK1/2 reversed EMT induced by EML4-ALK in H1299 cells. Taken together, these results suggested that EML4-ALK induced ERK activation is mechanistically associated with EMT phenotype. Thus, inhibition of ERK signaling pathway could be a potential strategy in treatment of NSCLC patients with EML4-ALK translocation. - Highlights: • EML4-ALK induced epithelial–mesenchymal transition in H1299 cells. • Expression of EML4-ALK promotes invasion and migration in vitro. • EML4-ALK enhanced sphere formation and stem cell-like properties in H1299 cells. • Blockage of ERK1/2 reverse Epithelial–Mesenchymal transition induced by EML4-ALK.

  2. Viral membrane fusion

    International Nuclear Information System (INIS)

    Harrison, Stephen C.

    2015-01-01

    Membrane fusion is an essential step when enveloped viruses enter cells. Lipid bilayer fusion requires catalysis to overcome a high kinetic barrier; viral fusion proteins are the agents that fulfill this catalytic function. Despite a variety of molecular architectures, these proteins facilitate fusion by essentially the same generic mechanism. Stimulated by a signal associated with arrival at the cell to be infected (e.g., receptor or co-receptor binding, proton binding in an endosome), they undergo a series of conformational changes. A hydrophobic segment (a “fusion loop” or “fusion peptide”) engages the target-cell membrane and collapse of the bridging intermediate thus formed draws the two membranes (virus and cell) together. We know of three structural classes for viral fusion proteins. Structures for both pre- and postfusion conformations of illustrate the beginning and end points of a process that can be probed by single-virion measurements of fusion kinetics. - Highlights: • Viral fusion proteins overcome the high energy barrier to lipid bilayer merger. • Different molecular structures but the same catalytic mechanism. • Review describes properties of three known fusion-protein structural classes. • Single-virion fusion experiments elucidate mechanism

  3. Determination of the minimal fusion peptide of bovine leukemia virus gp30

    International Nuclear Information System (INIS)

    Lorin, Aurelien; Lins, Laurence; Stroobant, Vincent; Brasseur, Robert; Charloteaux, Benoit

    2007-01-01

    In this study, we determined the minimal N-terminal fusion peptide of the gp30 of the bovine leukemia virus on the basis of the tilted peptide theory. We first used molecular modelling to predict that the gp30 minimal fusion peptide corresponds to the 15 first residues. Liposome lipid-mixing and leakage assays confirmed that the 15-residue long peptide induces fusion in vitro and that it is the shortest peptide inducing optimal fusion since longer peptides destabilize liposomes to the same extent but not shorter ones. The 15-residue long peptide can thus be considered as the minimal fusion peptide. The effect of mutations reported in the literature was also investigated. Interestingly, mutations related to glycoproteins unable to induce syncytia in cell-cell fusion assays correspond to peptides predicted as non-tilted. The relationship between obliquity and fusogenicity was also confirmed in vitro for one tilted and one non-tilted mutant peptide

  4. Nanosilica induced dose-dependent cytotoxicity and cell type-dependent multinucleation in HepG2 and L-02 cells

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Yongbo [Capital Medical University, Beijing Key Laboratory for Pediatric Diseases of Otolaryngology, Head and Neck Surgery, Beijing Pediatric Research Institute, Beijing Children’s Hospital (China); Duan, Junchao; Li, Yang; Yu, Yang; Hu, Hejing; Wu, Jing; Zhang, Yannan; Li, Yanbo; CaixiaGuo; Zhou, Xianqing; Sun, Zhiwei, E-mail: zwsun@ccmu.edu.cn [Capital Medical University, School of Public Health (China)

    2016-11-15

    The prevalent exposure to nanosilica gained concerns about health effects of these particles on human beings. Although nanosilica-induced multinucleation has been confirmed previously, the underlying mechanism was still not clear; this study was to investigate the origination of multinucleated cells caused by nanosilica (62 nm) in both HepG2 and L-02 cells. Cell viability and cellular uptake was determined by MTT assay and transmission electron microscope (TEM), respectively. Giemsa staining was applied to detect multinucleation. To clarify the origination of multinucleated cells, fluorescent probes, PKH26 and PKH67, time-lapse observation were further conducted by confocal microscopy. Results indicated that nanosilica particles were internalized into cells and induced cytotoxicity in a dose-dependent manner. Quantification analysis showed that nanosilica significantly increased the rates of binucleated and multinucleated cells, which suggested mitotic catastrophe induction. Moreover, dynamic visualization verified that multinucleation resulted from cell fusion in HepG2 cells not in L-02 cells after nanosilica exposure, suggesting cell type-dependent multinucleation formation. Both multinucleation and cell fusion were involved in genetic instability, which emphasized the significance to explore the multinucleation induced by nanosilica via environmental, occupational and consumer product exposure.

  5. [Standard protocol of ALK fusion gene assessment by fluorescent in situ hybridization in non-small cell lung cancer].

    Science.gov (United States)

    Guo, Lei; Zheng, Shan; Xie, Yong-qiang

    2013-08-01

    To investigate the standard protocol for anaplastic lymphoma kinase (ALK) fusion gene assessment by fluorescent in situ hybridization (FISH) in non-small cell lung cancer (NSCLC). Tissue specimens of NSCLC cases were retrospectively collected from Jan. 2011 to July 2012. ALK fusion gene was examined by FISH using break-apart ALK gene probes (Vysis company). The identification of ALK fusion gene was determined by fluorescent signals under a fluorescence microscope. One hundred and forty-six eligible NSCLC tumor specimens were tested for ALK fusion gene by FISH. The specimens included 110 cases (75.4%) of surgically-removed tissues, 11 cases (7.5%) of biopsy, 19 cases (13.0%) of lymph node and 6 cases (4.1%) of other metastatic tissues. The positivity of ALK fusion gene was 8.9% (13/146). The assessment of ALK fusing gene by FISH using standard protocol for formalin-fixed, paraffin-embedded (FFPE) tissue is feasible. The protocol can used to test in surgically-removed tissues, biopsies, metastatic lymph nodes and other metastastic specimens.

  6. Analysis of EML4-ALK Gene Fusion Mutation in Patients 
with Non-small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Xuzhou WANG

    2015-02-01

    Full Text Available Background and objective Non-small cell lung cancer (NSCLC is the main type of lung cancer, and the related locus mutation detection research has become a hot direction of molecular targeted therapy, studying on gene mutation status of echinodem microtubule associated protein like 4-Anaplastic lymphoma kinase (EML4-ALK and epidermal growth factor receptor (EGFR, detecting the sensitivity of EML4-ALK gene fusion and gene mutation of EGFR. Methods EML4-ALK gene fusion in 85 cases of paraffin embedded tumor tissue and adjacent lung tissue was detected with the application of immunohistochemistry (IHC, Scorpions amplification refractory mutation system (Scorpions ARMS fluorescence quantitative PCR and fluorescence in situ hybridization (FISH technology, and EGFR gene in 18, 19, 20 and 21 exon mutation status was detected with the application of ARMS method. Results In 115 cases of NSCLC, IHC showed 32 cases with ALK (D5F3 expression, the expression rate was 27.8%; ARMS showed 27 cases with EML4-ALK fusion gene mutation, the mutation detection rate was 23.5%; 53 cases were detected with EGFR mutation, the mutation rate was 46%. While FISH showed 23 cases with EML4-ALK fusion gene mutation, the detection rate was 20%, slightly lower than the ARMS detection results, suggesting that ARMS more sensitive. Conclusion The application of IHC, ARMS fluorescence quantitative PCR and FISH technology can make a rapid and accurate evaluation of EML4-ALK gene fusion.

  7. HAM-5 Functions As a MAP Kinase Scaffold during Cell Fusion in Neurospora crassa

    Science.gov (United States)

    Jonkers, Wilfried; Leeder, Abigail C.; Ansong, Charles; Wang, Yuexi; Yang, Feng; Starr, Trevor L.; Camp, David G.; Smith, Richard D.; Glass, N. Louise

    2014-01-01

    Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC-1, MEK-2 and MAK-2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every ∼8 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a recently identified scaffold for the MAK-1 MAP kinase pathway in Sordaria macrospora. How the MAK-2 oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) that can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK-2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM-5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM-5-GFP co-localized with NRC-1, MEK-2 and MAK-2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM-5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK-2 activity influences HAM-5 function/localization. However, MAK-2-GFP showed cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta. Via co-immunoprecipitation experiments, HAM-5 was shown to physically interact with NRC-1, MEK-2 and MAK-2, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members for oscillation and chemotropic interactions during germling and hyphal fusion in N. crassa. The identification of HAM-5 as a scaffold-like protein will help to link the activation of MAK-2 cascade to upstream factors and proteins involved in this intriguing process of

  8. Foxa2-venus fusion reporter mouse line allows live-cell analysis of endoderm-derived organ formation.

    Science.gov (United States)

    Burtscher, Ingo; Barkey, Wenke; Lickert, Heiko

    2013-08-01

    The Foxa2-winged helix/forkhead box transcription factor (TF) is absolutely required for endoderm formation and organogenesis. Foxa2 plays essential roles during lung, liver, pancreas, and gastrointestinal tract development and regulates cell-type specific programs in the adult organism. To specifically address Foxa2 function during organ development and homeostasis, we generated a Foxa2-Venus fusion (FVF) reporter protein by gene targeting in embryonic stem (ES) cells. The FVF knock-in reporter is expressed under endogenous Foxa2 control and the fluorescent protein fusion does not interfere with TF function, as homozygous mice are viable and fertile. Moreover, the FVF protein localizes to the nucleus, associates with chromatin during mitosis, and reflects the endogenous Foxa2 protein distribution pattern in several tissues in heterozygous animals. Importantly, live-cell imaging on single-cell level of the FVF and Sox17-Cherry fusion double knock-in reporter ES cell line reveals the dynamics of endoderm TF accumulation during ES cell differentiation. The FVF reporter also allowed us to identify the endoderm progenitors during gastrulation and to visualize the different branching morphogenesis modes of the lung and pancreas epithelium in ex vivo embryo and organ cultures. In summary, the generation of the FVF reporter line adds an important new tool to visualize and analyse endoderm-derived organ development and homeostasis on the cellular and molecular level. Copyright © 2013 Wiley Periodicals, Inc.

  9. Fusion to GFP blocks intercellular trafficking of the sucrose transporter SUT1 leading to accumulation in companion cells

    Directory of Open Access Journals (Sweden)

    Walsh Rama

    2003-12-01

    Full Text Available Abstract Background Plant phloem consists of an interdependent cell pair, the sieve element / companion cell complex. Sucrose transporters are localized to enucleate sieve elements (SE, despite being transcribed in companion cells (CC. Due to the high turnover of SUT1, sucrose transporter mRNA or protein must traffic from CC to SE via the plasmodesmata. Localization of SUT mRNA at plasmodesmatal orifices connecting CC and SE suggests RNA transport, potentially mediated by RNA binding proteins. In many organisms, polar RNA transport is mediated through RNA binding proteins interacting with the 3'-UTR and controlling localized protein synthesis. To study mechanisms for trafficking of SUT1, GFP-fusions with and without 3'-UTR were expressed in transgenic plants. Results In contrast to plants expressing GFP from the strong SUC2 promoter, in RolC-controlled expression GFP is retained in companion cells. The 3'-UTR of SUT1 affected intracellular distribution of GFP but was insufficient for trafficking of SUT1, GFP or their fusions to SEs. Fusion of GFP to SUT1 did however lead to accumulation of SUT1-GFP in the CC, indicating that trafficking was blocked while translational inhibition of SUT1 mRNA was released in CCs. Conclusion A fusion with GFP prevents targeting of the sucrose transporter SUT1 to the SE while leading to accumulation in the CC. The 3'-UTR of SUT1 is insufficient for mobilization of either the fusion or GFP alone. It is conceivable that SUT1-GFP protein transport through PD to SE was blocked due to the presence of GFP, resulting in retention in CC particles. Alternatively, SUT1 mRNA transport through the PD could have been blocked due to insertion of GFP between the SUT1 coding sequence and 3'-UTR.

  10. 5'/ 3' imbalance strategy to detect ALK fusion genes in circulating tumor RNA from patients with non-small cell lung cancer.

    Science.gov (United States)

    Tong, Yongqing; Zhao, Zhijun; Liu, Bei; Bao, Anyu; Zheng, Hongyun; Gu, Jian; McGrath, Mary; Xia, Ying; Tan, Bihua; Song, Chunhua; Li, Yan

    2018-03-27

    Detecting an ALK fusion gene in patients with non-small cell lung cancer (NSCLC) could provide evidence to guide individualized therapy. The 5'/3' imbalance strategy for quantitative reverse transcription-PCR (RT-qPCR) was developed to detect ALK fusion genes in circulating tumor RNA (ctRNA) of NSCLC patients. This method was validated in patients with the ALK fusion gene confirmed by next generation sequencing (NGS). The amount of the ALK fusion gene detected by the new method ranged from 33.2 to 987.4, (mean 315.2), in the patients confirmed to have the ALK fusion gene (+). This is much higher than the amount of fusion gene detected in the patients who are negative for the ALK fusion gene (-). The amount detected in the ALK fusion gene (-) samples ranged from 0.36 to 13.04, (mean 4.58). In 188 NSCLC patients, the specificity and sensitivity of the method was compared to that of the FISH method. About 10.64% of the patients showed higher ALK fusion gene expression, and were classified as ALK fusion gene (+). This is identical to the percentage of patients detected by the FISH method to be ALK fusion gene (+). The cutoff value for diagnosis of ALK fusion (+) is 32.9 as determined by this method. A new RT-PCR method using a 5'/3' imbalance strategy was developed, with high specificity and sensitivity, for detection of the ALK fusion gene in ctRNA of NSCLC patients. This method can rapidly detect ALK fusion genes in patients, which will be helpful for guiding targeted therapy, particularly the individualized usage of TKIs in these patients.

  11. Fusion of Semliki Forest virus with cholesterol-containing liposomes at low pH

    DEFF Research Database (Denmark)

    Wilschut, J; Corver, J; Nieva, J L

    1995-01-01

    . This, and the remarkably low levels of sphingolipid required for half-maximal fusion (1-2 mol%), suggest that the sphingolipid does not play a structural role in SFV fusion, but rather acts as a cofactor, possibly through activation of the viral fusion protein. Domain formation between cholesterol......Semliki Forest virus (SFV) utilizes a membrane fusion strategy to introduce its genome into the host cell. After binding to cell-surface receptors, virus particles are internalized through receptor-mediated endocytosis and directed to the endosomal cell compartment. Subsequently, triggered...... by the acid pH in the lumen of the endosomes, the viral envelope fuses with the endosomal membrane. As a result of this fusion reaction the viral RNA gains access to the cell cytosol. Low-pH-induced fusion of SFV, in model systems as well as in cells, has been demonstrated previously to be strictly dependent...

  12. Trophoblast lineage cells derived from human induced pluripotent stem cells

    International Nuclear Information System (INIS)

    Chen, Ying; Wang, Kai; Chandramouli, Gadisetti V.R.; Knott, Jason G.; Leach, Richard

    2013-01-01

    Highlights: •Epithelial-like phenotype of trophoblast lineage cells derived from human iPS cells. •Trophoblast lineage cells derived from human iPS cells exhibit trophoblast function. •Trophoblasts from iPS cells provides a proof-of-concept in regenerative medicine. -- Abstract: Background: During implantation, the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient’s placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. Methods and results: Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. Conclusion: Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro

  13. Trophoblast lineage cells derived from human induced pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Ying, E-mail: ying.chen@hc.msu.edu [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Wang, Kai; Chandramouli, Gadisetti V.R. [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Knott, Jason G. [Developmental Epigenetics Laboratory, Department of Animal Science, Michigan State University (United States); Leach, Richard, E-mail: Richard.leach@hc.msu.edu [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Department of Obstetrics, Gynecology and Women’s Health, Spectrum Health Medical Group (United States)

    2013-07-12

    Highlights: •Epithelial-like phenotype of trophoblast lineage cells derived from human iPS cells. •Trophoblast lineage cells derived from human iPS cells exhibit trophoblast function. •Trophoblasts from iPS cells provides a proof-of-concept in regenerative medicine. -- Abstract: Background: During implantation, the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient’s placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. Methods and results: Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. Conclusion: Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro.

  14. Perspective for special Gurdon issue for differentiation: can cell fusion inform nuclear reprogramming?

    Science.gov (United States)

    Burns, David; Blau, Helen M

    2014-07-01

    Nuclear reprogramming was first shown to be possible by Sir John Gurdon over a half century ago. The process has been revolutionized by the production of induced pluripotent cells by overexpression of the four transcription factors discovered by Shinya Yamanaka, which now enables mammalian applications. Yet, reprogramming by a few transcription factors remains incomplete and inefficient, whether to pluripotent or differentiated cells. We propose that a better understanding of mechanistic insights based on developmental principles gained from heterokaryon studies may inform the process of directing cell fate, fundamentally and clinically. Copyright © 2014 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  15. Induced Pluripotent Stem Cells in Cardiovascular Medicine

    Directory of Open Access Journals (Sweden)

    Toru Egashira

    2011-01-01

    Full Text Available Induced pluripotent stem (iPS cells are generated by reprogramming human somatic cells through the forced expression of several embryonic stem (ES cell-specific transcription factors. The potential of iPS cells is having a significant impact on regenerative medicine, with the promise of infinite self-renewal, differentiation into multiple cell types, and no problems concerning ethics or immunological rejection. Human iPS cells are currently generated by transgene introduction principally through viral vectors, which integrate into host genomes, although the associated risk of tumorigenesis is driving research into nonintegration methods. Techniques for pluripotent stem cell differentiation and purification to yield cardiomyocytes are also advancing constantly. Although there remain some unsolved problems, cardiomyocyte transplantation may be a reality in the future. After those problems will be solved, applications of human iPS cells in human cardiovascular regenerative medicine will be envisaged for the future. Furthermore, iPS cell technology has generated new human disease models using disease-specific cells. This paper summarizes the progress of iPS cell technology in cardiovascular research.

  16. A cell-permeable fusion protein based on Clostridium botulinum C2 toxin for delivery of p53 tumorsuppressor into cancer cells.

    Directory of Open Access Journals (Sweden)

    Jörg Fahrer

    Full Text Available Genetically engineered bacterial protein toxins are attractive systems for delivery of exogenous proteins into the cytosol of mammalian cells. The binary C2 toxin from C. botulinum has emerged as powerful delivery vehicle, which rests on its binding/translocation component C2IIa and the genetically modified adaptor domain C2IN that act in concert to trigger cell uptake. The p53 tumor suppressor protein has a crucial function in suppressing carcinogenesis and is frequently inactivated by diverse mechanisms in human tumor cells. Therefore, we constructed a C2IN-p53 fusion protein, which is internalized into cancer cells by C2IIa. To this end, the C2IN-p53 fusion construct was overexpressed in E. coli with good solubility, purified by heparin affinity chromatography and protein identity was confirmed by immunoblotting. We demonstrated that the fusion protein is capable of binding to the p53 consensus-DNA with high affinity in a p53-specific manner in vitro. Next, the internalization of C2IN-p53 was monitored in HeLa cells by cell fractionation and immunoblot analysis, which revealed a C2IIa-mediated translocation of the fusion protein into the cytosol. The uptake was also shown in A549 and Saos-2 cells with similar efficiency. These findings were further corroborated by confocal immunofluorescence analyses of C2IN-p53/C2IIa-treated HeLa and A549 cells, displaying predominantly cytoplasmic localization of the fusion construct.

  17. Fusions of Breast Carcinoma and Dendritic Cells as a Vaccine for the Treatment of Metatastic Breast Cancer.

    Science.gov (United States)

    2013-07-01

    calculation of SI as explained in Materials and Meth - ods section. 812 FUSION OF DCs WITH BREAST CARCINOMA CELLS were isolated by FACS gating of cells...tumour-in- duced dendritic-cell defects. Nat. Rev. Immunol. 4: 941–952. 10. Asavaroengchai, W., Y. Kotera, and J. J. Mule . 2002. Tumor lysate-pulsed...J. D. Geiger, and J. J. Mule . 2002. A phase I trial of tumor lysate-pulsed dendritic cells in the treatment of advanced cancer. Clin. Cancer Res. 8

  18. The rGel/BLyS Fusion Toxin Inhibits Diffuse Large B-cell Lymphoma Growth In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Mi-Ae Lyu

    2010-05-01

    Full Text Available Diffuse large B-cell lymphoma (DLBCL is an aggressive subtype of B-cell non-Hodgkin lymphoma (NHL and accounts for 30%to 40%of NHL. Molecules targeting nuclear factor-κB (NF-κB are expected to be of therapeutic value in those tumors where NF-κB seems to play a unique survival role such as activated B-cell (ABC-subtype DLBCL. We previously generated a rGel/BLyS fusion toxin for receptor-mediated delivery of the rGel toxin specifically to malignant B cells. In this study, we examined this fusion toxin for its ability to suppress DLBCL growth in vitro and in vivo. rGel/BLyS was specifically cytotoxic to DLBCL lines expressing all three BLyS receptors and constitutively active NF-κB. Treatment with rGel/BLyS induced down-regulation of the phosphorylation of inhibitory subunit of NF-κB (IκB-α, inhibition of NF-κB DNA-binding activity, and accumulation of IκB-α. In agreement with these results, we additionally found that rGel/BLyS downregulated levels of several NF-κB targets including Bcl-xL, Mcl-1, survivin, and x-chromosome linked inhibitor-of-apoptosis. Treatment also induced up-regulation of Bax and apoptosis through caspase-3 activation and poly ADP-ribose polymerase cleavage. Importantly, rGel/BLyS significantly inhibited tumor growth (P < .05 in a DLBCL xenograft model. Thus, our results indicate that rGel/BLyS is an excellent candidate for the treatment of aggressive NHLs that are both dependent on NF-κB and are resistant to conventional chemotherapeutic regimens.

  19. Bone marrow-derived mesenchymal stem cells assembled with low-dose BMP-2 in a three-dimensional hybrid construct enhances posterolateral spinal fusion in syngeneic rats.

    Science.gov (United States)

    Hu, Tao; Abbah, Sunny Akogwu; Toh, Soo Yein; Wang, Ming; Lam, Raymond Wing Moon; Naidu, Mathanapriya; Bhakta, Gajadhar; Cool, Simon M; Bhakoo, Kishore; Li, Jun; Goh, James Cho-Hong; Wong, Hee-Kit

    2015-12-01

    The combination of potent osteoinductive growth factor, functional osteoblastic cells, and osteoconductive materials to induce bone formation is a well-established concept in bone tissue engineering. However, supraphysiological dose of growth factor, such as recombinant human bone morphogenetic protein 2 (rhBMP-2), which is necessary in contemporary clinical application, have been reported to result in severe side effects. We hypothesize that the synergistic osteoinductive capacity of low-dose bone morphogenetic protein 2 (BMP-2) combined with undifferentiated bone marrow-derived stromal cells (BMSCs) is comparable to that of osteogenically differentiated BMSCs when used in a rodent model of posterolateral spinal fusion. A prospective study using a rodent model of posterolateral spinal fusion was carried out. Thirty-six syngeneic Fischer rats comprised the patient sample. Six groups of implants were evaluated as follows (n=6): (1) 10 µg BMP-2 with undifferentiated BMSCs; (2) 10 µg BMP-2 with osteogenic-differentiated BMSCs; (3) 2.5 µg BMP-2 with undifferentiated BMSCs; (4) 2.5 µg BMP-2 with osteogenic-differentiated BMSCs; (5) 0.5 µg BMP-2 with undifferentiated BMSCs; and (6) 0.5 µg BMP-2 with osteogenic-differentiated BMSCs. Optimal in vitro osteogenic differentiation of BMSCs was determined by quantitative real-time polymerase chain reaction (qRT-PCR) gene analysis whereas in vivo bone formation capacity was evaluated by manual palpation, micro-computed tomography, and histology. Rat BMSCs cultured in fibrin matrix that was loaded into the pores of medical-grade poly epsilon caprolactone tricalcium phosphate scaffolds differentiated toward osteogenic lineage by expressing osterix, runt-related transcription factor 2, and osteocalcium mRNA when supplemented with dexamethasone, ascorbic acid, and β-glycerophosphate. Whereas qRT-PCR revealed optimal increase in osteogenic genes expression after 7 days of in vitro culture, in vivo transplantation study showed

  20. Structural analysis of the genome of breast cancer cell line ZR-75-30 identifies twelve expressed fusion genes

    Directory of Open Access Journals (Sweden)

    Schulte Ina

    2012-12-01

    Full Text Available Abstract Background It has recently emerged that common epithelial cancers such as breast cancers have fusion genes like those in leukaemias. In a representative breast cancer cell line, ZR-75-30, we searched for fusion genes, by analysing genome rearrangements. Results We first analysed rearrangements of the ZR-75-30 genome, to around 10kb resolution, by molecular cytogenetic approaches, combining array painting and array CGH. We then compared this map with genomic junctions determined by paired-end sequencing. Most of the breakpoints found by array painting and array CGH were identified in the paired end sequencing—55% of the unamplified breakpoints and 97% of the amplified breakpoints (as these are represented by more sequence reads. From this analysis we identified 9 expressed fusion genes: APPBP2-PHF20L1, BCAS3-HOXB9, COL14A1-SKAP1, TAOK1-PCGF2, TIAM1-NRIP1, TIMM23-ARHGAP32, TRPS1-LASP1, USP32-CCDC49 and ZMYM4-OPRD1. We also determined the genomic junctions of a further three expressed fusion genes that had been described by others, BCAS3-ERBB2, DDX5-DEPDC6/DEPTOR and PLEC1-ENPP2. Of this total of 12 expressed fusion genes, 9 were in the coamplification. Due to the sensitivity of the technologies used, we estimate these 12 fusion genes to be around two-thirds of the true total. Many of the fusions seem likely to be driver mutations. For example, PHF20L1, BCAS3, TAOK1, PCGF2, and TRPS1 are fused in other breast cancers. HOXB9 and PHF20L1 are members of gene families that are fused in other neoplasms. Several of the other genes are relevant to cancer—in addition to ERBB2, SKAP1 is an adaptor for Src, DEPTOR regulates the mTOR pathway and NRIP1 is an estrogen-receptor coregulator. Conclusions This is the first structural analysis of a breast cancer genome that combines classical molecular cytogenetic approaches with sequencing. Paired-end sequencing was able to detect almost all breakpoints, where there was adequate read depth. It supports

  1. Directly Observing Micelle Fusion and Growth in Solution by Liquid-Cell Transmission Electron Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Parent, Lucas R. [Department; amp, Biochemistry, University of California, San Diego, La Jolla, California 92093, United States; Bakalis, Evangelos [Dipartimento; Ramírez-Hernández, Abelardo [Materials; Institute; Kammeyer, Jacquelin K. [Department; amp, Biochemistry, University of California, San Diego, La Jolla, California 92093, United States; Park, Chiwoo [Department; de Pablo, Juan [Materials; Institute; Zerbetto, Francesco [Dipartimento; Patterson, Joseph P. [Department; amp, Biochemistry, University of California, San Diego, La Jolla, California 92093, United States; Laboratory; Gianneschi, Nathan C. [Department; amp, Biochemistry, University of California, San Diego, La Jolla, California 92093, United States

    2017-11-16

    Amphiphilic small molecules and polymers form commonplace nanoscale macromolecular compartments and bilayers, and as such are truly essential components in all cells and in many cellular processes. The nature of these architectures, including their formation, phase changes, and stimuli-response behaviors, is necessary for the most basic functions of life, and over the past half-century, these natural micellar structures have inspired a vast diversity of industrial products, from biomedicines to detergents, lubricants, and coatings. The importance of these materials and their ubiquity have made them the subject of intense investigation regarding their nanoscale dynamics with increasing interest in obtaining sufficient temporal and spatial resolution to directly observe nanoscale processes. However, the vast majority of experimental methods involve either bulk-averaging techniques including light, neutron, and X-ray scattering, or are static in nature including even the most advanced cryogenic transmission electron microscopy techniques. Here, we employ in situ liquid-cell transmission electron microscopy (LCTEM) to directly observe the evolution of individual amphiphilic block copolymer micellar nanoparticles in solution, in real time with nanometer spatial resolution. These observations, made on a proof-of-concept bioconjugate polymer amphiphile, revealed growth and evolution occurring by unimer addition processes and by particle-particle collision-and-fusion events. The experimental approach, combining direct LCTEM observation, quantitative analysis of LCTEM data, and correlated in silico simulations, provides a unique view of solvated soft matter nanoassemblies as they morph and evolve in time and space, enabling us to capture these phenomena in solution.

  2. Fusion of intestinal epithelial cells with bone marrow derived cells is dispensable for tissue homeostasis

    NARCIS (Netherlands)

    de Jong, Joan H.; Rodermond, Hans M.; Zimberlin, Cheryl D.; Lascano, Valeria; de Sousa E Melo, Felipe; Richel, Dick J.; Medema, Jan Paul; Vermeulen, Louis

    2012-01-01

    The epithelial lining of the intestine is characterized by an immense cellular turn-over ascertaining an extensive regenerative capacity. Multiple reports suggest that besides the local intestinal stem cell pool, circulating cells of bone marrow origin (BMDCs) contribute to this process by fusing

  3. Comparison of immune responses against foot-and-mouth disease virus induced by fusion proteins using the swine IgG heavy chain constant region or β-galactosidase as a carrier of immunogenic epitopes

    International Nuclear Information System (INIS)

    Li Guangjin; Chen Weizao; Yan Weiyao; Zhao Kai; Liu Mingqiu; Zhang Jun; Fei Liang; Xu Quanxing; Sheng Zutian; Lu Yonggan; Zheng Zhaoxin

    2004-01-01

    Previously, we demonstrated that a fusion protein (Gal-FMDV) consisting of β-galactosidase and an immunogenic peptide, amino acids (141-160)-(21-40)-(141-160), of foot-and-mouth disease virus (FMDV) VP1 protein induced protective immune responses in guinea pigs and swine. We now designed a new potential recombinant protein vaccine against FMDV in swine. The immunogenic peptide, amino acids (141-160)-(21-40)-(141-160) from the VP1 protein of serotype O FMDV, was fused to the carboxy terminus of a swine immunoglobulin G single heavy chain constant region and expressed in Escherichia coli. The expressed fusion protein (IgG-FMDV) was purified and emulsified with oil adjuvant. Vaccination twice at an interval of 3 weeks with the emulsified IgG-FMDV fusion protein induced an FMDV-specific spleen proliferative T-cell response in guinea pigs and elicited high levels of neutralizing antibody in guinea pigs and swine. All of the immunized animals were efficiently protected against FMDV challenge. There was no significant difference between IgG-FMDV and Gal-FMDV in eliciting immunity after vaccination twice in swine. However, when evaluating the efficacy of a single inoculation of the fusion proteins, we found that IgG-FMDV could elicit a protective immune response in swine, while Gal-FMDV only elicited a weak neutralizing activity and could not protect the swine against FMDV challenge. Our results suggest that the IgG-FMDV fusion protein is a promising vaccine candidate for FMD in swine

  4. Heat induces gene amplification in cancer cells

    International Nuclear Information System (INIS)

    Yan, Bin; Ouyang, Ruoyun; Huang, Chenghui; Liu, Franklin; Neill, Daniel; Li, Chuanyuan; Dewhirst, Mark

    2012-01-01

    Highlights: ► This study discovered that heat exposure (hyperthermia) results in gene amplification in cancer cells. ► Hyperthermia induces DNA double strand breaks. ► DNA double strand breaks are considered to be required for the initiation of gene amplification. ► The underlying mechanism of heat-induced gene amplification is generation of DNA double strand breaks. -- Abstract: Background: Hyperthermia plays an important role in cancer therapy. However, as with radiation, it can cause DNA damage and therefore genetic instability. We studied whether hyperthermia can induce gene amplification in cancer cells and explored potential underlying molecular mechanisms. Materials and methods: (1) Hyperthermia: HCT116 colon cancer cells received water-submerged heating treatment at 42 or 44 °C for 30 min; (2) gene amplification assay using N-(phosphoacetyl)-L-aspartate (PALA) selection of cabamyl-P-synthetase, aspartate transcarbarmylase, dihydro-orotase (cad) gene amplified cells; (3) southern blotting for confirmation of increased cad gene copies in PALA-resistant cells; (4) γH2AX immunostaining to detect γH2AX foci as an indication for DNA double strand breaks. Results: (1) Heat exposure at 42 or 44 °C for 30 min induces gene amplification. The frequency of cad gene amplification increased by 2.8 and 6.5 folds respectively; (2) heat exposure at both 42 and 44 °C for 30 min induces DNA double strand breaks in HCT116 cells as shown by γH2AX immunostaining. Conclusion: This study shows that heat exposure can induce gene amplification in cancer cells, likely through the generation of DNA double strand breaks, which are believed to be required for the initiation of gene amplification. This process may be promoted by heat when cellular proteins that are responsible for checkpoints, DNA replication, DNA repair and telomere functions are denatured. To our knowledge, this is the first study to provide direct evidence of hyperthermia induced gene amplification.

  5. Spinal fusion using adipose stem cells seeded on a radiolucent cage filler : a feasibility study of a single surgical procedure in goats

    NARCIS (Netherlands)

    Kroeze, Robert J.; Smit, Theo H.; Vergroesen, Pieter P.; Bank, Ruud A.; Stoop, Reinout; van Rietbergen, Bert; van Royen, Barend J.; Helder, Marco N.

    2015-01-01

    To assess the feasibility of a one-step surgical concept, employing adipose stem cells (ASCs) and a novel degradable radiolucent cage filler (poly-L-lactide-co-caprolactone; PLCL), within polyetheretherketone cages in a stand-alone caprine spinal fusion model. A double-level fusion study was

  6. Spinal fusion using adipose stem cells seeded on a radiolucent cage filler: a feasibility study of a single surgical procedure in goats

    NARCIS (Netherlands)

    Kroeze, R.J.; Smit, T.H.; Vergroesen, P.P.A.; Bank, R.A.; Stoop, R.; van Rietbergen, B.; van Royen, B.J.; Helder, M.N.

    2015-01-01

    Purpose: To assess the feasibility of a one-step surgical concept, employing adipose stem cells (ASCs) and a novel degradable radiolucent cage filler (poly-L-lactide-co-caprolactone; PLCL), within polyetheretherketone cages in a stand-alone caprine spinal fusion model. Methods: A double-level fusion

  7. Spinal fusion using adipose stem cells seeded on a radiolucent cage filler: a feasibility study of a single surgical procedure in goats

    NARCIS (Netherlands)

    Kroeze, R.J.; Smit, T.H.; Vergroesen, P.P.; Bank, R.A.; Stoop, R.; Rietbergen, B. van; Royen, B.J. van; Helder, M.N.

    2015-01-01

    PURPOSE: To assess the feasibility of a one-step surgical concept, employing adipose stem cells (ASCs) and a novel degradable radiolucent cage filler (poly-L-lactide-co-caprolactone; PLCL), within polyetheretherketone cages in a stand-alone caprine spinal fusion model. METHODS: A double-level fusion

  8. Spinal fusion using adipose stem cells seeded on a radiolucent cage filler: a feasibility study of a single surgical procedure in goats

    NARCIS (Netherlands)

    Kroeze, Robert J.; Smit, Theo H.; Vergroesen, Pieter P.; Bank, Ruud A.; Stoop, Reinout; van Rietbergen, Bert; van Royen, Barend J.; Helder, Marco N.

    2015-01-01

    To assess the feasibility of a one-step surgical concept, employing adipose stem cells (ASCs) and a novel degradable radiolucent cage filler (poly-L-lactide-co-caprolactone; PLCL), within polyetheretherketone cages in a stand-alone caprine spinal fusion model. A double-level fusion study was

  9. Ionizing radiation induces stemness in cancer cells.

    Directory of Open Access Journals (Sweden)

    Laura Ghisolfi

    Full Text Available The cancer stem cell (CSC model posits the presence of a small number of CSCs in the heterogeneous cancer cell population that are ultimately responsible for tumor initiation, as well as cancer recurrence and metastasis. CSCs have been isolated from a variety of human cancers and are able to generate a hierarchical and heterogeneous cancer cell population. CSCs are also resistant to conventional chemo- and radio-therapies. Here we report that ionizing radiation can induce stem cell-like properties in heterogeneous cancer cells. Exposure of non-stem cancer cells to ionizing radiation enhanced spherogenesis, and this was accompanied by upregulation of the pluripotency genes Sox2 and Oct3/4. Knockdown of Sox2 or Oct3/4 inhibited radiation-induced spherogenesis and increased cellular sensitivity to radiation. These data demonstrate that ionizing radiation can activate stemness pathways in heterogeneous cancer cells, resulting in the enrichment of a CSC subpopulation with higher resistance to radiotherapy.

  10. D-D nuclear fusion processes induced in polyethylene foams by TW Laser-generated plasma

    Directory of Open Access Journals (Sweden)

    Torrisi L.

    2015-01-01

    Full Text Available Deuterium-Deuterium fusion processes were generated by focusing the 3 TW PALS Laser on solid deuterated polyethylene targets placed in vacuum. Deuterium ion acceleration of the order of 4 MeV was obtained using laser irradiance Iλ2 ∼ 5 × 1016 W μm2/cm2 on the target. Thin and thick targets, at low and high density, were irradiated and plasma properties were monitored “on line” and “off line”. The ion emission from plasma was monitored with Thomson Parabola Spectrometer, track detectors and ion collectors. Fast semiconductor detectors based on SiC and fast plastic scintillators, both employed in time-of-flight configuration, have permitted to detect the characteristic 3.0 MeV protons and 2.45 MeV neutrons emission from the nuclear fusion reactions. From massive absorbent targets we have evaluated the neutron flux by varying from negligible values up to about 5 × 107 neutrons per laser shot in the case of foams targets, indicating a reaction rate of the order of 108 fusion events per laser shot using “advanced targets”.

  11. Menstruum induces changes in mesothelial cell morphology.

    Science.gov (United States)

    Koks, C A; Demir Weusten, A Y; Groothuis, P G; Dunselman, G A; de Goeij, A F; Evers, J L

    2000-01-01

    In previous studies, we have shown that menstrual endometrium preferentially adheres to the subepithelial lining of the peritoneum. It remains to be elucidated, however, whether this damage is preexisting or inflicted by the menstrual tissue itself. We hypothesized that the menstrual tissue itself damages the peritoneum. To investigate this, the viability of menstrual endometrial tissue in peritoneal fluid (PF) was evaluated and the morphologic changes in the mesothelial cells were studied by in vitro cocultures of menstruum with mesothelial cell monolayers. Menstruum was collected with a menstrual cup. Endometrial tissue was isolated from the menstruum, resuspended in culture medium or in the cell-free fraction of PF and cultured for 24, 48 or 72 h. A 3(4, 5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed to obtain a relative measure of viable adhered endometrial cells. Mesothelial cells isolated from human omental tissue were cultured on Matrigel or uncoated plastic. At confluence, overnight cocultures were performed and scanning electron microscopy was used to evaluate the morphologic changes. The viability of endometrial fragments was 84% (n = 36, p Menstrual endometrial fragments or menstrual serum added to and cocultured with mesothelial cells induced severe morphologic alterations of the latter, including retraction, shrinking and gap formation. Similar morphologic changes were observed when mesothelial cells were cocultured with menstrual endometrial fragments in PF or in culture inserts. Incubation with conditioned medium from cultured menstrual endometrium induced similar but less pronounced changes in morphology. In conclusion, menstrual endometrial fragments remain viable in PF in vitro for at least 72 h. Antegradely shed menstruum induces changes in mesothelial cell morphology, including retraction and shrinking with exposure of the underlying surface. These findings suggest that menstruum is harmful to the peritoneal

  12. Pro-inflammatory activated Kupffer cells by lipids induce hepatic NKT cells deficiency through activation-induced cell death.

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    Tongfang Tang

    Full Text Available BACKGROUND: Dietary lipids play an important role in the progression of non-alcoholic fatty liver disease (NAFLD through alternation of liver innate immune response. AIMS: The present study was to investigate the effect of lipid on Kupffer cells phenotype and function in vivo and in vitro. And further to investigate the impact of lipid on ability of Kupffer cell lipid antigen presentation to activate NKT cells. METHODS: Wild type male C57BL/6 mice were fed either normal or high-fat diet. Hepatic steatosis, Kupffer cell abundance, NKT cell number and cytokine gene expression were evaluated. Antigen presentation assay was performed with Kupffer cells treated with certain fatty acids in vitro and co-cultured with NKT cells. RESULTS: High-fat diet induced hepatosteatosis, significantly increased Kupffer cells and decreased hepatic NKT cells. Lipid treatment in vivo or in vitro induced increase of pro-inflammatory cytokines gene expression and toll-like receptor 4 (TLR4 expression in Kupffer cells. Kupffer cells expressed high levels of CD1d on cell surface and only presented exogenous lipid antigen to activate NKT cells. Ability of Kupffer cells to present antigen and activate NKT cells was enhanced after lipid treatment. In addition, pro-inflammatory activated Kupffer cells by lipid treatment induced hepatic NKT cells activation-induced apoptosis and necrosis. CONCLUSION: High-fat diet increase Kupffer cells number and induce their pro-inflammatory status. Pro-inflammatory activated Kupfffer cells by lipid promote hepatic NKT cell over-activation and cell death, which lead to further hepatic NKT cell deficiency in the development of NAFLD.

  13. Pro-inflammatory activated Kupffer cells by lipids induce hepatic NKT cells deficiency through activation-induced cell death.

    Science.gov (United States)

    Tang, Tongfang; Sui, Yongheng; Lian, Min; Li, Zhiping; Hua, Jing

    2013-01-01

    Dietary lipids play an important role in the progression of non-alcoholic fatty liver disease (NAFLD) through alternation of liver innate immune response. The present study was to investigate the effect of lipid on Kupffer cells phenotype and function in vivo and in vitro. And further to investigate the impact of lipid on ability of Kupffer cell lipid antigen presentation to activate NKT cells. Wild type male C57BL/6 mice were fed either normal or high-fat diet. Hepatic steatosis, Kupffer cell abundance, NKT cell number and cytokine gene expression were evaluated. Antigen presentation assay was performed with Kupffer cells treated with certain fatty acids in vitro and co-cultured with NKT cells. High-fat diet induced hepatosteatosis, significantly increased Kupffer cells and decreased hepatic NKT cells. Lipid treatment in vivo or in vitro induced increase of pro-inflammatory cytokines gene expression and toll-like receptor 4 (TLR4) expression in Kupffer cells. Kupffer cells expressed high levels of CD1d on cell surface and only presented exogenous lipid antigen to activate NKT cells. Ability of Kupffer cells to present antigen and activate NKT cells was enhanced after lipid treatment. In addition, pro-inflammatory activated Kupffer cells by lipid treatment induced hepatic NKT cells activation-induced apoptosis and necrosis. High-fat diet increase Kupffer cells number and induce their pro-inflammatory status. Pro-inflammatory activated Kupfffer cells by lipid promote hepatic NKT cell over-activation and cell death, which lead to further hepatic NKT cell deficiency in the development of NAFLD.

  14. Characterization of the Neurospora crassa cell fusion proteins, HAM-6, HAM-7, HAM-8, HAM-9, HAM-10, AMPH-1 and WHI-2.

    Directory of Open Access Journals (Sweden)

    Ci Fu

    Full Text Available Intercellular communication of vegetative cells and their subsequent cell fusion is vital for different aspects of growth, fitness, and differentiation of filamentous fungi. Cell fusion between germinating spores is important for early colony establishment, while hyphal fusion in the mature colony facilitates the movement of resources and organelles throughout an established colony. Approximately 50 proteins have been shown to be important for somatic cell-cell communication and fusion in the model filamentous fungus Neurospora crassa. Genetic, biochemical, and microscopic techniques were used to characterize the functions of seven previously poorly characterized cell fusion proteins. HAM-6, HAM-7 and HAM-8 share functional characteristics and are proposed to function in the same signaling network. Our data suggest that these proteins may form a sensor complex at the cell wall/plasma membrane for the MAK-1 cell wall integrity mitogen-activated protein kinase (MAPK pathway. We also demonstrate that HAM-9, HAM-10, AMPH-1 and WHI-2 have more general functions and are required for normal growth and development. The activation status of the MAK-1 and MAK-2 MAPK pathways are altered in mutants lacking these proteins. We propose that these proteins may function to coordinate the activities of the two MAPK modules with other signaling pathways during cell fusion.

  15. Characterization of the Neurospora crassa cell fusion proteins, HAM-6, HAM-7, HAM-8, HAM-9, HAM-10, AMPH-1 and WHI-2.

    Science.gov (United States)

    Fu, Ci; Ao, Jie; Dettmann, Anne; Seiler, Stephan; Free, Stephen J

    2014-01-01

    Intercellular communication of vegetative cells and their subsequent cell fusion is vital for different aspects of growth, fitness, and differentiation of filamentous fungi. Cell fusion between germinating spores is important for early colony establishment, while hyphal fusion in the mature colony facilitates the movement of resources and organelles throughout an established colony. Approximately 50 proteins have been shown to be important for somatic cell-cell communication and fusion in the model filamentous fungus Neurospora crassa. Genetic, biochemical, and microscopic techniques were used to characterize the functions of seven previously poorly characterized cell fusion proteins. HAM-6, HAM-7 and HAM-8 share functional characteristics and are proposed to function in the same signaling network. Our data suggest that these proteins may form a sensor complex at the cell wall/plasma membrane for the MAK-1 cell wall integrity mitogen-activated protein kinase (MAPK) pathway. We also demonstrate that HAM-9, HAM-10, AMPH-1 and WHI-2 have more general functions and are required for normal growth and development. The activation status of the MAK-1 and MAK-2 MAPK pathways are altered in mutants lacking these proteins. We propose that these proteins may function to coordinate the activities of the two MAPK modules with other signaling pathways during cell fusion.

  16. Characterization of the Neurospora crassa Cell Fusion Proteins, HAM-6, HAM-7, HAM-8, HAM-9, HAM-10, AMPH-1 and WHI-2

    Science.gov (United States)

    Fu, Ci; Ao, Jie; Dettmann, Anne; Seiler, Stephan; Free, Stephen J.

    2014-01-01

    Intercellular communication of vegetative cells and their subsequent cell fusion is vital for different aspects of growth, fitness, and differentiation of filamentous fungi. Cell fusion between germinating spores is important for early colony establishment, while hyphal fusion in the mature colony facilitates the movement of resources and organelles throughout an established colony. Approximately 50 proteins have been shown to be important for somatic cell-cell communication and fusion in the model filamentous fungus Neurospora crassa. Genetic, biochemical, and microscopic techniques were used to characterize the functions of seven previously poorly characterized cell fusion proteins. HAM-6, HAM-7 and HAM-8 share functional characteristics and are proposed to function in the same signaling network. Our data suggest that these proteins may form a sensor complex at the cell wall/plasma membrane for the MAK-1 cell wall integrity mitogen-activated protein kinase (MAPK) pathway. We also demonstrate that HAM-9, HAM-10, AMPH-1 and WHI-2 have more general functions and are required for normal growth and development. The activation status of the MAK-1 and MAK-2 MAPK pathways are altered in mutants lacking these proteins. We propose that these proteins may function to coordinate the activities of the two MAPK modules with other signaling pathways during cell fusion. PMID:25279949

  17. P2X-selective purinergic antagonists are strong inhibitors of HIV-1 fusion during both cell-to-cell and cell-free infection.

    Science.gov (United States)

    Swartz, Talia H; Esposito, Anthony M; Durham, Natasha D; Hartmann, Boris M; Chen, Benjamin K

    2014-10-01

    Human immunodeficiency virus type 1 (HIV-1) infection is chronic and presently still incurable. Antiretroviral drugs effectively suppress replication; however, persistent activation of inflammatory pathways remains a key cause of morbidity. Recent studies proposed that purinergic signaling is required for HIV-1 infection. Purinergic receptors are distributed throughout a wide variety of tissue types and detect extracellular ATP as a danger signal released from dying cells. We have explored how these pathways are involved in the transmission of HIV-1 from cell to cell through virological synapses. Infection of CD4+ T lymphocytes with HIV-1 in the presence of an inhibitor of P2X receptors effectively inhibited HIV-1 infection through both cell-free and cell-to-cell contact in a dose-dependent manner. Inhibition of direct cell-to-cell infection did not affect the formation of virological synapses or the subsequent cell-to-cell transfer of HIV-1. During both cell-free and cell-to-cell CD4+ T lymphocyte infection, purinergic antagonists blocked infection at the level of viral membrane fusion. During cell-to-cell transmission, we observed CXCR4 colocalization with the newly internalized virus particles within target lymphocytes and found that the purinergic antagonists did not impair the recruitment of the coreceptor CXCR4 to the site of Gag internalization in the target cell. In a screen of a library of purinergic antagonists, we found that the most potent inhibitors of HIV-1 fusion were those that target P2X receptors, while P2Y-selective receptor antagonists or adenosine receptor antagonists were ineffective. Our results suggest that P2X receptors may provide a therapeutic target and that purinergic antagonists may have potent activity against viral infection of CD4+ T lymphocytes by both cell-free and cell-to-cell transmission. This study identifies purinergic antagonists to be potent inhibitors of HIV-1 cell-free and cell-to-cell-mediated infection and provides a

  18. Cell cycle regulation and radiation-induced cell death

    International Nuclear Information System (INIS)

    Favaudon, V.

    2000-01-01

    Tight control of cell proliferation is mandatory to prevent cancer formation as well as to normal organ development and homeostasis. This occurs through checkpoints that operate in both time and space and are involved in the control of numerous pathways including DNA replication and transcription, cell cycle progression, signal transduction and differentiation. Moreover, evidence has accumulated to show that apoptosis is tightly connected with the regulation of cell cycle progression. In this paper we describe the main pathways that determine checkpoints in the cell cycle and apoptosis. It is also recalled that in solid tumors radiation-induced cell death occurs most frequently through non-apoptotic mechanisms involving oncosis, and mitotic or delayed cell death. (author)

  19. Cell-autonomous correction of ring chromosomes in human induced pluripotent stem cells

    Science.gov (United States)

    Bershteyn, Marina; Hayashi, Yohei; Desachy, Guillaume; Hsiao, Edward C.; Sami, Salma; Tsang, Kathryn M.; Weiss, Lauren A.; Kriegstein, Arnold R.; Yamanaka, Shinya; Wynshaw-Boris, Anthony

    2014-03-01

    Ring chromosomes are structural aberrations commonly associated with birth defects, mental disabilities and growth retardation. Rings form after fusion of the long and short arms of a chromosome, and are sometimes associated with large terminal deletions. Owing to the severity of these large aberrations that can affect multiple contiguous genes, no possible therapeutic strategies for ring chromosome disorders have been proposed. During cell division, ring chromosomes can exhibit unstable behaviour leading to continuous production of aneuploid progeny with low viability and high cellular death rate. The overall consequences of this chromosomal instability have been largely unexplored in experimental model systems. Here we generated human induced pluripotent stem cells (iPSCs) from patient fibroblasts containing ring chromosomes with large deletions and found that reprogrammed cells lost the abnormal chromosome and duplicated the wild-type homologue through the compensatory uniparental disomy (UPD) mechanism. The karyotypically normal iPSCs with isodisomy for the corrected chromosome outgrew co-existing aneuploid populations, enabling rapid and efficient isolation of patient-derived iPSCs devoid of the original chromosomal aberration. Our results suggest a fundamentally different function for cellular reprogramming as a means of `chromosome therapy' to reverse combined loss-of-function across many genes in cells with large-scale aberrations involving ring structures. In addition, our work provides an experimentally tractable human cellular system for studying mechanisms of chromosomal number control, which is of critical relevance to human development and disease.

  20. Flagellar membrane fusion and protein exchange in trypanosomes; a new form of cell-cell communication? [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Simon Imhof

    2016-04-01

    Full Text Available Diverse structures facilitate direct exchange of proteins between cells, including plasmadesmata in plants and tunnelling nanotubes in bacteria and higher eukaryotes.  Here we describe a new mechanism of protein transfer, flagellar membrane fusion, in the unicellular parasite Trypanosoma brucei. When fluorescently tagged trypanosomes were co-cultured, a small proportion of double-positive cells were observed. The formation of double-positive cells was dependent on the presence of extracellular calcium and was enhanced by placing cells in medium supplemented with fresh bovine serum. Time-lapse microscopy revealed that double-positive cells arose by bidirectional protein exchange in the absence of nuclear transfer.  Furthermore, super-resolution microscopy showed that this process occurred in ≤1 minute, the limit of temporal resolution in these experiments. Both cytoplasmic and membrane proteins could be transferred provided they gained access to the flagellum. Intriguingly, a component of the RNAi machinery (Argonaute was able to move between cells, raising the possibility that small interfering RNAs are transported as cargo. Transmission electron microscopy showed that shared flagella contained two axonemes and two paraflagellar rods bounded by a single membrane. In some cases flagellar fusion was partial and interactions between cells were transient. In other cases fusion occurred along the entire length of the flagellum, was stable for several hours and might be irreversible. Fusion did not appear to be deleterious for cell function: paired cells were motile and could give rise to progeny while fused. The motile flagella of unicellular organisms are related to the sensory cilia of higher eukaryotes, raising the possibility that protein transfer between cells via cilia or flagella occurs more widely in nature.

  1. Intratumoral vaccination of adenoviruses expressing fusion protein RM4/tumor necrosis factor (TNF)-alpha induces significant tumor regression.

    Science.gov (United States)

    Wright, P; Zheng, C; Moyana, T; Xiang, J

    1998-01-01

    Recombinant adenovirus (AdV) vectors are highly efficient at in vitro and in vivo gene delivery. VKCK is a murine myeloma cell line expressing the light chain of the fusion protein RM4/tumor necrosis factor (TNF)-alpha. The in vitro transfection of VKCK cells with the AdV AdV5LacZ, which contains the marker gene beta-galactosidase, can reach a maximal 75% at a multiplicity of infection of 1000. Intratumoral injections of AdV5LacZ (2 x 10(9) plaque-forming units) resulted in substantial gene transfer in nearly 50% of VKCK tumors. The AdV pLpA/M4-TNF-alpha, which contains a fused gene M4-TNF-alpha that codes for the heavy chain of fusion protein RM4/TNF-alpha, was constructed. After the in vitro transfection of pLpA/M4-TNF-alpha at a multiplicity of infection of 1000, transfected VKCK cells showed significant secretion of RM4/TNF-alpha (36 ng/mL/10(6) cells) containing the functional TNF-alpha moiety in tissue culture. The secretion peaks at day 3 and is diminished at day 6 following the viral infection. These transfected VKCK cells also became more immunogenic with enhanced expression of major histocompatibility complex class I antigen. Intratumoral injections of 2 x 10(9) plaque-forming units of pLpA/M4-TNF-alpha virus with a repeated booster resulted in significant VKCK tumor regression in immune-competent mice, but not in athymic nude mice with a mean tumor weight of 0.07 g that were compared with 1.58 g and 1.70 g for tumors injected with AdV5LacZ and phosphate-buffered saline, respectively (P management of solid human tumors.

  2. Production of cloned dogs by decreasing the interval between fusion and activation during somatic cell nuclear transfer.

    Science.gov (United States)

    Kim, Sue; Park, Sun Woo; Hossein, Mohammad Shamim; Jeong, Yeon Woo; Kim, Joung Joo; Lee, Eugine; Kim, Yeun Wook; Hyun, Sang Hwan; Shin, Taeyoung; Hwang, Woo Suk

    2009-05-01

    To improve the efficiency of somatic cell nuclear transfer (SCNT) in dogs, we evaluated whether or not the interval between fusion and activation affects the success rate of SCNT. Oocytes retrieved from outbred dogs were reconstructed with adult somatic cells from a male or female Golden Retriever. In total, 151 and 225 reconstructed oocytes were transferred to 9 and 14 naturally synchronized surrogates for male and female donor cells, respectively. Chromosomal morphology was evaluated in 12 oocytes held for an interval of 2 hr between fusion and activation and 14 oocytes held for an interval of 4 hr. Three hundred seventy-six and 288 embryos were transferred to 23 and 16 surrogates for the 2 and 4 hr interval groups, respectively. Both the male (two pregnant surrogates gave birth to three puppies) and female (one pregnant surrogate gave birth to one puppy) donor cells gave birth to live puppies (P > 0.05). In the 2 hr group, significantly more reconstructed oocytes showed condensed, metaphase-like chromosomes compared to the 4 hr group (P dogs carried pregnancies to term and four puppies were born. These results demonstrate that decreasing the interval between fusion and activation increases the success rate of clone production and pregnancy. These results may increase the overall efficiency of SCNT in the canine family. (c) 2008 Wiley-Liss, Inc.

  3. Expression and purification of bioactive soluble murine stem cell factor from recombinant Escherichia coli using thioredoxin as fusion partner.

    Science.gov (United States)

    Bals, Carola; Schambach, Axel; Meyer, Johann; Scheper, Thomas; Rinas, Ursula

    2011-03-10

    Stem cell factor (SCF) known as the c-kit ligand, plays important roles in spermatogenesis, melanogenesis and early stages of hematopoiesis. As for the latter, SCF is essential for growth and expansion of hematopoietic stem and progenitor cells. We herein describe the production of recombinant murine SCF from Escherichia coli as soluble thioredoxin-fusion protein. The formation of insoluble and inactive inclusion bodies, usually observed when SCF is expressed in E. coli, was almost entirely prevented. After purification based on membrane adsorber technology, the fusion protein was subsequently cleaved by TEV protease in order to release mature mSCF. Following dialysis and a final purification step, the target protein was isolated in high purity. Bioactivity of mSCF was proven by different tests (MTT analogous assay, long-term proliferation assay) applying a human megakaryocytic cell line. Furthermore, the biological activity of the uncleaved fusion protein was tested as well. We observed a significant activity, even though it was less than the activity displayed by the purified mSCF. In summary, avoiding inclusion body formation we present an efficient production procedure for mSCF, one of the most important stem cell cytokines. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. Generation of a panel of somatic cell hybrids containing fragments of human chromosome 12P by X-ray irradiation and cell fusion

    NARCIS (Netherlands)

    Sinke, R J; Suijkerbuijk, R F; Herbergs, J; Janssen, H; Cassiman, J J; Geurts van Kessel, A

    We have employed an irradiation and fusion procedure to generate somatic cell hybrids containing various fragments of the short arm of human chromosome 12 using a 12p-only hybrid (M28) as starting material. For the initial identification of hybrids retaining human DNA, nonradioactive in situ

  5. IGF Binding Protein-5 Induces Cell Senescence

    Directory of Open Access Journals (Sweden)

    Fumihiro Sanada

    2018-02-01

    Full Text Available Cellular senescence is the complex process of deterioration that drives the aging of an organism, resulting in the progressive loss of organ function and eventually phenotypic aging. Senescent cells undergo irreversible growth arrest, usually by inducing telomere shortening. Alternatively, senescence may also occur prematurely in response to various stress stimuli, such as oxidative stress, DNA damage, or activated oncogenes. Recently, it has been shown that IGF binding protein-5 (IGFBP-5 with the induction of the tumor suppressor p53 is upregulated during cellular senescence. This mechanism mediates interleukin-6/gp130-induced premature senescence in human fibroblasts, irradiation-induced premature senescence in human endothelial cells (ECs, and replicative senescence in human ECs independent of insulin-like growth factor I (IGF-I and IGF-II. Additionally, a link between IGFBP-5, hyper-coagulation, and inflammation, which occur with age, has been implicated. Thus, IGFBP-5 seems to play decisive roles in controlling cell senescence and cell inflammation. In this review, we describe the accumulating evidence for this role of IGFBP-5 including our new finding.

  6. Recombinant ESAT-6-CFP10 Fusion Protein Induction of Th1/Th2 Cytokines and FoxP3 Expressing Treg Cells in Pulmonary TB.

    Directory of Open Access Journals (Sweden)

    Dolly Jackson-Sillah

    Full Text Available Early secretory antigenic target 6 (ESAT-6 and culture filtrate protein 10 (CFP-10 are Mycobacterium tuberculosis (Mtb-specific antigens that are secreted by actively metabolising bacteria and contribute to the virulence of the bacteria. Their ability to induce Treg and Th2 responses, particularly during the first two weeks of treatment, has not been comprehensively examined to date. The purpose of this work was to characterise Th1, Th2 and Treg response