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Sample records for cell fusion induced

  1. Optically-Induced Cell Fusion on Cell Pairing Microstructures

    Science.gov (United States)

    Yang, Po-Fu; Wang, Chih-Hung; Lee, Gwo-Bin

    2016-02-01

    Cell fusion is a critical operation for numerous biomedical applications including cell reprogramming, hybridoma formation, cancer immunotherapy, and tissue regeneration. However, unstable cell contact and random cell pairings have limited efficiency and yields when utilizing traditional methods. Furthermore, it is challenging to selectively perform cell fusion within a group of cells. This study reports a new approach called optically-induced cell fusion (OICF), which integrates cell-pairing microstructures with an optically-induced, localized electrical field. By projecting light patterns onto a photoconductive film (hydrogen-rich, amorphous silicon) coated on an indium-tin-oxide (ITO) glass while an alternating current electrical field was applied between two such ITO glass slides, “virtual” electrodes could be generated that could selectively fuse pairing cells. At 10 kHz, a 57% cell paring rate and an 87% fusion efficiency were successfully achieved at a driving voltage of 20  Vpp, suggesting that this new technology could be promising for selective cell fusion within a group of cells.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

    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.

  4. Globular adiponectin induces differentiation and fusion of skeletal muscle cells

    Institute of Scientific and Technical Information of China (English)

    Tania Fiaschi; Domenico Cirelli; Giuseppina Comito; Stefania Gelmini; Giampietro Ramponi; Maria Serio; Paola Chiarugi

    2009-01-01

    The growing interest in skeletal muscle regeneration is associated with the opening of new therapeutic strategies for muscle injury after trauma, as well as several muscular degenerative pathologies, including dystrophies, muscu-lar atrophy, and cachexia. Studies focused on the ability of extracellular factors to promote myogenesis are therefore highly promising. We now report that an adipocyte-derived factor, globular adiponectin (gAd), is able to induce mus-cle gene expression and cell differentiation, gAd, besides its well-known ability to regulate several metabolic func-tions in muscle, including glucose uptake and consumption and fatty acid catabolism, is able to block cell cycle entry of myoblasts, to induce the expression of specific skeletal muscle markers such as myosin heavy chain or eaveolin-3, as well as to provoke cell fusion into multinucleated syneytia and, finally, muscle fibre formation, gAd exerts its pro-differentiative activity through redox-dependent activation of p38, Akt and 5'-AMP-activated protein kinase path-ways. Interestingly, differentiating myoblasts are autocrine for adiponectiu, and the mimicking of pro-inflammatory settings or exposure to oxidative stress strongly increases the production of the hormone from differentiating cells. These data suggest a novel function of adiponectin, directly coordinating the myogenic differentiation program and serving an autocrine function during skeletal myogenesis.

  5. Estimating intercellular surface tension by laser-induced cell fusion

    International Nuclear Information System (INIS)

    Intercellular surface tension is a key variable in understanding cellular mechanics. However, conventional methods are not well suited for measuring the absolute magnitude of intercellular surface tension because these methods require determination of the effective viscosity of the whole cell, a quantity that is difficult to measure. In this study, we present a novel method for estimating the intercellular surface tension at single-cell resolution. This method exploits the cytoplasmic flow that accompanies laser-induced cell fusion when the pressure difference between cells is large. Because the cytoplasmic viscosity can be measured using well-established technology, this method can be used to estimate the absolute magnitudes of tension. We applied this method to two-cell-stage embryos of the nematode Caenorhabditis elegans and estimated the intercellular surface tension to be in the 30–90 µN m−1 range. Our estimate was in close agreement with cell–medium surface tensions measured at single-cell resolution. (communication)

  6. 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

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    and Purkinje neurons. However, through lineage fate-mapping we demonstrate that such in vivo fusion of lymphoid and myeloid blood cells does not occur to an appreciable extent in steady-state adult tissues or during normal development. Rather, fusion of blood cells with different non-haematopoietic cell types...... 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......Recent studies have suggested that regeneration of non-haematopoietic cell lineages can occur through heterotypic cell fusion with haematopoietic cells of the myeloid lineage. Here we show that lymphocytes also form heterotypic-fusion hybrids with cardiomyocytes, skeletal muscle, hepatocytes...

  8. In vitro antitumor immune response induced by fusion of dendritic cells and colon cancer cells

    Institute of Scientific and Technical Information of China (English)

    Feng Xu; Ying-Jiang Ye; Shan Wang

    2004-01-01

    AIM: The prevention of recurrence of colon cancer (CC)after operation is very important for improvement of the prognosis of CC patients, especially those with micrometastasis. The generation of fused cells between dendritic cells (DCs) and tumor cells maybe an effective approach for tumor antigen presentation in immunotherapy. In this study,we fused human colon caner SW480 cells and human peripheral blood - derived DCs to induce an antitumor activity against human CC.METHODS: CC SW480 cells and human peripheral blood derived DCs were fused with 500 mL/L polyethylene glycol (PEG).RESULTS: The specific T cell responses activated by fusion cells (FCs), were observed. About 100 mL/L to 160 mL/L of the PEG-treated non-adherent cells with fluorescences were considered to be dendritomas that highly expressed the key molecules for antigen presentation in our five cases. In vitro studies showed that fusions effectively activated CD8+ T lymphocytes to secrete interferon-γ. The early apoptotic ratio of the colon cancer SW480 cells was higher than that of controls, which was affected by cytotoxic T lymphocytes (CTLs) stimulated by dendritomas.CONCLUSION: The data indicate that fusion of tumor cells with DCs is an attractive strategy to induce tumor rejection.

  9. 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.

  10. 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.

  11. Cell fusion induced by ERVWE1 or measles virus causes cellular senescence

    OpenAIRE

    Chuprin, Anna; Gal, Hilah; BIRON-SHENTAL, Tal; Biran, Anat; Amiel, Aliza; Rozenblatt, Shmuel; Krizhanovsky, Valery

    2013-01-01

    Cellular senescence limits proliferation of potentially detrimental cells, preventing tumorigenesis and restricting tissue damage. However, the function of senescence in nonpathological conditions is unknown. Here, Krizhanovsky and colleagues discover a new pathway to activate senescence cell fusion. The authors find that fusion-induced senescence occurs during embryonic development in the placenta. A counterpart of this process is also observed after infection by the measles virus. The resul...

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

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    Tiwari, Vaibhav [Department of Ophthalmology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific and College of Optometry, Western University of Health Sciences, Pomona, CA 91766 (United States); Darmani, Nissar A.; Thrush, Gerald R. [Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific and College of Optometry, Western University of Health Sciences, Pomona, CA 91766 (United States); Shukla, Deepak, E-mail: dshukla@uic.edu [Department of Ophthalmology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612 (United States)

    2009-12-18

    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.

  13. APOPTOSIS INDUCTION BY THE RECOMBINANT FUSION APOPTOSIS INDUCING FACTOR ON HELA CELLS

    Institute of Scientific and Technical Information of China (English)

    于翠娟; 孟艳玲; 桂俊豪; 赵晶; 金明; 王智; 王成济; 杨安钢

    2003-01-01

    Objective: To obtain the recombinant fusion AIF genes inserted into the eukaryotic expression vector Pires2-EGFP, to observe the expression and location of the fusion AIF genes (3NE: PE(280-358)-AIFΔ1-120, and 4NE: PE(280-364)-AIFΔ1-120), and to detect and compare their apoptosis inducing effects on the transfected HeLa cells. Methods: Full-length human AIF gene was cloned by RT-PCR, and its N-terminal mitochondrial localization sequence (MLS) was replaced by part sequence of Psuedomonas exotoxin A (PE) translocation domain (PEII(280-358/364)), then the recombinant fusion genes were inserted into the Pires2-EGFP eukaryotic expression vector. After these genes were transiently transfected into HeLa cells with LipofectAmine, the expression of the recombinant fusion AIF genes and their effects on HeLa cells were detected by fluorescent microscopy, laser confocal microscopy and electron microscopy. Results: The eukaryotic expression vectors containing the recombinant fusion AIF genes (Pires2-EGFP-PEII(280-358/364)- AIFΔ1- 120) were constructed successfully. It was demonstrated that the fusion AIF protein genes were expressed effectively in the transfected cells, with the GFP comco-expressed in cells by indirect immunofluorescence staining analysis. After transfection, expression of the genes could induce HeLa cells to exhibit the typical apoptosis features: such as plasma membrane blebbing and peripheral chromatin condensation. As compared with control groups, the untreated cells and the void vector transfected cells, the living cell number of the AIF gene transfected cells reduced distinctly. Conclusion: Our data prove that the expression of the recombinant human AIF fusion genes could induce apoptosis in transfected HeLa cells, which provides new strategy for cancer killing.

  14. Dependence of herpes simplex virus type 1-induced cell fusion on cell type

    Energy Technology Data Exchange (ETDEWEB)

    Bzik, D.J.; Person, S.

    1981-04-15

    Syncytial mutants of herpes simplex virus type 1 (HSV-1), such as syn20, cause extensive fusion of human embryonic lung (HEL) cells but only a small amount of fusion of human epidermoid carcinoma No. 2 (HEp-2) cells. In order to determine the cellular basis of this difference in fusion, sparse cultures of syn20-infected HEL or HEp-2 cells, previously labeled with (/sup 3/H)thymidine, were surrounded with uninfected, unlabeled HEL or HEp-2 cells. The fusion of radioactive with nonradioactive cells was determined at different times after infection using radioautography. The major difference in the fusion capacity of HEL and HEp-2 cells was not due to a difference in cell-surface receptors for a fusion factor in the two cell types. The process of infection of HEp-2 cells did not cause the plasma membranes of the cells to become refractory to fusion, because syn20-infected HEL cells fused equally well with either uninfected or infected HEp-2 cells. In a mixed infection with equal numbers of MP and its nonsyncytial parent, mP, extensive fusion was observed for infected HEL cells and significantly less fusion was observed for infected African green monkey (CV-1), baby hamster kidney (BHK-21), and HEp-2 cells.

  15. Laser-induced tobacco protoplast fusion

    Institute of Scientific and Technical Information of China (English)

    李银妹; 关力劼; 楼立人; 崔国强; 姚湲; 王浩威; 操传顺; 鲁润龙; 陈曦

    1999-01-01

    Laser tweezers can manipulate small particles, such as cells and organdies. When coupling them with laser microbeam selective fusion of two tobacco protoplasts containing some chloroplast was achieved. Physical and biological variables that affect laser trapping and laser-induced fusion were also discussed. The results show that the effect of chloroplast content and distribution on the yield of cell fusion is remarkable.

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

    Directory of Open Access Journals (Sweden)

    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.

  17. Active inhibition of herpes simplex virus type 1-induced cell fusion

    Energy Technology Data Exchange (ETDEWEB)

    Bzik, D.J.; Person, S.; Read, G.S.

    1982-01-01

    Previous studies have demonstrated that syn mutant-infected cells fuse less well with nonsyncytial virus-infected cells than with uninfected cells, a phenomenon defined as function inhibition. The present study characterizes the kinetics as well as the requirements for expression of fusion inhibition. Initially, the capacity of sparse syn mutant-infected cells to fuse with uninfected surrounding cells was determined throughout infection. Of seven syn mutants examined, including representatives with alterations in two different viral genes that affect cell fusion, all showed an increase in fusion capacity up to 12 hr after infection and a decrease at later times. Fusion inhibition was examined in experiments employing sparse syn20-infected cells which had been incubated to a maximum fusion capacity; it was shown that surrounding cells infected with KOS, the parent of syn20, began to inhibit fusion by the syn20-infected cells at about 4 hr after infection, and that the maximum ability to inhibit fusion was attained at about 6 hr after infection. The metabolic blocking agents actinomycin D (RNA), cycloheximide (protein), 2-deoxyglucose, and tunicamycin (glycoslyation of glycoproteins) all showed the ability to inhibit the expression of fusion inhibition by KOS-infected cells if added shortly after infection. It is concluded that fusion inhibition is an active process that requires the synthesis of RNA, proteins, and glycoproteins. 17 references, 3 figures, 2 tables.

  18. A generic screening platform for inhibitors of virus induced cell fusion using cellular electrical impedance.

    Science.gov (United States)

    Watterson, Daniel; Robinson, Jodie; Chappell, Keith J; Butler, Mark S; Edwards, David J; Fry, Scott R; Bermingham, Imogen M; Cooper, Matthew A; Young, Paul R

    2016-01-01

    Fusion of the viral envelope with host cell membranes is an essential step in the life cycle of all enveloped viruses. Despite such a clear target for antiviral drug development, few anti-fusion drugs have progressed to market. One significant hurdle is the absence of a generic, high-throughput, reproducible fusion assay. Here we report that real time, label-free measurement of cellular electrical impedance can quantify cell-cell fusion mediated by either individually expressed recombinant viral fusion proteins, or native virus infection. We validated this approach for all three classes of viral fusion and demonstrated utility in quantifying fusion inhibition using antibodies and small molecule inhibitors specific for dengue virus and respiratory syncytial virus. PMID:26976324

  19. Combined TLR2/4-activated dendritic/tumor cell fusions induce augmented cytotoxic T lymphocytes.

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    Shigeo Koido

    Full Text Available Induction of antitumor immunity by dendritic cell (DC-tumor fusion cells (DC/tumor can be modulated by their activation status. In this study, to address optimal status of DC/tumor to induce efficient antigen-specific cytotoxic T lymphocytes (CTLs, we have created various types of DC/tumor: 1 un-activated DC/tumor; 2 penicillin-killed Streptococcus pyogenes (OK-432; TLR4 agonist-activated DC/tumor; 3 protein-bound polysaccharides isolated from Coriolus versicolor (PSK; TLR2 agonist-activated DC/tumor; and 4 Combined OK-432- and PSK-activated DC/tumor. Moreover, we assessed the effects of TGF-β1 derived from DC/tumor on the induction of MUC1-specific CTLs. Combined TLR2- and TLR4-activated DC/tumor overcame immune-suppressive effect of TGF-β1 in comparison to those single activated or un-activated DC/tumor as demonstrated by: 1 up-regulation of MHC class II and CD86 expression on DC/tumor; 2 increased fusion efficiency; 3 increased production of fusions derived IL-12p70; 4 activation of CD4(+ and CD8(+ T cells that produce high levels of IFN-γ; 5 augmented induction of CTL activity specific for MUC1; and 6 superior efficacy in inhibiting CD4(+CD25(+Foxp3(+ T cell generation. However, DC/tumor-derived TGF-β1 reduced the efficacy of DC/tumor vaccine in vitro. Incorporating combined TLRs-activation and TGF-β1-blockade of DC/tumor may enhance the effectiveness of DC/tumor-based cancer vaccines and have the potential applicability to the field of adoptive immunotherapy.

  20. Wnt signaling induces transcription, spatial proximity, and translocation of fusion gene partners in human hematopoietic cells.

    Science.gov (United States)

    Ugarte, Giorgia D; Vargas, Macarena F; Medina, Matías A; León, Pablo; Necuñir, David; Elorza, Alvaro A; Gutiérrez, Soraya E; Moon, Randall T; Loyola, Alejandra; De Ferrari, Giancarlo V

    2015-10-01

    Chromosomal translocations are frequently associated with a wide variety of cancers, particularly hematologic malignancies. A recurrent chromosomal abnormality in acute myeloid leukemia is the reciprocal translocation t(8;21) that fuses RUNX1 and ETO genes. We report here that Wnt/β-catenin signaling increases the expression of ETO and RUNX1 genes in human hematopoietic progenitors. We found that β-catenin is rapidly recruited into RNA polymerase II transcription factories (RNAPII-Ser5) and that ETO and RUNX1 genes are brought into close spatial proximity upon Wnt3a induction. Notably, long-term treatment of cells with Wnt3a induces the generation a frequent RUNX1-ETO translocation event. Thus, Wnt/β-catenin signaling induces transcription and translocation of RUNX1 and ETO fusion gene partners, opening a novel window to understand the onset/development of leukemia. PMID:26333776

  1. Genetically modified mesenchymal stem cells induce mechanically stable posterior spine fusion

    OpenAIRE

    Sheyn, D; Rüthemann, M; Mizrahi, O; Kallai, I; Zilberman, Y.; Tawackoli, W; Kanim, L E A; Zhao, L; Bae, H; Pelled, G.; Snedeker, J G; Gazit, D.

    2010-01-01

    Most spine fusion procedures involve the use of prosthetic fixation devices combined with autologous bone grafts rather than biological treatment. We had shown that spine fusion could be achieved by injection of bone morphogenetic protein-2 (BMP-2)-expressing mesenchymal stem cells (MSCs) into the paraspinal muscle. In this study, we hypothesized that posterior spinal fusion achieved using genetically modified MSCs would be mechanically comparable to that realized using a mechanical fixation....

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

    Science.gov (United States)

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

    2016-04-01

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

  3. 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.

  4. A gold nanoparticle pentapeptide: gene fusion to induce therapeutic gene expression in mesenchymal stem cells.

    Science.gov (United States)

    Muroski, Megan E; Morgan, Thomas J; Levenson, Cathy W; Strouse, Geoffrey F

    2014-10-22

    Mesenchymal stem cells (MSC) have been identified as having great potential as autologous cell therapeutics to treat traumatic brain injury and spinal injury as well as neuronal and cardiac ischemic events. All future clinical applications of MSC cell therapies must allow the MSC to be harvested, transfected, and induced to express a desired protein or selection of proteins to have medical benefit. For the full potential of MSC cell therapy to be realized, it is desirable to systematically alter the protein expression of therapeutically beneficial biomolecules in harvested MSC cells with high fidelity in a single transfection event. We have developed a delivery platform on the basis of the use of a solid gold nanoparticle that has been surface modified to produce a fusion containing a zwitterionic, pentapeptide designed from Bax inhibiting peptide (Ku70) to enhance cellular uptake and a linearized expression vector to induce enhanced expression of brain-derived neurotrophic factor (BDNF) in rat-derived MSCs. Ku70 is observed to effect >80% transfection following a single treatment of femur bone marrow isolated rat MSCs with efficiencies for the delivery of a 6.6 kbp gene on either a Au nanoparticle (NP) or CdSe/ZnS quantum dot (QD). Gene expression is observed within 4 d by optical measurements, and secretion is observed within 10 d by Western Blot analysis. The combination of being able to selectively engineer the NP, to colocalize biological agents, and to enhance the stability of those agents has provided the strong impetus to utilize this novel class of materials to engineer primary MSCs. PMID:25198921

  5. Studies on virus-induced cell fusion. Progress report, May 1, 1976--July 31, 1977

    Energy Technology Data Exchange (ETDEWEB)

    Person, S.

    1977-01-01

    The cell fusion process of Herpes simplex virus has been characterized as regards macromolecular synthesis and post-macromolecular synthesis events. Studies with actinomycin D revealed that the critical period for RNA synthesis associated with cell fusion was 2 to 4 hr after infection, studies with cycloheximide showed that the critical period for protein synthesis was 3 to 5.5 hr after infection, and studies with 2-deoxyglucose showed that the critical period for glycosylation was 5 to 7 hr after infection. NH/sub 4/Cl and 2-adamantanone block events that occur subsequently (5.5 to 8 hr after infection). A temperature-sensitive step in fusion occurs from 4 to 6 hr after infection. Phosphonoacetic acid, a specific inhibitor of viral DNA synthesis, inhibits fusion only at a low MOI. Even in this case it decreases the rate but not the amount of fusion (if the kinetics curves are extended to sufficiently long times). There is no effect of phosphonoacetic acid at MOIs greater than 10 PFU/cell. Presumably the transcription and translation of 10 viral genomes/cell are required to achieve the maximum rate of fusion. 2-adamantanone partitions efficiently into cellular membranes and increases their fluidity. The effect of 2-adamantanone on cell fusion and on virus growth as well as its effect on the physical state of membranes has been studied extensively during the past year. Although 2-adamantanone does not inactivate mature virions, it does inhibit virus growth almost completely at the same concentration (5 mM). The molecular event sensitive to 2-adamantanone occurs late in viral infection and is at least partially reversible.

  6. Cell fusions in mammals

    DEFF Research Database (Denmark)

    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...

  7. Effect of the ionophore monensin on herpes simplex virus type 1-induced cell fusion, glycoprotein synthesis, and virion infectivity.

    Science.gov (United States)

    Kousoulas, K G; Bzik, D J; Person, S

    1983-01-01

    The ionophore monensin inhibited the formation of mature, fully glycosylated glycoproteins gB, gC, and gD during herpes simplex virus type 1 infection of human embryonic lung cells. Underglycosylated forms, including the apparent high-mannose precursor forms of the major glycoproteins, appeared. Monensin inhibited virus-induced cell fusion. Infectious virions produced in the presence of monensin appeared to contain predominantly underglycosylated glycoproteins. PMID:6307921

  8. Fusion with stem cell makes the hepatocellular carcinoma cells similar to liver tumor-initiating cells

    OpenAIRE

    Wang, Ran; Chen, Shuxun; Li, Changxian; Ng, Kevin Tak Pan; Kong, Chi-Wing; Cheng, Jinping; Cheng, Shuk Han; Li, Ronald A.; Lo, Chung Mau; Man, Kwan; Sun, Dong

    2016-01-01

    Background Cell fusion is a fast and highly efficient technique for cells to acquire new properties. The fusion of somatic cells with stem cells can reprogram somatic cells to a pluripotent state. Our research on the fusion of stem cells and cancer cells demonstrates that the fused cells can exhibit stemness and cancer cell-like characteristics. Thus, tumor-initiating cell-like cells are generated. Methods We employed laser-induced single-cell fusion technique to fuse the hepatocellular carci...

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

    International Nuclear Information System (INIS)

    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

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

    International Nuclear Information System (INIS)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  12. Cavitation-Induced Fusion: Proof of Concept

    OpenAIRE

    Fomitchev-Zamilov, Max I.

    2012-01-01

    Cavitation-induced fusion (also known as bubble fusion or sonofusion) has been a topic of much debate and controversy and is generally (albeit incorrectly) perceived as unworkable. In this paper we present the theoretical foundations of cavitation-induced fusion and summarize the experimental results of the research conducted in the past 20 years. Based on the systematic study of all available data we conclude that the cavitation-induced fusion is feasible, doable, and can be used for commerc...

  13. A Fusion Protein between Streptavidin and the Endogenous TLR4 Ligand EDA Targets Biotinylated Antigens to Dendritic Cells and Induces T Cell Responses In Vivo

    Directory of Open Access Journals (Sweden)

    Laura Arribillaga

    2013-01-01

    Full Text Available The development of tools for efficient targeting of antigens to antigen presenting cells is of great importance for vaccine development. We have previously shown that fusion proteins containing antigens fused to the extra domain A from fibronectin (EDA, an endogenous TLR4 ligand, which targets antigens to TLR4-expressing dendritic cells (DC, are highly immunogenic. To facilitate the procedure of joining EDA to any antigen of choice, we have prepared the fusion protein EDAvidin by linking EDA to the N terminus of streptavidin, allowing its conjugation with biotinylated antigens. We found that EDAvidin, as streptavidin, forms tetramers and binds biotin or biotinylated proteins with a Kd ~ 2.6 × 10−14 mol/L. EDAvidin favours the uptake of biotinylated green fluorescent protein by DC. Moreover, EDAvidin retains the proinflammatory properties of EDA, inducing NF-κβ by TLR4-expressing cells, as well as the production of TNF-α by the human monocyte cell line THP1 and IL-12 by DC. More importantly, immunization of mice with EDAvidin conjugated with the biotinylated nonstructural NS3 protein from hepatitis C virus induces a strong anti-NS3 T cell immune response. These results open a new way to use the EDA-based delivery tool to target any antigen of choice to DC for vaccination against infectious diseases and cancer.

  14. A fusion protein between streptavidin and the endogenous TLR4 ligand EDA targets biotinylated antigens to dendritic cells and induces T cell responses in vivo.

    Science.gov (United States)

    Arribillaga, Laura; Durantez, Maika; Lozano, Teresa; Rudilla, Francesc; Rehberger, Federico; Casares, Noelia; Villanueva, Lorea; Martinez, Marta; Gorraiz, Marta; Borrás-Cuesta, Francisco; Sarobe, Pablo; Prieto, Jesús; Lasarte, Juan José

    2013-01-01

    The development of tools for efficient targeting of antigens to antigen presenting cells is of great importance for vaccine development. We have previously shown that fusion proteins containing antigens fused to the extra domain A from fibronectin (EDA), an endogenous TLR4 ligand, which targets antigens to TLR4-expressing dendritic cells (DC), are highly immunogenic. To facilitate the procedure of joining EDA to any antigen of choice, we have prepared the fusion protein EDAvidin by linking EDA to the N terminus of streptavidin, allowing its conjugation with biotinylated antigens. We found that EDAvidin, as streptavidin, forms tetramers and binds biotin or biotinylated proteins with a Kd ~ 2.6 × 10(-14) mol/L. EDAvidin favours the uptake of biotinylated green fluorescent protein by DC. Moreover, EDAvidin retains the proinflammatory properties of EDA, inducing NF- κβ by TLR4-expressing cells, as well as the production of TNF- α by the human monocyte cell line THP1 and IL-12 by DC. More importantly, immunization of mice with EDAvidin conjugated with the biotinylated nonstructural NS3 protein from hepatitis C virus induces a strong anti-NS3 T cell immune response. These results open a new way to use the EDA-based delivery tool to target any antigen of choice to DC for vaccination against infectious diseases and cancer. PMID:24093105

  15. Rabies Virus-Induced Membrane Fusion Pathway

    OpenAIRE

    Gaudin, Yves

    2000-01-01

    Fusion of rabies virus with membranes is triggered at low pH and is mediated by the viral glycoprotein (G). The rabies virus-induced fusion pathway was studied by investigating the effects of exogenous lipids having various dynamic molecular shapes on the fusion process. Inverted cone-shaped lysophosphatidylcholines (LPCs) blocked fusion at a stage subsequent to fusion peptide insertion into the target membrane. Consistent with the stalk-hypothesis, LPC with shorter alkyl chains inhibited fus...

  16. 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.

  17. 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.

  18. A TLR4/MD2 fusion protein inhibits LPS-induced pro-inflammatory signaling in hepatic stellate cells

    International Nuclear Information System (INIS)

    Activated hepatic stellate cells (HSCs) play a key role in hepatic fibrogenesis. In injured liver they are the main extracellular matrix protein producing cell type and further perpetuate hepatic injury by secretion of pro-inflammatory mediators. Since LPS-mediated signaling through toll-like receptor 4 (TLR4) has been identified as key fibrogenic signal in HSCs we aimed to test TLR4 as potential target of therapy via ligand-binding soluble receptors. Incubation of human HSCs with a fusion protein between the extracellular domain of TLR4 and MD2 which binds LPS inhibited LPS-induced NFκB and JNK activation. TLR4/MD2 abolished LPS-induced secretion of IL-6, IL-8, MCP1, and RANTES in HSCs. In addition, TLR4/MD2 fused to human IgG-Fc neutralized LPS activity. Since TLR4 mutant mice are resistant to liver fibrosis, the TLR4/MD2 soluble receptor might represent a new therapeutic molecule for liver fibrogenesis in vivo

  19. 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.

  20. Differential Roles of Cell Death-inducing DNA Fragmentation Factor-α-like Effector (CIDE) Proteins in Promoting Lipid Droplet Fusion and Growth in Subpopulations of Hepatocytes.

    Science.gov (United States)

    Xu, Wenyi; Wu, Lizhen; Yu, Miao; Chen, Feng-Jung; Arshad, Muhammad; Xia, Xiayu; Ren, Hao; Yu, Jinhai; Xu, Li; Xu, Dijin; Li, John Zhong; Li, Peng; Zhou, Linkang

    2016-02-26

    Lipid droplets (LDs) are dynamic subcellular organelles whose growth is closely linked to obesity and hepatic steatosis. Cell death-inducing DNA fragmentation factor-α-like effector (CIDE) proteins, including Cidea, Cideb, and Cidec (also called Fsp27), play important roles in lipid metabolism. Cidea and Cidec are LD-associated proteins that promote atypical LD fusion in adipocytes. Here, we find that CIDE proteins are all localized to LD-LD contact sites (LDCSs) and promote lipid transfer, LD fusion, and growth in hepatocytes. We have identified two types of hepatocytes, one with small LDs (small LD-containing hepatocytes, SLHs) and one with large LDs (large LD-containing hepatocytes, LLHs) in the liver. Cideb is localized to LDCSs and promotes lipid exchange and LD fusion in both SLHs and LLHs, whereas Cidea and Cidec are specifically localized to the LDCSs and promote lipid exchange and LD fusion in LLHs. Cideb-deficient SLHs have reduced LD sizes and lower lipid exchange activities. Fasting dramatically induces the expression of Cidea/Cidec and increases the percentage of LLHs in the liver. The majority of the hepatocytes from the liver of obese mice are Cidea/Cidec-positive LLHs. Knocking down Cidea or Cidec significantly reduced lipid storage in the livers of obese animals. Our data reveal that CIDE proteins play differential roles in promoting LD fusion and lipid storage; Cideb promotes lipid storage under normal diet conditions, whereas Cidea and Cidec are responsible for liver steatosis under fasting and obese conditions.

  1. Cavitation-Induced Fusion: Proof of Concept

    CERN Document Server

    Fomitchev-Zamilov, Max I

    2012-01-01

    Cavitation-induced fusion (also known as bubble fusion or sonofusion) has been a topic of much debate and controversy and is generally (albeit incorrectly) perceived as unworkable. In this paper we present the theoretical foundations of cavitation-induced fusion and summarize the experimental results of the research conducted in the past 20 years. Based on the systematic study of all available data we conclude that the cavitation-induced fusion is feasible, doable, and can be used for commercial power generation. We present the results of our own research and disclose a commercial reactor prototype.

  2. Effects of GM-CSF, IL-3, and GM-CSF/IL-3 fusion protein on apoptosis of human myeloid leukemic cell line Tf-1 induced by irradiation

    Institute of Scientific and Technical Information of China (English)

    Su-rongYANG; LiWEN; Ying-qingLU; Qin-yanGONG; RongYU; Ming-huiYAO

    2004-01-01

    AIM: To observe the effects of three cytokines on the apoptosis of Tf-1 cells induced by γ irradiation and investigate the relationship between apoptosis and caspase-3 activity. METHODS: Different cytokines GM-CSF, IL-3 and GM-CS/IL-3 fusion protein were added into the irradiated Tf-1 cells. MTT assay, morphology, flow cytometry,and DNA fragmentation assay were used to observe the effects of cytokines on apoptosis. The caspase-3 activity was determined with a fluorocytometer. RESULTS: Irradiated Tf-1 cells showed typical morphological characteristic of apoptosis demonstrated by transmission electron microscopy and were accumulated in G0/G1 phase. In the groups treated with growth factors after irradiation, three cytokines significantly increased the viability rate, distinctly decreased the apoptosis rate and the proportion of DNA fragmentation. When Tf-1 cells were irradiated by γ irradiation, caspase-3 activity was increased at different time points. In comparison with the control group in which no growth factor was added after the cells were irradiated, the caspase-3 activity of irradiated Tf-1 cells was significantly inhibited by addition of the above cytokines. Thirty-six hours after irradiation, in the control group,GM-CSF, IL-3, GM-CSF and IL-3 in combination, and two GM-CSF/IL-3 fusion protein groups, the apoptosis ratewas 73 %, 11%, 15 %, 13 %, 12 %, and 13 %. The percent of fragmented DNA was 36 %, 19 %, 18 %, 14 %,13 %, and 14 %. The fluorescence intensity was 16923, 5529, 6581, 5322, 5426, and 5485. CONCLUSION:GM-CSF, IL-3, and GM-CSF/IL-3 fusion protein could protect Tf-1 cells from apoptosis induced by γ irradiation.After Tf-1 cells were irradiated, the caspase-3 activity was significantly increased but was dramatically decreased by the above cytokines. The remarkable inhibition of caspase-3 activity may be one of the mechanisms of these hematopoietic growth factors exerting their anti-apoptotic effects.

  3. [La(3+)-induced fusion of plant protoplasts].

    Science.gov (United States)

    Sheremet'ev, Iu A; Smirnova, D V; Sheremet'eva, A V

    2009-01-01

    The effect of La(3+) on the fusion of plant protoplasts has been studied. It was shown that La(3+) induced the aggregation of plant protoplasts. The incubation of a suspension of aggregated protoplasts at 42 degrees C for 30 min resulted in their fusion.

  4. 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.

  5. Dissipative Particle Dynamics of Tension-induced Membrane Fusion

    OpenAIRE

    Grafmueller, Andrea; Lipowsky, Reinhard; Shillcock, Julian C.

    2009-01-01

    Abstract 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...

  6. Mechanical tension drives cell membrane fusion

    OpenAIRE

    Kim, Ji Hoon; Ren, Yixin; Ng, Win Pin; Li, Shuo; Son, Sungmin; Kee, Yee-Seir; Zhang, Shiliang; Zhang, Guofeng; Fletcher, Daniel A.; Robinson, Douglas N.; Chen, Elizabeth H.

    2015-01-01

    Membrane fusion is an energy-consuming process that requires tight juxtaposition of two lipid bilayers. Little is known about how cells overcome energy barriers to bring their membranes together for fusion. Previously, we have shown that cell-cell fusion is an asymmetric process in which an “attacking” cell drills finger-like protrusions into the “receiving” cell to promote cell fusion. Here we show that the receiving cell mounts a Myosin II (MyoII)-mediated mechanosensory response to its inv...

  7. 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)

  8. Visualization of radiation-induced cell cycle-associated events in tumor cells expressing the fusion protein of Azami Green and the destruction box of human Geminin

    International Nuclear Information System (INIS)

    Ionizing radiation (IR) influences cell cycle-associated events in tumor cells. We expressed the fusion protein of Azami Green (AG) and the destruction box plus nuclear localization signal of human Geminin, an inhibitor of DNA replication licensing factor, in oral tumor cells. This approach allowed us to visualize G2 arrest in living cells following irradiation. The combination of time-lapse imaging analysis allowed us to observe the nuclear envelope break down (NEBD) at early M phase, and disappearance of fluorescence (DF) at the end of M phase. The duration from NEBD to DF was not much affected in irradiated cells; however, most of daughter cells harbored double-strand breaks. Complete DF was also observed in cells exhibiting abnormal mitosis or cytokinesis. We conclude that the fluorescent Geminin probe could function as a stable cell cycle indicator irrespective of genome integrity.

  9. Cancer Cell Fusion: Mechanisms Slowly Unravel

    Science.gov (United States)

    Noubissi, Felicite K.; Ogle, Brenda M.

    2016-01-01

    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. PMID:27657058

  10. Study on biological characters of SGC7901 gastric cancer cell-dendritic cell fusion vaccines

    Institute of Scientific and Technical Information of China (English)

    Kun Zhang; Peng-Fen Gao; Pei-Wu Yu; Yun Rao; Li-Xin Zhou

    2006-01-01

    AIM: To detect the biological characters of the SGC7901 gastric cancer cell-dendritic cell fusion vaccines.METHODS: The suspending living SGC7901 gastric cancer cells and dendritic cells were induced to be fusioned by polyethylene glycol. Pure fusion cells were obtained by selective culture with the HAT/HT culture systems.The fusion cells were counted at different time points of culture and their growth curves were drawn to reflect their proliferative activities. The fusion cells were also cultured in culture medium to investigate whether they could grow into cell clones. MTT method was used to test the stimulating abilities of the fusion cells on T lymphocytes' proliferations. Moreover, the fusion cells were planted into nude mice to observe whether they could grow into new planted tumors in this kind of immunodeficiency animals.RESULTS: The fusion cells had weaker proliferative activity and clone abilities than their parental cells. When they were cultured, the counts of cells did not increase remarkably, nor could they grow into cell clones in culture medium. The fusion cells could not grow into new planted tumors after planted into nude mice. The stimulating abilities of the fusion cells on T lymphocytes' proliferations were remarkably increased than their parental dendritic cells.CONCLUSION: The SGC7901 gastric cancer cell-dendritic cell fusion vaccines have much weaker proliferative abilities than their parental cells, but they keep strong abilities to irritate the T lymphocytes and have no abilities to grow into new planted tumors in immunodeficiency animals. These are the biological basis for their antitumor biotherapies.

  11. From Adult Bone Marrow Cells to Other Cell Lineages:Transdifferentiation or Cells Fusion

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Recent studies have demonstrated that intravenous transplantation or local injection of bone marrow cells can induce unexpected changes of their fate. The results of these experiments showed that after transplantation or injecton, some of tissue specific somatic cells such as hepatocytes, skeleton, cardiac muscle cells and brain cells expressed the donor cell-specific genes, such as Y chromosome. There are two hypotheses that can explain this phenomenon. One is bone marrow stem cell transdifferentiation and the other is spontaneous cell fusion.

  12. A suicidal DNA vaccine expressing the fusion protein of peste des petits ruminants virus induces both humoral and cell-mediated immune responses in mice.

    Science.gov (United States)

    Wang, Yong; Yue, Xiaolin; Jin, Hongyan; Liu, Guangqing; Pan, Ling; Wang, Guijun; Guo, Hao; Li, Gang; Li, Yongdong

    2015-12-01

    Peste des petits ruminants (PPR), a highly contagious disease induced by PPR virus (PPRV), affects sheep and goats. PPRV fusion (F) protein is important for the induction of immune responses against PPRV. We constructed a Semliki Forest virus (SFV) replicon-vectored DNA vaccine ("suicidal DNA vaccine") and evaluated its immunogenicity in BALB/c mice. The F gene of PPRV was cloned and inserted into the SFV replicon-based vector pSCA1. The antigenicity of the resultant plasmid pSCA1/F was identified by indirect immunofluorescence and western blotting. BALB/c mice were then intramuscularly injected with pSCA1/F three times at 14-d intervals. Specific antibodies and virus-neutralizing antibodies against PPRV were quantified by indirect ELISA and microneutralization tests, respectively. Cell-mediated immune responses were examined by cytokine and lymphocyte proliferation assays. The pSCA1/F expressed F protein in vitro and induced specific and neutralizing antibody production, and lymphocyte proliferation in mice. Mice vaccinated with pSCA1/F had increased IL-2 and IL-10 levels after 24-h post first immunization. IFN-γ and TNF-α levels increased from that time point and gradually decreased thereafter. Thus, the Semliki Forest virus replicon-vectored DNA vaccine expressing the F protein of PPRV induced both humoral and cell-mediated immune responses in mice. This could be considered as a novel strategy for vaccine development against PPR. PMID:26343487

  13. Rapid assessment of high-dose radiation exposures through scoring of cell-fusion-induced premature chromosome condensation and ring chromosomes.

    Science.gov (United States)

    Lamadrid Boada, A I; Romero Aguilera, I; Terzoudi, G I; González Mesa, J E; Pantelias, G; García, O

    2013-09-18

    Analysis of premature chromosome condensation (PCC) mediated by fusion of G0-lymphocytes with mitotic CHO cells in combination with rapid visualization and quantification of rings (PCC-Rf) is proposed as an alternative technique for dose assessment of radiation-exposed individuals. Isolated lymphocytes or whole blood from six individuals were γ-irradiated with 5, 10, 15 and 20Gy at a dose rate of 0.5Gy/min. Following either 8- or 24-h post-exposure incubation of irradiated samples at 37°C, chromosome spreads were prepared by standard PCC cytogenetic procedures. The protocol for PCC fusion proved to be effective at doses as high as 20Gy, enabling the analysis of ring chromosomes and excess PCC fragments. The ring frequencies remained constant during the 8-24-h repair time; the pooled dose relationship between ring frequency (Y) and dose (D) was linear: Y=(0.088±0.005)×D. During the repair time, excess fragments decreased from 0.91 to 0.59 chromatid pieces per Gy, revealing the importance of information about the exact time of exposure for dose assessment on the basis of fragments. Compared with other cytogenetic assays to estimate radiation dose, the PCC-Rf method has the following benefits: a 48-h culture time is not required, allowing a much faster assessment of dose in comparison with conventional scoring of dicentrics and rings in assays for chemically-induced premature chromosome condensation (PCC-Rch), and it allows the analysis of heavily irradiated lymphocytes that are delayed or never reach mitosis, thus avoiding the problem of saturation at high doses. In conclusion, the use of the PCC fusion assay in conjunction with scoring of rings in G0-lymphocytes offers a suitable alternative for fast dose estimation following accidental exposure to high radiation doses.

  14. 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...

  15. 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...

  16. A novel control scheme for inducing angiostatin-human IgG fusion protein production using recombinant CHO cells in a oscillating bioreactor.

    Science.gov (United States)

    Wang, Ing-Kae; Hsieh, Sing-Ying; Chang, King-Ming; Wang, Yu-Chi; Chu, Andy; Shaw, Shyh-Yu; Ou, Jung-Jung; Ho, Lewis

    2006-02-10

    In this study, a novel control scheme for inducing protein production using a recombinant CHO cell line in a BelloCell bioreactor was developed. This control scheme was applied in a simple regular semi-batch process. Production of angiostatin-human IgG fusion protein in a suspension recombinant CHO cell culture and a protein-free medium was used for this study. The bottom holding time (BH) was the sole operating variable to control the exposure time of the cells immobilized on the carriers to the air and allow the nutrient remained on the liquid film of the carriers to be consumed to a threshold level so that the cells can be arrested and promoted for protein production. In the cell cultures with various BH (1.5-90 min), final cell densities of 1.6-4.0 x 10(9) have been obtained in 20 days while total angiostatin-human IgG production of 228-388 mg have been harvested. In general, low BH will minimize the nutrient limitation and favor the cell growth, while high BH will restrict the nutrient and promote the production in this type of non-growth associated production systems. It was found that specific production rate was generally inversely proportional to the specific growth rate. In this case of study, BH of 30 and 60 min were found to be about 72% better than BH of 1.5 min and 35% better than BH of 9 and 90 min in term of the total angiostatin-human IgG production. In comparison to a conventional spinner flask study, a 3.8-fold increase of the total angiostatin-human IgG production was realized in a 35-day culture. This study illustrated that a simple method of using BH in a semi-batch process can effectively control the apparent nutrient concentration to the cells, and thus regulate the cell growth and protein production in a novel oscillating bioreactor. PMID:16162365

  17. Methodologies in the study of cell-cell fusion.

    Science.gov (United States)

    Cohen, F S; Melikyan, G B

    1998-10-01

    The process of membrane fusion has been profitably studied by fusing cells that express fusion proteins on their surfaces to the membranes of target cells. Primary methods for monitoring the occurrence of fusion between cells are measurement of formation of heterokaryons, measurement of activation of reporter genes, measurement of transfer of lipidic and aqueous fluorescent dyes, and electrophysiological recording of fusion pores. Fluorescence and electrical methods have been well developed for fusion of a nucleated cell expressing viral fusion proteins to red blood cell targets. These techniques are now being extended to the study of fusion between two nucleated cells. Microscopic observation of spread of fluorescent dyes from one cell to another is a sensitive and convenient means of detecting fusion on the level of single events. In such studies, both the membrane and the aqueous continuities that occur as a result of fusion can be measured in the same experiment. By following spread of aqueous dyes of different sizes from one cell to another, the growth of a fusion pore can also be followed. By labeling cells with fluorescent probes, a state of hemifusion can be identified if probes in outer membrane leaflets transfer but probes in inner leaflets or aqueous spaces do not. Electrical measurements-both capacitance and double-whole-cell voltage-clamp techniques-are the most sensitive methods yet developed for detecting the formation of pores and for quantifying their growth. These powerful single-event methodologies should be directly applicable to further advances in expressing nonviral fusion proteins on cell surfaces. PMID:9790869

  18. Flavivirus cell entry and membrane fusion

    NARCIS (Netherlands)

    Smit, Jolanda M.; Moesker, Bastiaan; Rodenhuis-Zybert, Izabela; Wilschut, Jan

    2011-01-01

    Flaviviruses, such as dengue virus and West Nile virus, are enveloped viruses that infect cells through receptor-mediated endocytosis and fusion from within acidic endosomes. The cell entry process of flaviviruses is mediated by the viral E glycoprotein. This short review will address recent advance

  19. Induction of Cell-Cell Fusion from Without by Human Herpesvirus 6B

    OpenAIRE

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

    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 onl...

  20. Convenient cell fusion assay for rapid screening for HIV entry inhibitors

    Science.gov (United States)

    Jiang, Shibo; Radigan, Lin; Zhang, Li

    2000-03-01

    Human immunodeficiency viruses (HIV)-induced cell fusion is a critical pathway of HIV spread from infected cells to uninfected cells. A rapid and simple assay was established to measure HIV-induce cell fusion. This study is particularly useful to rapid screen for HIV inhibitors that block HIV cell-to-cell transmission. Present study demonstrated that coculture of HIV-infected cells with uninfected cells at 37 degree(s)C for 2 hours resulted in the highest cell fusion rate. Using this cell fusion assay, we have identified several potent HIV inhibitors targeted to the HIV gp41 core. These antiviral agents can be potentially developed as antiviral drugs for chemotherapy and prophylaxis of HIV infection and AIDS.

  1. Effects of ROCK inhibitor Y-27632 on cell fusion through a microslit.

    Science.gov (United States)

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

    2015-11-01

    We previously reported a direct cytoplasmic transfer method using a microfluidic device, in which cell fusion was induced through a microslit (slit-through-fusion) by the Sendai virus envelope (HVJ-E) to prevent nuclear mixing. However, the method was impractical due to low efficiency of slit-through-fusion formation and insufficient prevention of nuclear mixing. The purpose of this study was to establish an efficient method for inducing slit-through-fusion without nuclear mixing. We hypothesized that modulation of cytoskeletal component can decrease nuclear migration through the microslit considering its functions. Here we report that supplementation with Y-27632, a specific ROCK inhibitor, significantly enhances cell fusion induction and prevention of nuclear mixing. Supplementation with Y-27632 increased the formation of slit-through-fusion efficiency by more than twofold. Disruption of F-actin by Y-27632 prevented nuclear migration between fused cells through the microslit. These two effects of Y-27632 led to promotion of the slit-through-fusion without nuclear mixing with a 16.5-fold higher frequency compared to our previous method (i.e., cell fusion induction by HVJ-E without supplementation with Y-27632). We also confirmed that mitochondria were successfully transferred to the fusion partner under conditions of Y-27632 supplementation. These findings demonstrate the practicality of our cell fusion system in producing direct cytoplasmic transfer between live cells.

  2. 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.

  3. Calpastatin overexpression reduces oxidative stress-induced mitochondrial impairment and cell death in human neuroblastoma SH-SY5Y cells by decreasing calpain and calcineurin activation, induction of mitochondrial fission and destruction of mitochondrial fusion.

    Science.gov (United States)

    Tangmansakulchai, Kulvadee; Abubakar, Zuroida; Kitiyanant, Narisorn; Suwanjang, Wilasinee; Leepiyasakulchai, Chaniya; Govitrapong, Piyarat; Chetsawang, Banthit

    2016-09-01

    Calpain is an intracellular Ca(2+)-dependent protease, and the activation of calpain has been implicated in neurodegenerative diseases. Calpain activity can be regulated by calpastatin, an endogenous specific calpain inhibitor. Several lines of evidence have demonstrated a potential role of calpastatin in preventing calpain-mediated pathogenesis. Additionally, several studies have revealed that calpain activation and mitochondrial damage are involved in the cell death process; however, recent evidence has not clearly indicated a neuroprotective mechanism of calpastatin against calpain-dependent mitochondrial impairment in the process of neuronal cell death. Therefore, the purpose of this study was to investigate the potential ability of calpastatin to inhibit calpain activation and mitochondrial impairment in oxidative stress-induced neuron degeneration. Calpastatin was stably overexpressed in human neuroblastoma SH-SY5Y cells. In non-calpastatin overexpressing SH-SY5Y cells, hydrogen peroxide significantly decreased cell viability, superoxide dismutase activity, mitochondrial membrane potential, ATP production and mitochondrial fusion protein (Opa1) levels in the mitochondrial fraction but increased reactive oxygen species formation, calpain and calcineurin activation, mitochondrial fission protein (Fis1 and Drp1) levels in the mitochondrial fraction and apoptotic cells. Nevertheless, these toxic effects were abolished in hydrogen peroxide-treated calpastatin-overexpressing SH-SY5Y cells. The results of the present study demonstrate the potential ability of calpastatin to diminish calpain and calcineurin activation and mitochondrial impairment in neurons that are affected by oxidative damage. PMID:27453331

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

    Science.gov (United States)

    Markosyan, Ruben M; Miao, Chunhui; Zheng, Yi-Min; Melikyan, Gregory B; Liu, Shan-Lu; Cohen, Fredric S

    2016-01-01

    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. PMID:26730950

  5. Cell-surface expression of a mutated Epstein–Barr virus glycoprotein B allows fusion independent of other viral proteins

    OpenAIRE

    McShane, Marisa P.; Longnecker, Richard

    2004-01-01

    Epstein–Barr virus (EBV) infects human B lymphocytes and epithelial cells. We have compared the requirements for EBV glycoprotein-induced cell fusion between Chinese hamster ovary effecter cells and human B lymphoblasts or epithelial cells by using a virus-free cell fusion assay. EBV-encoded gB, gH, gL, and gp42 glycoproteins were required for efficient B cell fusion, whereas EBV gB, gH, and gL glycoproteins were required for Chinese hamster ovary effecter cell fusion with epithelial cell lin...

  6. 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...... 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....

  7. 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.

  8. Membrane Fusion Induced by Small Molecules and Ions

    OpenAIRE

    Sutapa Mondal Roy; Munna Sarkar

    2011-01-01

    Membrane fusion is a key event in many biological processes. These processes are controlled by various fusogenic agents of which proteins and peptides from the principal group. The fusion process is characterized by three major steps, namely, inter membrane contact, lipid mixing forming the intermediate step, pore opening and finally mixing of inner contents of the cells/vesicles. These steps are governed by energy barriers, which need to be overcome to complete fusion. Structural reorganizat...

  9. Epigenetics changes caused by the fusion of human embryonic stem cell and ovarian cancer cells.

    Science.gov (United States)

    He, Ke; Qu, Hu; Xu, Li-Nan; Gao, Jun; Cheng, Fu-Yi; Xiang, Peng; Zhou, Can-Quan

    2016-10-01

    To observe the effect of gene expression and tumorigenicity in hybrid cells of human embryonic stem cells (hESCs) and ovarian cancer cells in vitro and in vivo using a mouse model, and to determine its feasibility in reprogramming tumour cells growth and apoptosis, for a potential exploration of the role of hESCs and tumour cells fusion in the management of ovarian cancer. Stable transgenic hESCs (H1) and ovarian cancer cell line OVCAR-3 were established before fusion, and cell fusion system was established to analyse the related indicators. PTEN expression in HO-H1 cells was higher than those in the parental stem cells and lower than those in parental tumour cells; the growth of OV-H1 (RFP+GFP) hybrid cells with double fluorescence expressions were obviously slower than that of human embryonic stem cells and OVCAR-3 ovarian cancer cells. The apoptosis signal of the OV-H1 hybrid cells was significantly higher than that of the hESCs and OVCAR-3 ovarian cancer cells. In vivo results showed that compared with 7 days, 28 days and 35 days after inoculation of OV-H1 hybrid cells; also, apoptotic cell detection indicated that much stronger apoptotic signal was found in OV-H1 hybrid cells inoculated mouse. The hESCs can inhibit the growth of OVCAR-3 cells in vitro by suppressing p53 and PTEN expression to suppress the growth of tumour that may be achieved by inducing apoptosis of OVCAR-3 cells. The change of epigenetics after fusion of ovarian cancer cells and hESCs may become a novel direction for treatment of ovarian cancer.

  10. Epigenetics changes caused by the fusion of human embryonic stem cell and ovarian cancer cells

    Science.gov (United States)

    He, Ke; Qu, Hu; Xu, Li-Nan; Gao, Jun; Cheng, Fu-Yi; Xiang, Peng; Zhou, Can-Quan

    2016-01-01

    To observe the effect of gene expression and tumorigenicity in hybrid cells of human embryonic stem cells (hESCs) and ovarian cancer cells in vitro and in vivo using a mouse model, and to determine its feasibility in reprogramming tumour cells growth and apoptosis, for a potential exploration of the role of hESCs and tumour cells fusion in the management of ovarian cancer. Stable transgenic hESCs (H1) and ovarian cancer cell line OVCAR-3 were established before fusion, and cell fusion system was established to analyse the related indicators. PTEN expression in HO-H1 cells was higher than those in the parental stem cells and lower than those in parental tumour cells; the growth of OV-H1 (RFP+GFP) hybrid cells with double fluorescence expressions were obviously slower than that of human embryonic stem cells and OVCAR-3 ovarian cancer cells. The apoptosis signal of the OV-H1 hybrid cells was significantly higher than that of the hESCs and OVCAR-3 ovarian cancer cells. In vivo results showed that compared with 7 days, 28 days and 35 days after inoculation of OV-H1 hybrid cells; also, apoptotic cell detection indicated that much stronger apoptotic signal was found in OV-H1 hybrid cells inoculated mouse. The hESCs can inhibit the growth of OVCAR-3 cells in vitro by suppressing p53 and PTEN expression to suppress the growth of tumour that may be achieved by inducing apoptosis of OVCAR-3 cells. The change of epigenetics after fusion of ovarian cancer cells and hESCs may become a novel direction for treatment of ovarian cancer. PMID:27377320

  11. Antibodies against analogous heptad repeat peptide HR212 of Newcastle Disease Virus inhibit virus-cell membrane fusion

    Institute of Scientific and Technical Information of China (English)

    LI Ying; TIEN Po

    2007-01-01

    Membrane fusion is a key step in enveloped virus entry. Highly conserved heptad repeat regions (HR1 and HR2) of Newcastle disease virus (NDV) fusion protein (F) are critical functional domains for viral membrane fusion. They display different conformations in the membrane fusion states and are viewed as candidate targets for neutralizing antibody responses. We previously reported that an analog of heptad repeat peptides HR2-HR1-HR2(HR212) and HR2 could inhibit NDV induced cell-cell membrane fusion. Here, we show that HR212 can induce the production of highly potent antibody in immunized rabbits, which could recognize full length peptides of both HR1 and HR2, and inhibit NDV hemagglutination and NDV entry. These suggest that either HR212 or its antibody could be an inhibitor of virus-induced cell-cell membrane fusion.

  12. Human Herpesvirus 6 Variant A but Not Variant B Induces Fusion from Without in a Variety of Human Cells through a Human Herpesvirus 6 Entry Receptor, CD46

    OpenAIRE

    Mori, Yasuko; Seya, Tsukasa; Huang, Hong Lan; Akkapaiboon, Pilailuk; Dhepakson, Panadda; Yamanishi, Koichi

    2002-01-01

    Human herpesvirus 6 (HHV-6) is a lymphotropic betaherpesvirus that productively infects T cells and monocytes. HHV-6 isolates can be differentiated into two groups, variants A and B (HHV-6A and HHV-6B). Here, we show a functional difference between HHV-6A and -6B in that HHV-6A induced syncytium formation of diverse human cells but HHV-6B did not. The syncytium formation induced by HHV-6A was observed 2 h after infection; moreover, it was found in the presence of cycloheximide, indicating tha...

  13. Implication des peptides de fusion des glycoprotéines de fusion virales de classe I dans la fusion membranaire

    OpenAIRE

    Brasseur R.; Charloteaux B.; Lins L.; Lorin A.

    2007-01-01

    The implication of fusion peptides of class I viral fusion glycoproteins in the membrane fusion. Viral infection involves fusion between the viral envelope and the target cell plasmic membrane. The fusion is induced by a glycoprotein anchored in the viral envelope. After activation, the glycoprotein undergoes a conformational change inducing the exposure of a region named « fusion peptide » essential for the fusion process. Studies on glycoproteins and on isolated fusion peptides have allowed...

  14. 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.

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

    International Nuclear Information System (INIS)

    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

  16. Thermally-induced aggregation and fusion of protein-free lipid vesicles.

    Science.gov (United States)

    Ibarguren, Maitane; Bomans, Paul H H; Ruiz-Mirazo, Kepa; Frederik, Peter M; Alonso, Alicia; Goñi, Félix M

    2015-12-01

    Membrane fusion is an important phenomenon in cell biology and pathology. This phenomenon can be modeled using vesicles of defined size and lipid composition. Up to now fusion models typically required the use of chemical (polyethyleneglycol, cations) or enzymatic catalysts (phospholipases). We present here a model of lipid vesicle fusion induced by heat. Large unilamellar vesicles consisting of a phospholipid (dioleoylphosphatidylcholine), cholesterol and diacylglycerol in a 43:57:3 mol ratio were employed. In this simple system, fusion was the result of thermal fluctuations, above 60 °C. A similar system containing phospholipid and cholesterol but no diacylglycerol was observed to aggregate at and above 60 °C, in the absence of fusion. Vesicle fusion occurred under our experimental conditions only when (31)P NMR and cryo-transmission electron microscopy of the lipid mixtures used in vesicle preparation showed non-lamellar lipid phase formation (hexagonal and cubic). Non-lamellar structures are probably the result of lipid reassembly of the products of individual fusion events, or of fusion intermediates. A temperature-triggered mechanism of lipid reassembly might have occurred at various stages of protocellular evolution.

  17. Restricted movement of lipid and aqueous dyes through pores formed by influenza hemagglutinin during cell fusion

    OpenAIRE

    1994-01-01

    The fusion of cells by influenza hemagglutinin (HA) is the best characterized example of protein-mediated membrane fusion. In simultaneous measurements of pairs of assays for fusion, we determined the order of detectable events during fusion. Fusion pore formation in HA-triggered cell-cell fusion was first detected by changes in cell membrane capacitance, next by a flux of fluorescent lipid, and finally by flux of aqueous fluorescent dye. Fusion pore conductance increased by small steps. A re...

  18. Repair-deficient xeroderma pigmentosum cells made UV light resistant by fusion with X-ray-inactivated Chinese hamster cells.

    OpenAIRE

    Karentz, D; Cleaver, J.E.

    1986-01-01

    Xeroderma pigmentosum (XP) is an autosomal recessive human disease, characterized by an extreme sensitivity to sunlight, caused by the inability of cells to repair UV light-induced damage to DNA. Cell fusion was used to transfer fragments of Chinese hamster ovary (CHO) chromosomes into XP cells. The hybrid cells exhibited UV resistance and DNA repair characteristics comparable to those expressed by CHO cells, and their DNA had greater homology with CHO DNA than did the DNA from XP cells. Cont...

  19. 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.

  20. Dendritic-tumor fusion cells in cancer immunotherapy.

    Science.gov (United States)

    Takakura, Kazuki; Kajihara, Mikio; Ito, Zensho; Ohkusa, Toshifumi; Gong, Jianlin; Koido, Shigeo

    2015-03-01

    A promising area of clinical investigation is the use of cancer immunotherapy to treat cancer patients. Dendritic cells (DCs) operate as professional antigen-presenting cells (APCs) and play a critical role in the induction of antitumor immune responses. Thus, DC-based cancer immunotherapy represents a powerful strategy. One DC-based cancer immunotherapy strategy that has been investigated is the administration of fusion cells generated with DCs and whole tumor cells (DC-tumor fusion cells). The DC-tumor fusion cells can process a broad array of tumor-associated antigens (TAAs), including unidentified molecules, and present them through major histocompatibility complex (MHC) class I and II pathways in the context of co-stimulatory signals. Improving the therapeutic efficacy of DC-tumor fusion cell-based cancer immunotherapy requires increased immunogenicity of DCs and whole tumor cells. We discuss the potential ability of DC-tumor fusion cells to activate antigen-specific T cells and strategies to improve the immunogenicity of DC-tumor fusion cells as anticancer vaccines.

  1. Relativistic-electron-beam-induced fusion

    International Nuclear Information System (INIS)

    The interaction of a focussed relativistic electron beam (REB) with a solid target has been investigated. The beam performance of the REB generator ''REIDEN III'' is 500 kV, 80 kA at a focal spot of 1.5 mm diameter, which gives 2X1012Wcm-2. High-temperature dense plasmas are produced at the focal point on the solid target. It expands radially along the target surface. The measured electron temperature (1-2 keV) and the ion energy (approximately 2 keV) endorse the existence of enhanced REB absorption in a dense plasma. The neutrons observed (approximately 109/shot, 2.45 MeV) in the case of a CD2 target are of thermonuclear origin and compatible with the plasma temperature. On the assumption that the electron beam of radius r is stopped at a length Λ and deposits its energy, the energy balance equation is approximately given by πr2Λn1kT=IVtau. On inserting beam current I, voltage V, pulse time tau and density n1, the energy deposition distance Λ can be estimated. For a fusion temperature of 1 keV, the distance Λ must be two orders of magnitude shorter than the simple classical stopping length, which seems to be due to non-linear coupling. A pellet implosion experiment of a multi-structure target has been performed. (author)

  2. Adhesion and Fusion of Muscle Cells Are Promoted by Filopodia.

    Science.gov (United States)

    Segal, Dagan; Dhanyasi, Nagaraju; Schejter, Eyal D; Shilo, Ben-Zion

    2016-08-01

    Indirect flight muscles (IFMs) in Drosophila are generated during pupariation by fusion of hundreds of myoblasts with larval muscle templates (myotubes). Live observation of these muscles during the fusion process revealed multiple long actin-based protrusions that emanate from the myotube surface and require Enabled and IRSp53 for their generation and maintenance. Fusion is blocked when formation of these filopodia is compromised. While filopodia are not required for the signaling process underlying critical myoblast cell-fate changes prior to fusion, myotube-myoblast adhesion appears to be filopodia dependent. Without filopodia, close apposition between the cell membranes is not achieved, the cell-adhesion molecule Duf is not recruited to the myotube surface, and adhesion-dependent actin foci do not form. We therefore propose that the filopodia are necessary to prime the heterotypic adhesion process between the two cell types, possibly by recruiting the cell-adhesion molecule Sns to discrete patches on the myoblast cell surface.

  3. Fusion of conformal interfaces and bulk induced boundary RG flows

    CERN Document Server

    Konechny, Anatoly

    2015-01-01

    We consider the basic radius changing conformal interface for a free compact boson. After investigating different theoretical aspects of this object we focus on the fusion of this interface with conformal boundary conditions. At fractions of the self-dual radius there exist exceptional D-branes. It was argued in [1] that changing the radius in the bulk induces a boundary RG flow. Following [2] we conjecture that fusing the basic radius changing interface (that changes the radius from a fraction of the self-dual radius) with the exceptional boundary conditions gives the boundary condition which is the end point of the RG flow considered in [1]. By studying the fusion singularities we recover RG logarithms and see, in particular instances, how they get resummed into power singularities. We discuss what quantities need to be calculated to gain full non-perturbative control over the fusion.

  4. 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...

  5. Elcatonin prevents bone loss caused by skeletal unloading by inhibiting preosteoclast fusion through the unloading-induced high expression of calcitonin receptors in bone marrow cells.

    Science.gov (United States)

    Tsukamoto, Manabu; Menuki, Kunitaka; Murai, Teppei; Hatakeyama, Akihisa; Takada, Shinichiro; Furukawa, Kayoko; Sakai, Akinori

    2016-04-01

    This study aimed to clarify whether elcatonin (EL) has a preventive action on bone dynamics in skeletal unloading. Seven-week-old male C57BL/6J mice with either ground control (GC) or tail suspension (TS) were administered EL 20U/kg or a vehicle (veh) three times per week and assigned to one of the following four groups: GCEL, GCveh, TSEL, and TSveh. Blood samples and bilateral femurs and tibias of the mice were obtained for analysis. After 7days of unloading, the trabecular bone mineral density in the distal femur obtained via peripheral quantitative computed tomography and the trabecular bone volume were significantly higher in the TSEL group than in the TSveh group. The bone resorption histomorphometric parameters, such as the osteoclast surface and osteoclast number, were significantly suppressed in the TSEL mice, whereas the number of preosteoclasts was significantly increased. The plasma level of tartrate-resistant acid phosphatase-5b (TRACP-5b) was significantly lower in the TSEL group than in all other groups. In the bone marrow cell culture, the number of TRACP-positive (TRACP(+)) multinucleated cells was significantly lower in the TSEL mice than in the TSveh mice, whereas the number of TRACP(+) mononucleated cells was higher in the TSEL mice. On day 4, the expression of nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1), cathepsin K and d2 isoform of vacuolar ATPase V0 domain (ATP6V0D2) mRNA in the bone marrow cells in the TSEL mice was suppressed, and the expression of calcitonin receptor (Calcr) mRNA on day 1 and Calcr antigen on day 4 were significantly higher in the TSveh mice than in the GCveh mice. EL prevented the unloading-induced bone loss associated with the high expression of Calcr in the bone marrow cells of mouse hindlimbs after tail suspension, and it suppressed osteoclast development from preosteoclasts to mature osteoclasts through bone-resorbing activity. This study of EL-treated unloaded mice provides the

  6. Recombinant fusion protein of albumin-retinol binding protein inactivates stellate cells

    International Nuclear Information System (INIS)

    Highlights: ► We designed novel recombinant albumin-RBP fusion proteins. ► Expression of fusion proteins inactivates pancreatic stellate cells (PSCs). ► Fusion proteins are successfully internalized into and inactivate PSCs. ► RBP moiety mediates cell specific uptake of fusion protein. -- Abstract: Quiescent pancreatic- (PSCs) and hepatic- (HSCs) stellate cells store vitamin A (retinol) in lipid droplets via retinol binding protein (RBP) receptor and, when activated by profibrogenic stimuli, they transform into myofibroblast-like cells which play a key role in the fibrogenesis. Despite extensive investigations, there is, however, currently no appropriate therapy available for tissue fibrosis. We previously showed that the expression of albumin, composed of three homologous domains (I–III), inhibits stellate cell activation, which requires its high-affinity fatty acid-binding sites asymmetrically distributed in domain I and III. To attain stellate cell-specific uptake, albumin (domain I/III) was coupled to RBP; RBP-albumindomainIII (R-III) and albumindomainI-RBP-albuminIII (I-R-III). To assess the biological activity of fusion proteins, cultured PSCs were used. Like wild type albumin, expression of R-III or I-R-III in PSCs after passage 2 (activated PSCs) induced phenotypic reversal from activated to fat-storing cells. On the other hand, R-III and I-R-III, but not albumin, secreted from transfected 293 cells were successfully internalized into and inactivated PSCs. FPLC-purified R-III was found to be internalized into PSCs via caveolae-mediated endocytosis, and its efficient cellular uptake was also observed in HSCs and podocytes among several cell lines tested. Moreover, tissue distribution of intravenously injected R-III was closely similar to that of RBP. Therefore, our data suggest that albumin-RBP fusion protein comprises of stellate cell inactivation-inducing moiety and targeting moiety, which may lead to the development of effective anti

  7. Deuterium Clusters Fusion Induced by the Intense Femtosecond Laser Pulse

    Institute of Scientific and Technical Information of China (English)

    LIU Hong-Jie; CHEN Jia-Bin; WANG Hong-Bin; JIAO Chun-Ye; HE Ying-Ling; WEN Tian-Shu; WEN Xian-Lun; CHEN Ming; ZHENG Zhi-Jian; GU Yu-Qiu; ZHANG Bao-Han; RHEE Yong-Joo; NAM Sung-Mo; HAN Jae-Min; RHEE Yong-Woo; YEA Kwon-Hae

    2007-01-01

    Neutrons (2.45 MeV) from deuterium cluster fusion induced by the intense femtosecond (30 fs) laser pulse are experimentally demonstrated. The average neutron yield 103 per shot is obtained. It is found that the yield slightly increases with the increasing laser spot size. No neutron can be observed when the laser intensity Ⅰ <4.3×1015 W/cm2.

  8. Vesicle transfer and cell fusion: Emerging concepts of cell-cell communication in the tumor microenvironment.

    Science.gov (United States)

    Howcroft, T Kevin; Zhang, Huang-Ge; Dhodapkar, Madhav; Mohla, Suresh

    2011-08-01

    Cell-cell fusion and vesicle-mediated transfer are fundamental biological processes that are emerging as novel mechanisms for re-programming cells in the tumor microenvironment. Both cell-cell fusion and intercellular transfer of vesicles (including microvesicles and exosomes) allow for the transfer of information among tumor cells, between tumor cells and tumor stroma, and between tumor cells and the host immune system, which could have profound implications for our understanding of tumor initiation and progression. The National Cancer Institute's Division of Cancer Biology sponsored a recent workshop (December 4-6, 2010) entitled, Vesicle Transfer and Cell Fusion: Emerging Concepts of Cell-Cell Communication in the Tumor Microenvironment to assess the current state of the science in these two scientific areas. Co-chaired by Drs. Huang-Ge Zhang (University of Louisville) and Madhav Dhodapkar (Yale University) this workshop brought together, for the first time at the NIH, leaders in the field to assess the effects of vesicle transfer and cell-cell fusion on cancer initiation, progression and metastasis. This meeting report includes brief summaries of the presentations and identifies the major questions, roadblocks, and opportunities. The meeting report is presented here to highlight research priorities and to stimulate basic and translational research efforts to better understand the contributions of cell-cell fusion and vesicle transfer to cancer. PMID:21725211

  9. 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.

  10. 3D analysis of Founder Cell and Fusion Competent Myoblasts arrangements outlines a new model of myoblast fusion

    OpenAIRE

    Beckett, Karen; Baylies, Mary K.

    2007-01-01

    Formation of the Drosophila larval body wall muscles requires the specification, coordinated cellular behaviors and fusion of two cell types: Founder Cells (FCs) that control the identity of the individual muscle and Fusion Competent Myoblasts (FCMs) that provide mass. These two cell types come together to control the final size, shape and attachment of individual muscles. However, the spatial arrangement of these cells over time, the sequence of fusion events and the contribution of these ce...

  11. Genetic analysis of the SARS-coronavirus spike glycoprotein functional domains involved in cell-surface expression and cell-to-cell fusion

    International Nuclear Information System (INIS)

    The SARS-coronavirus (SARS-CoV) is the etiological agent of 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. To delineate functional domains of the SARS-CoV S glycoprotein, single point mutations, cluster-to-lysine and cluster-to-alanine mutations, as well as carboxyl-terminal truncations were investigated in transient expression experiments. Mutagenesis of either the coiled-coil domain of the S glycoprotein amino terminal heptad repeat, the predicted fusion peptide, or an adjacent but distinct region, severely compromised S-mediated cell-to-cell fusion, while intracellular transport and cell-surface expression were not adversely affected. Surprisingly, a carboxyl-terminal truncation of 17 amino acids substantially increased S glycoprotein-mediated cell-to-cell fusion suggesting that the terminal 17 amino acids regulated the S fusogenic properties. In contrast, truncation of 26 or 39 amino acids eliminating either one or both of the two endodomain cysteine-rich motifs, respectively, inhibited cell fusion in comparison to the wild-type S. The 17 and 26 amino-acid deletions did not adversely affect S cell-surface expression, while the 39 amino-acid truncation inhibited S cell-surface expression suggesting that the membrane proximal cysteine-rich motif plays an essential role in S cell-surface expression. Mutagenesis of the acidic amino-acid cluster in the carboxyl terminus of the S glycoprotein as well as modification of a predicted phosphorylation site within the acidic cluster revealed that this amino-acid motif may play a functional role in the retention of S at cell surfaces. This genetic analysis reveals that the SARS-CoV S glycoprotein contains extracellular domains that regulate cell fusion as well as distinct endodomains that function in intracellular transport, cell-surface expression, and cell fusion

  12. 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.

  13. On Stability of Targets for Plasma Jet Induced Magnetoinertial Fusion

    CERN Document Server

    Samulyak, Roman; Kim, Hyoungekun

    2015-01-01

    The compression and stability of plasma targets for the plasma jet-induced magneto-inertial fusion (PJMIF) have been investigated via large scale simulations using the FronTier code capable of explicit tracking of material interfaces. In the PJMIF concept, a plasma liner, formed by the merger of a large number of radial, highly supersonic plasma jets, implodes on a magnetized plasma target and compresses it to conditions of the fusion ignition. A multi-stage computational approach for simulations of the liner-target interaction and the compression of plasma targets has been developed to minimize computing time. Simulations revealed important features of the target compression process, including instability and disintegration of targets. The non-uniformity of the leading edge of the liner, caused by plasma jets as well as oblique shock waves between them, leads to instabilities during the target compression. By using front tracking, the evolution of targets has been studied in 3-dimensional simulations. Optimi...

  14. 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.

  15. 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 ...

  16. 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.

  17. 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

  18. Staphylococcus aureus Cell Wall Stress Stimulon Gene-lacZ Fusion Strains: Potential for Use in Screening for Cell Wall-Active Antimicrobials▿

    OpenAIRE

    Steidl, Rebecca; Pearson, Stacy; Stephenson, Robert E.; Ledala, Nagender; Sitthisak, Sutthirat; Wilkinson, Brian J; Jayaswal, Radheshyam K.

    2008-01-01

    lacZ fusion strains were constructed using the promoters of five cell wall stress stimulon genes: pbp2, tcaA, vraSR, sgtB, and lytR. All fusion strains were induced only in the presence of cell wall-active antibiotics, suggesting the potential of these strains for use in high-throughput screening for new cell wall-active agents.

  19. Human cord blood CD34+ progenitor cells acquire functional cardiac properties through a cell fusion process.

    Science.gov (United States)

    Avitabile, Daniele; Crespi, Alessia; Brioschi, Chiara; Parente, Valeria; Toietta, Gabriele; Devanna, Paolo; Baruscotti, Mirko; Truffa, Silvia; Scavone, Angela; Rusconi, Francesca; Biondi, Andrea; D'Alessandra, Yuri; Vigna, Elisa; Difrancesco, Dario; Pesce, Maurizio; Capogrossi, Maurizio C; Barbuti, Andrea

    2011-05-01

    The efficacy of cardiac repair by stem cell administration relies on a successful functional integration of injected cells into the host myocardium. Safety concerns have been raised about the possibility that stem cells may induce foci of arrhythmia in the ischemic myocardium. In a previous work (36), we showed that human cord blood CD34(+) cells, when cocultured on neonatal mouse cardiomyocytes, exhibit excitation-contraction coupling features similar to those of cardiomyocytes, even though no human genes were upregulated. The aims of the present work are to investigate whether human CD34(+) cells, isolated after 1 wk of coculture with neonatal ventricular myocytes, possess molecular and functional properties of cardiomyocytes and to discriminate, using a reporter gene system, whether cardiac differentiation derives from a (trans)differentiation or a cell fusion process. Umbilical cord blood CD34(+) cells were isolated by a magnetic cell sorting method, transduced with a lentiviral vector carrying the enhanced green fluorescent protein (EGFP) gene, and seeded onto primary cultures of spontaneously beating rat neonatal cardiomyocytes. Cocultured EGFP(+)/CD34(+)-derived cells were analyzed for their electrophysiological features at different time points. After 1 wk in coculture, EGFP(+) cells, in contact with cardiomyocytes, were spontaneously contracting and had a maximum diastolic potential (MDP) of -53.1 mV, while those that remained isolated from the surrounding myocytes did not contract and had a depolarized resting potential of -11.4 mV. Cells were then resuspended and cultured at low density to identify EGFP(+) progenitor cell derivatives. Under these conditions, we observed single EGFP(+) beating cells that had acquired an hyperpolarization-activated current typical of neonatal cardiomyocytes (EGFP(+) cells, -2.24 ± 0.89 pA/pF; myocytes, -1.99 ± 0.63 pA/pF, at -125 mV). To discriminate between cell autonomous differentiation and fusion, EGFP(+)/CD34

  20. ULTRASOUND INDUCED AND LASER ENHANCED COLD FUSION CHEMISTRY

    Institute of Scientific and Technical Information of China (English)

    T.V.Prevenslik

    1995-01-01

    The standard model of sonoluminescence suggests that the coulomb barrer to deuterium fusion may be overcome by high bubble gas temperatures caused by compression heating if the bubble diameter remains spherical during bubble collapse.However,in the more likely collapse geometry of a pancake shape,the temperature rise in the bubbles is negligible.But the collapsing pancake bubble is fund to significantly increase the frequency of the infrared energy available in the vibrational state of the water molecules at ambient temperature.For a collapse to liquied density,ultraviolet radiation at about 10eV is fund.Although the ultraviolet radiation is of a low intensity,higher intensities may be possible if the bubble collapse is enhanced by visible and infrared lases.Neither hot nor cold fusion is predicted in bubble collapse but the ultraviolet energy at about 10eV developed in the bubble is sufficient to provide the basis for a new field of chemistry called ultrasound induced and laser enhanced cold fusion chemistry.

  1. 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.

  2. Tissue Regeneration in the Chronically Inflamed Tumor Environment: Implications for Cell Fusion Driven Tumor Progression and Therapy Resistant Tumor Hybrid Cells

    Directory of Open Access Journals (Sweden)

    Thomas Dittmar

    2015-12-01

    Full Text Available The biological phenomenon of cell fusion in a cancer context is still a matter of controversial debates. Even though a plethora of in vitro and in vivo data have been published in the past decades the ultimate proof that tumor hybrid cells could originate in (human cancers and could contribute to the progression of the disease is still missing, suggesting that the cell fusion hypothesis is rather fiction than fact. However, is the lack of this ultimate proof a valid argument against this hypothesis, particularly if one has to consider that appropriate markers do not (yet exist, thus making it virtually impossible to identify a human tumor cell clearly as a tumor hybrid cell. In the present review, we will summarize the evidence supporting the cell fusion in cancer concept. Moreover, we will refine the cell fusion hypothesis by providing evidence that cell fusion is a potent inducer of aneuploidy, genomic instability and, most likely, even chromothripsis, suggesting that cell fusion, like mutations and aneuploidy, might be an inducer of a mutator phenotype. Finally, we will show that “accidental” tissue repair processes during cancer therapy could lead to the origin of therapy resistant cancer hybrid stem cells.

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

    DEFF Research Database (Denmark)

    Shillcock, Julian C.

    2008-01-01

    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...

  4. 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.

  5. 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 fusio

  6. Cell fusion and intramembrane particle distribution in polyethylene glycol-resistant cells

    OpenAIRE

    1983-01-01

    The distribution of intramembrane particles (IMP) as revealed by freeze- fracture electron microscopy has been analyzed following treatment of mouse L cells and fusion-deficient L cell derivatives with several concentrations of polyethylene glycol (PEG). In cell cultures treated with concentrations of PEG below the critical level for fusion, no aggregation of IMP was observed. When confluent cultures of the parental cells are treated with 50% PEG, greater than 90% of the cells fuse, and cold-...

  7. Embryonic stem cells contribute to mouse chimeras in the absence of detectable cell fusion.

    Science.gov (United States)

    Kidder, Benjamin L; Oseth, Leann; Miller, Shanna; Hirsch, Betsy; Verfaillie, Catherine; Coucouvanis, Electra

    2008-06-01

    Embryonic stem (ES) cells are capable of differentiating into all embryonic and adult cell types following mouse chimera production. Although injection of diploid ES cells into tetraploid blastocysts suggests that tetraploid cells have a selective disadvantage in the developing embryo, tetraploid hybrid cells, formed by cell fusion between ES cells and somatic cells, have been reported to contribute to mouse chimeras. In addition, other examples of apparent stem cell plasticity have recently been shown to be the result of cell fusion. Here we investigate whether ES cells contribute to mouse chimeras through a cell fusion mechanism. Fluorescence in situ hybridization (FISH) analysis for X and Y chromosomes was performed on dissociated tissues from embryonic, neonatal, and adult wild-type, and chimeric mice to follow the ploidy distributions of cells from various tissues. FISH analysis showed that the ploidy distributions in dissociated tissues, notably the tetraploid cell number, did not differ between chimeric and wild-type tissues. To address the possibility that early cell fusion events are hidden by subsequent reductive divisions or other changes in cell ploidy, we injected Z/EG (lacZ/EGFP) ES cells into ACTB-cre blastocysts. Recombination can only occur as the result of cell fusion, and the recombined allele should persist through any subsequent changes in cell ploidy. We did not detect evidence of fusion in embryonic chimeras either by direct fluorescence microscopy for GFP or by PCR amplification of the recombined Z/EG locus on genomic DNA from ACTB-cre::Z/EG chimeric embryos. Our results argue strongly against cell fusion as a mechanism by which ES cells contribute to chimeras.

  8. Heme-mediated apoptosis and fusion damage in BeWo trophoblast cells

    Science.gov (United States)

    Liu, Mingli; Hassana, Salifu; Stiles, Jonathan K.

    2016-01-01

    Placental malaria (PM) is a complication associated with malaria infection during pregnancy that often leads to abortion, premature delivery, intrauterine growth restriction and low birth weight. Increased levels of circulating free heme, a by-product of Plasmodium-damaged erythrocytes, is a major contributor to inflammation, tissue damage and loss of blood brain barrier integrity associated with fatal experimental cerebral malaria. However, the role of heme in PM remains unknown. Proliferation and apoptosis of trophoblasts and fusion of the mononucleated state to the syncytial state are of major importance to a successful pregnancy. In the present study, we examined the effects of heme on the viability and fusion of a trophoblast-derived cell line (BeWo). Results indicate that heme induces apoptosis in BeWo cells by activation of the STAT3/caspase-3/PARP signaling pathway. In the presence of forskolin, which triggers trophoblast fusion, heme inhibits BeWo cell fusion through activation of STAT3. Understanding the effects of free plasma heme in pregnant women either due to malaria, sickle cell disease or other hemolytic diseases, will enable identification of high-risk women and may lead to discovery of new drug targets against associated adverse pregnancy outcome. PMID:27796349

  9. Alpha-latrotoxin Triggers Extracellular Ca2+-dependent Exocytosis and Sensitizes Fusion Machinery in Endocrine Cells

    Institute of Scientific and Technical Information of China (English)

    Zhi-Tao HU; Ping ZHAO; Jie LIU; Zheng-Xing WU; Tao XU

    2006-01-01

    α-Latrotoxin from the venom of black widow spider induces and augments neurotransmitter and hormone release by way of extracellular Ca2+ influx and cellular signal transduction pathways. By using whole cell current and capacitance recording, the photolysis of caged Ca2+, and Ca2+ microfluorometry and amperometry, we investigated the stimulating effect and mechanism of o-latrotoxin on exocytosis in rat pancreatic β cells, LβT2 cells and latrophilin plasmid-transfected INS-1 cells. Our data indicated that: (1) α-latrotoxin increased cytosolic Ca2+ concentration through the formation of cation-permitting pores and subsequent Ca2+ influx with the presence of extracellular Ca2+; (2) α-latrotoxin stimulated exocytosis in normal bath solution and its stimulating effect on secretion was eradicated in Ca2+-free bath solution; and (3) α-latrotoxin sensitized the molecular machinery of fusion through activation of protein kinase C and increased the response of cells to Ca2+ photolysed by a flash of ultraviolet light. In summary, α-latrotoxin induced exocytosis by way of Ca2+ influx and accelerated vesicle fusion by the sensitization of fusion machinery.

  10. The molecular role of connexin 43 in human trophoblast cell fusion.

    Science.gov (United States)

    Dunk, Caroline E; Gellhaus, Alexandra; Drewlo, Sascha; Baczyk, Dora; Pötgens, Andy J G; Winterhager, Elke; Kingdom, John C P; Lye, Steven J

    2012-04-01

    Connexin expression and gap junctional intercellular communication (GJIC) mediated by connexin 43 (Cx43)/gap junction A1 (GJA1) are required for cytotrophoblast fusion into the syncytium, the outer functional layer of the human placenta. Cx43 also impacts intracellular signaling through protein-protein interactions. The transcription factor GCM1 and its downstream target ERVW-1/SYNCYTIN-1 are key players in trophoblast fusion and exert their actions through the ERVW-1 receptor SLC1A5/ASCT-2/RDR/ATB(0). To investigate the molecular role of the Cx43 protein and its interaction with this fusogenic pathway, we utilized stable Cx43-transfected cell lines established from the choriocarcinoma cell line Jeg3: wild-type Jeg3, alphahCG/Cx43 (constitutive Cx43 expression), JpUHD/Cx43 (doxycyclin-inducible Cx43 expression), or JpUHD/trCx43 (doxycyclin-inducible Cx43 carboxyterminal deleted). We hypothesized that truncation of Cx43 at its C-terminus would inhibit trophoblast fusion and protein interaction with either ERVW-1 or SLC1A5. In the alphahCG/Cx43 and JpUHD/Cx43 lines, stimulation with cAMP caused 1) increase in GJA1 mRNA levels, 2) increase in percentage of fused cells, and 3) downregulation of SLC1A5 expression. Cell fusion was inhibited by GJIC blockade using carbenoxylone. Neither Jeg3, which express low levels of Cx43, nor the JpUHD/trCx43 cell line demonstrated cell fusion or downregulation of SLC1A5. However, GCM1 and ERVW-1 mRNAs were upregulated by cAMP treatment in both Jeg3 and all Cx43 cell lines. Silencing of GCM1 prevented the induction of GJA1 mRNA by forskolin in BeWo choriocarcinoma cells, demonstrating that GCM1 is upstream of Cx43. All cell lines and first-trimester villous explants also demonstrated coimmunoprecipitation of SLC1A5 and phosphorylated Cx43. Importantly, SLC1A5 and Cx43 gap junction plaques colocalized in situ to areas of fusing cytotrophoblast, as demonstrated by the loss of E-cadherin staining in the plasma membrane in first

  11. Recombinant fusion protein of albumin-retinol binding protein inactivates stellate cells

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Soyoung; Park, Sangeun; Kim, Suhyun [Laboratory of Cellular Oncology, Korea University Graduate School of Medicine, Ansan, Gyeonggi do 425-707 (Korea, Republic of); Lim, Chaeseung [Department of Laboratory Medicine, Korea University Guro Hospital, Seoul 152-703 (Korea, Republic of); Kim, Jungho [Department of Life Science, Sogang University, Seoul 121-742 (Korea, Republic of); Cha, Dae Ryong [Department of Internal Medicine, Korea University Ansan Hospital, Ansan, Gyeonggi do 425-020 (Korea, Republic of); Oh, Junseo, E-mail: ohjs@korea.ac.kr [Laboratory of Cellular Oncology, Korea University Graduate School of Medicine, Ansan, Gyeonggi do 425-707 (Korea, Republic of)

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer We designed novel recombinant albumin-RBP fusion proteins. Black-Right-Pointing-Pointer Expression of fusion proteins inactivates pancreatic stellate cells (PSCs). Black-Right-Pointing-Pointer Fusion proteins are successfully internalized into and inactivate PSCs. Black-Right-Pointing-Pointer RBP moiety mediates cell specific uptake of fusion protein. -- Abstract: Quiescent pancreatic- (PSCs) and hepatic- (HSCs) stellate cells store vitamin A (retinol) in lipid droplets via retinol binding protein (RBP) receptor and, when activated by profibrogenic stimuli, they transform into myofibroblast-like cells which play a key role in the fibrogenesis. Despite extensive investigations, there is, however, currently no appropriate therapy available for tissue fibrosis. We previously showed that the expression of albumin, composed of three homologous domains (I-III), inhibits stellate cell activation, which requires its high-affinity fatty acid-binding sites asymmetrically distributed in domain I and III. To attain stellate cell-specific uptake, albumin (domain I/III) was coupled to RBP; RBP-albumin{sup domain} {sup III} (R-III) and albumin{sup domain} {sup I}-RBP-albumin{sup III} (I-R-III). To assess the biological activity of fusion proteins, cultured PSCs were used. Like wild type albumin, expression of R-III or I-R-III in PSCs after passage 2 (activated PSCs) induced phenotypic reversal from activated to fat-storing cells. On the other hand, R-III and I-R-III, but not albumin, secreted from transfected 293 cells were successfully internalized into and inactivated PSCs. FPLC-purified R-III was found to be internalized into PSCs via caveolae-mediated endocytosis, and its efficient cellular uptake was also observed in HSCs and podocytes among several cell lines tested. Moreover, tissue distribution of intravenously injected R-III was closely similar to that of RBP. Therefore, our data suggest that albumin-RBP fusion protein comprises

  12. Raman spectroscopic evidence of tissue restructuring in heat-induced tissue fusion.

    Science.gov (United States)

    Su, Lei; Cloyd, Kristy L; Arya, Shobhit; Hedegaard, Martin A B; Steele, Joseph A M; Elson, Daniel S; Stevens, Molly M; Hanna, George B

    2014-09-01

    Heat-induced tissue fusion via radio-frequency (RF) energy has gained wide acceptance clinically and here we present the first optical-Raman-spectroscopy study on tissue fusion samples in vitro. This study provides direct insights into tissue constituent and structural changes on the molecular level, exposing spectroscopic evidence for the loss of distinct collagen fibre rich tissue layers as well as the denaturing and restructuring of collagen crosslinks post RF fusion. These findings open the door for more advanced optical feedback-control methods and characterization during heat-induced tissue fusion, which will lead to new clinical applications of this promising technology.

  13. Induced pluripotent stem cells

    Institute of Scientific and Technical Information of China (English)

    Siddhartha Bhowmik; LI Yong

    2011-01-01

    Induced pluripotent stem (iPS) cells are a recent development which has brought a promise of great therapeutic values. The previous technique of somatic cell nuclear transfer (SCNT) has been ineffective in humans. Recent discoveries show that human fibroblasts can be reprogrammed by a transient over expression of a small number of genes; they can undergo induced pluripotency. iPS were first produced in 2006. By 2008, work was underway to remove the potential oncogenes from their structure. In 2009, protein iPS (piPS) cells were discovered. Surface markers and reporter genes play an important role in stem cell research. Clinical applications include generation of self renewing stem cells, tissue replacement and many more. Stem cell therapy has the ability to dramatically change the treatment of human diseases.

  14. 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

  15. 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

    2014-12-16

    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

  16. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Wong-Baeza, Carlos; Bustos, Israel; Serna, Manuel; Tescucano, Alonso; Alcantara-Farfan, Veronica; Ibanez, Miguel [Biochemistry Department, National Polytechnic Institute (IPN), Mexico City 11340 (Mexico); Montanez, Cecilia [Department of Genetics and Molecular Biology, Centre for Research and Advanced Studies (CINVESTAV), IPN, Mexico City 07360 (Mexico); Wong, Carlos [Biochemistry Department, National Polytechnic Institute (IPN), Mexico City 11340 (Mexico); Baeza, Isabel, E-mail: ibaeza@encb.ipn.mx [Biochemistry Department, National Polytechnic Institute (IPN), Mexico City 11340 (Mexico)

    2010-05-28

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Gambhir, Sanjiv; Pritha, Ray

    2015-07-14

    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.

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

    Science.gov (United States)

    Gambhir; Sanjiv , Pritha; Ray

    2009-04-28

    Novel double and triple fusion reporter gene constructs harboring distinct imageable 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.

  20. Functional Analysis of Glycoprotein L (gL) from Rhesus Lymphocryptovirus in Epstein-Barr Virus-Mediated Cell Fusion Indicates a Direct Role of gL in gB-Induced Membrane Fusion▿

    OpenAIRE

    Plate, Aileen E.; Smajlović, Jasmina; Jardetzky, Theodore S.; Longnecker, Richard

    2009-01-01

    Glycoprotein L (gL), which complexes with gH, is a conserved herpesvirus protein that is essential for Epstein-Barr virus (EBV) entry into host cells. The gH/gL complex has a conserved role in entry among herpesviruses, yet the mechanism is not clear. To gain a better understanding of the role of gL in EBV-mediated fusion, chimeric proteins were made using rhesus lymphocryptovirus (Rh-LCV) gL (Rh gL), which shares a high sequence homology with EBV gL but does not complement EBV gL in mediatin...

  1. The effect of acute microgravity on mechanically-induced membrane damage and membrane-membrane fusion events

    Science.gov (United States)

    Clarke, M. S.; Vanderburg, C. R.; Feeback, D. L.; McIntire, L. V. (Principal Investigator)

    2001-01-01

    Although it is unclear how a living cell senses gravitational forces there is no doubt that perturbation of the gravitational environment results in profound alterations in cellular function. In the present study, we have focused our attention on how acute microgravity exposure during parabolic flight affects the skeletal muscle cell plasma membrane (i.e. sarcolemma), with specific reference to a mechanically-reactive signaling mechanism known as mechanically-induced membrane disruption or "wounding". Both membrane rupture and membrane resealing events mediated by membrane-membrane fusion characterize this response. We here present experimental evidence that acute microgravity exposure can inhibit membrane-membrane fusion events essential for the resealing of sarcolemmal wounds in individual human myoblasts. Additional evidence to support this contention comes from experimental studies that demonstrate acute microgravity exposure also inhibits secretagogue-stimulated intracellular vesicle fusion with the plasma membrane in HL-60 cells. Based on our own observations and those of other investigators in a variety of ground-based models of membrane wounding and membrane-membrane fusion, we suggest that the disruption in the membrane resealing process observed during acute microgravity is consistent with a microgravity-induced decrease in membrane order.

  2. Repair-deficient xeroderma pigmentosum cells made UV light resistant by fusion with X-ray-inactivated Chinese hamster cells

    International Nuclear Information System (INIS)

    Xeroderma pigmentosum (XP) is an autosomal recessive human disease, characterized by an extreme sensitivity to sunlight, caused by the inability of cells to repair UV light-induced damage to DNA. Cell fusion was used to transfer fragments of Chinese hamster ovary (CHO) chromosomes into XP cells. The hybrid cells exhibited UV resistance and DNA repair characteristics comparable to those expressed by CHO cells, and their DNA had greater homology with CHO DNA than did the DNA from XP cells. Control experiments consisted of fusion of irradiated and unirradiated XP cells and repeated exposure of unfused XP cells to UV doses used for hybrid selection. These treatments did not result in an increase in UV resistance, repair capability, or homology with CHO DNA. The hybrid cell lines do not, therefore, appear to be XP revertants. The establishment of these stable hybrid cell lines is an initial step toward identifying and cloning CHO DNA repair genes that complement the XP defect in human cells. The method should also be applicable to cloning genes for other diseases, such as ataxia-telangiectasia and Fanconi's anemia

  3. Fusion and metabolism of plant cells as affected by microgravity.

    Science.gov (United States)

    Hampp, R; Hoffmann, E; Schönherr, K; Johann, P; De Filippis, L

    1997-01-01

    Plant cell protoplasts derived from leaf tissue of two different tobacco species (Nicotiana tabacum., N. rustica L.) were exposed to short-term (sounding rocket experiments) and long-term (spacelab) microgravity environments in order to study both (electro) cell fusion and cell metabolism during early and later stages of tissue regeneration. The period of exposure to microgravity varied from 10 min (sounding rocket) to 10 d (space shuttle). The process of electro fusion of protoplasts was improved under conditions of microgravity: the time needed to establish close membrane contact between protoplasts (alignment time) was reduced (5 as compared to 15 s under 1 g) and numbers of fusion products between protoplasts of different specific density were increased by a factor of about 10. In addition, viability of fusion products, as shown by the ability to form callus, increased from about 60% to more than 90%. Regenerated fusion products obtained from both sounding-rocket and spacelab experiments showed a wide range of intermediate properties between the two parental plants. This was verified by isozyme analysis and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). In order to address potential metabolic responses, more general markers such as the overall energy state (ATP/ADP ratio), the redox charge of the diphosphopyridine nucleotide system (NADH/NAD ratio), and the pool size of fructose-2,6-bisphosphate (Fru 2,6 bisp), a regulator of the balance between glycolysis and gluconeogenesis, were determined. Responses of these parameters were different with regard to short-term and long-term exposure. Shortly after transition to reduced gravitation (sounding rocket) ratios of ATP/ADP exhibited strong fluctuation while the pool size of NAD decreased (indicating an increased NADH/NAD ratio) and that of Fru 2,6 bisp increased. As similar changes can be observed under stress conditions, this response is probably indicative of a metabolic stress

  4. Fusion and metabolism of plant cells as affected by microgravity.

    Science.gov (United States)

    Hampp, R; Hoffmann, E; Schönherr, K; Johann, P; De Filippis, L

    1997-01-01

    Plant cell protoplasts derived from leaf tissue of two different tobacco species (Nicotiana tabacum., N. rustica L.) were exposed to short-term (sounding rocket experiments) and long-term (spacelab) microgravity environments in order to study both (electro) cell fusion and cell metabolism during early and later stages of tissue regeneration. The period of exposure to microgravity varied from 10 min (sounding rocket) to 10 d (space shuttle). The process of electro fusion of protoplasts was improved under conditions of microgravity: the time needed to establish close membrane contact between protoplasts (alignment time) was reduced (5 as compared to 15 s under 1 g) and numbers of fusion products between protoplasts of different specific density were increased by a factor of about 10. In addition, viability of fusion products, as shown by the ability to form callus, increased from about 60% to more than 90%. Regenerated fusion products obtained from both sounding-rocket and spacelab experiments showed a wide range of intermediate properties between the two parental plants. This was verified by isozyme analysis and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). In order to address potential metabolic responses, more general markers such as the overall energy state (ATP/ADP ratio), the redox charge of the diphosphopyridine nucleotide system (NADH/NAD ratio), and the pool size of fructose-2,6-bisphosphate (Fru 2,6 bisp), a regulator of the balance between glycolysis and gluconeogenesis, were determined. Responses of these parameters were different with regard to short-term and long-term exposure. Shortly after transition to reduced gravitation (sounding rocket) ratios of ATP/ADP exhibited strong fluctuation while the pool size of NAD decreased (indicating an increased NADH/NAD ratio) and that of Fru 2,6 bisp increased. As similar changes can be observed under stress conditions, this response is probably indicative of a metabolic stress

  5. 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

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

    Science.gov (United States)

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

    2015-08-01

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

  7. The impact of dendritic cell-tumor fusion cells on cancer vaccines - past progress and future strategies.

    Science.gov (United States)

    Kajihara, Mikio; Takakura, Kazuki; Ohkusa, Toshifumi; Koido, Shigeo

    2015-01-01

    Dendritic cells (DCs) are potent antigen-presenting cells that can be used in cancer vaccines. Thus, various strategies have been developed to deliver tumor-associated antigens via DCs. One strategy includes administering DC-tumor fusion cells (DC-tumor FCs) to induce antitumor immune responses in cancer patients. However, clinical trials using this strategy have fallen short of expectations. Several factors might limit the efficacy of these anticancer vaccines. To induce efficient antitumor immune responses and enhance potential clinical benefits, DC-tumor FC-based cancer vaccines require manipulations that improve immunogenicity for both DCs and whole tumor cells. This review addresses recent progress in improving clinical outcomes using DC-tumor FC-based cancer vaccines. PMID:26507578

  8. pH-sensitive liposomes: acid-induced liposome fusion.

    OpenAIRE

    Connor, J.; Yatvin, M B; Huang, L.

    1984-01-01

    Sonicated unilamellar liposomes containing phosphatidylethanolamine and palmitoylhomocysteine fuse rapidly when the medium pH is lowered from 7 to 5. Liposome fusion was demonstrated by (i) mixing of the liposomal lipids as shown by resonance energy transfer, (ii) gel filtration, and (iii) electron microscopy. The pH-sensitive fusion of liposomes was observed only when palmitoylhomocysteine (greater than or equal to 20 mol%) was present in the liposomes. The presence of phosphatidyl-ethanolam...

  9. The nature of telomere fusion and a definition of the critical telomere length in human cells

    OpenAIRE

    Capper, Rebecca; Britt-Compton, Bethan; Tankimanova, Maira; Rowson, Jan; Letsolo, Boitelo; Man, Stephen; Haughton, Michele; Baird, Duncan M.

    2007-01-01

    The loss of telomere function can result in telomeric fusion events that lead to the types of genomic rearrangements, such as nonreciprocal translocations, that typify early-stage carcinogenesis. By using single-molecule approaches to characterize fusion events, we provide a functional definition of fusogenic telomeres in human cells. We show that approximately half of the fusion events contained no canonical telomere repeats at the fusion point; of those that did, the longest was 12.8 repeat...

  10. Chloroquine Increases Glucose Uptake via Enhancing GLUT4 Translocation and Fusion with the Plasma Membrane in L6 Cells

    Directory of Open Access Journals (Sweden)

    Qi Zhou

    2016-05-01

    Full Text Available Background/Aims: Chloroquine can induce an increase in the cellular uptake of glucose; however, the underlying mechanism is unclear. Methods: In this study, translocation of GLUT4 and intracellular Ca2+ changes were simultaneously observed by confocal microscope in L6 cells stably over-expressing IRAP-mOrange. The GLUT4 fusion with the plasma membrane (PM was traced using HA-GLUT4-GFP. Glucose uptake was measured using a cell-based glucose uptake assay. GLUT4 protein was detected by Western blotting and mRNA level was detected by RT-PCR. Results: We found that chloroquine induced significant increases in glucose uptake, glucose transporter GLUT4 translocation to the plasma membrane (GTPM, GLUT4 fusion with the PM, and intracellular Ca2+ in L6 muscle cells. Chloroquine-induced increases of GTPM and intracellular Ca2+ were inhibited by Gallein (Gβγ inhibitor and U73122 (PLC inhibitor. However, 2-APB (IP3R blocker only blocked the increase in intracellular Ca2+ but did not inhibit GTPM increase. These results indicate that chloroquine, via the Gβγ-PLC-IP3-IP3R pathway, induces elevation of Ca2+, and this Ca2+ increase does not play a role in chloroqui-ne-evoked GTPM increase. However, GLUT4 fusion with the PM and glucose uptake were significantly inhibited with BAPTA-AM. This suggests that Ca2+ enhances GLUT4 fusion with the PM resulting in glucose uptake increase. Conclusion: Our data indicate that chloroquine via Gβγ-PLC-IP3-IP3R induces Ca2+ elevation, which in turn promotes GLUT4 fusion with the PM. Moreover, chloroquine can enhance GLUT4 trafficking to the PM. These mechanisms eventually result in glucose uptake increase in control and insulin-resistant L6 cells. These findings suggest that chloroquine might be a potential drug for improving insulin tolerance in diabetic patients.

  11. Mitofusin-2 protects against cold stress-induced cell injury in HEK293 cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Wenbin; Chen, Yaomin; Yang, Qun; Che, Honglei; Chen, Xiangjun; Yao, Ting; Zhao, Fang; Liu, Mingchao; Ke, Tao [Department of Occupational and Environmental Health, School of Public Health, Fourth Military Medical University, Xi' an 710032 (China); Chen, Jingyuan, E-mail: jy_chen@fmmu.edu.cn [Department of Occupational and Environmental Health, School of Public Health, Fourth Military Medical University, Xi' an 710032 (China); Luo, Wenjing, E-mail: luowenj@fmmu.edu.cn [Department of Occupational and Environmental Health, School of Public Health, Fourth Military Medical University, Xi' an 710032 (China)

    2010-06-25

    Mitochondrial impairment is hypothesized to contribute to cell injury during cold stress. Mitochondria fission and fusion are closely related in the function of the mitochondria, but the precise mechanisms whereby these processes regulate cell injury during cold stress remain to be determined. HEK293 cells were cultured in a cold environment (4.0 {+-} 0.1 {sup o}C) for 2, 4, 8, or 12 h. Western blot analyses showed that these cells expressed decreased fission-related protein Drp1 and increased fusion-related protein Mfn2 at 4 h; meanwhile, electron microscopy analysis revealed large and long mitochondrial morphology within these cells, indicating increased mitochondrial fusion. With silencing of Mfn2 but not of Mfn1 by siRNA promoted cold-stress-induced cell death with decreased ATP production in HEK293 cells. Our results show that increased expression of Mfn2 and mitochondrial fusion are important for mitochondrial function as well as cell survival during cold stress. These findings have important implications for understanding the mechanisms of mitochondrial fusion and fission in cold-stress-induced cell injury.

  12. 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.

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

    OpenAIRE

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

    2011-01-01

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

  14. 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.

  15. Production of Induced Pluripotent Stem Cells by Reprogramming of Blood Cells

    Directory of Open Access Journals (Sweden)

    Sadia Zia

    2011-06-01

    Full Text Available Blood cells are the simple, efficient and economical source for the production of induced pluripotent cells. The discovery of induced pluripotent cells was not novel; it was pedestal on the scientific principals and technologies which have been developed over last six decades. These are nuclear transfer and the cloning of Animals, Pluripotent cell lines and fusion hybrids and Transcription Factors and lineage switching. The use of human embryonic stem cells in regenerative medicines was a breakthrough but make use of these cells arise ethical issues as they are obtained from human embryos. An alternative advancement using induced pluripotent stem cells, which mimics the embryonic stem cells has the significant gain that they replaced the embryonic stem cells. The pluripotent cells can be induced from terminally differentiated somatic cells by the Induction of only four defined factors including c-Myc, klf4, Oct4 and Sox2 which are enough to alter the fate of cell.

  16. Immunologic Monitoring of Cellular Responses by Dendritic/Tumor Cell Fusion Vaccines

    Directory of Open Access Journals (Sweden)

    Shigeo Koido

    2011-01-01

    Full Text Available Although dendritic cell (DC- based cancer vaccines induce effective antitumor activities in murine models, only limited therapeutic results have been obtained in clinical trials. As cancer vaccines induce antitumor activities by eliciting or modifying immune responses in patients with cancer, the Response Evaluation Criteria in Solid Tumors (RECIST and WHO criteria, designed to detect early effects of cytotoxic chemotherapy in solid tumors, may not provide a complete assessment of cancer vaccines. The problem may, in part, be resolved by carrying out immunologic cellular monitoring, which is one prerequisite for rational development of cancer vaccines. In this review, we will discuss immunologic monitoring of cellular responses for the evaluation of cancer vaccines including fusions of DC and whole tumor cell.

  17. Inhibition of mitochondrial fusion is an early and critical event in breast cancer cell apoptosis by dietary chemopreventative benzyl isothiocyanate.

    Science.gov (United States)

    Sehrawat, Anuradha; Croix, Claudette St; Baty, Catherine J; Watkins, Simon; Tailor, Dhanir; Singh, Rana P; Singh, Shivendra V

    2016-09-01

    Benzyl isothiocyanate (BITC) is a highly promising phytochemical abundant in cruciferous vegetables with preclinical evidence of in vivo efficacy against breast cancer in xenograft and transgenic mouse models. Mammary cancer chemoprevention by BITC is associated with apoptotic cell death but the underlying mechanism is not fully understood. Herein, we demonstrate for the first time that altered mitochondrial dynamics is an early and critical event in BITC-induced apoptosis in breast cancer cells. Exposure of MCF-7 and MDA-MB-231 cells to plasma achievable doses of BITC resulted in rapid collapse of mitochondrial filamentous network. BITC treatment also inhibited polyethyleneglycol-induced mitochondrial fusion. In contrast, a normal human mammary epithelial cell line (MCF-10A) that was derived from fibrocystic breast disease, was resistant to BITC-mediated alterations in mitochondrial dynamics as well as apoptosis. Transient or sustained decrease in levels of proteins engaged in regulation of mitochondrial fission and fusion was clearly evident after BITC treatment in both cancer cell lines. A trend for a decrease in the levels of mitochondrial fission- and fusion-related proteins was also observed in vivo in tumors of BITC-treated mice compared with control. Immortalized mouse embryonic fibroblasts from Drp1 knockout mice were resistant to BITC-induced apoptosis when compared with those from wild-type mice. Upon treatment with BITC, Bak dissociated from mitofusin 2 in both MCF-7 and MDA-MB-231 cells suggesting a crucial role for interaction of Bak and mitofusins in BITC-mediated inhibition of fusion and morphological dynamics. In conclusion, the present study provides novel insights into the molecular complexity of BITC-induced cell death. PMID:27374852

  18. Characterization of docking and fusion of synaptic-like microvesicles in PC12 cells using TIRFM

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Neurotransmitters are released by the fusion of synaptic vesicles with presynaptic membrane, which has been extensively studied. The analysis of single vesicle fusion kinetics reveals that there exist fusion modes of "kiss and run" and "kiss and stay" which may be favored by neurons especially during strong firing beside full fusion. Pre-fusion steps of translocation, docking and priming along the exocytotic pathway play important roles in neurotransmitter release and its regulation. In the present report, we used dual-color imaging of VAMP2-pHluorin and VAChT-TDimer2 under total internal reflection fluorescence microscope (TIRFM) to monitor the docking and fusion of synaptic-like microvesicles (SLMVs) in PC12 cells stimulated by high K+. Our results show that "kiss and run" is a dominative fusion mode in PC12 cells under high K+-challenge, and the dwell time of SLMVs is prolonged by the high K+ stimulation that suggests an enhancement of vesicle priming.

  19. Lysosome fusion to the cell membrane is mediated by the dysferlin C2A domain in coronary arterial endothelial cells

    OpenAIRE

    Han, Wei-Qing; Xia, Min; Xu, Ming; Krishna M Boini; Ritter, Joseph K.; Li, Ning-Jun; Li, Pin-Lan

    2012-01-01

    Dysferlin has recently been reported to participate in cell membrane repair in muscle and other cells through lysosome fusion. Given that lysosome fusion is a crucial mechanism that leads to membrane raft clustering, the present study attempted to determine whether dysferlin is involved in this process and its related signalling, and explores the mechanism underlying dysferlin-mediated lysosome fusion in bovine coronary arterial endothelial cells (CAECs). We found that dysferlin is clustered ...

  20. 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.

  1. Approximations in fusion and breakup reactions induced by radioactive beams

    Energy Technology Data Exchange (ETDEWEB)

    Cardenas, W.H.Z.; Carlin Filho, N.; Hussein, M.S. [Sao Paulo Univ., SP (Brazil). Inst. de Fisica; Canto, L.F.; Donangelo, R. [Universidade Federal, Rio de Janeiro, RJ (Brazil). Inst. de Fisica; Lubian, J. [Universidade Federal Fluminense, Niteroi, RJ (Brazil). Inst. de Fisica; Centro de Aplicaciones Tecnologicas y Desarrollo Nuclear (CEADEN), Havana (Cuba); Romanelli, A. [Facultad de Ingenieria, Montevideo (Uruguay). Inst. de Fisica

    2000-07-01

    Some commonly used approximations for complete fusion and breakup transmission coefficients in collisions of weakly bound projectiles at near barrier energies are assessed. We show that they strongly depend on the adopted classical trajectory and can be significantly improved with proper treatment of the incident and emergent currents in the WKB approximation. (author)

  2. Isolation, Regeneration and PEG-Induced Fusion of Protoplasts of Pleurotus pulmonarius and Pleurotus florida

    OpenAIRE

    Eyini, M.; Rajkumar, K.; Balaji, P.

    2006-01-01

    Inter-specific hybridization between Pleurotus pulmonarius and P. florida was attempted through PEG-induced protoplast fusion to select a fusant. The protocol for protoplast release, regeneration and fusion in these two Pleurotus species was standardized using the variables controlling the process. The mixture of mycolytic enzymes, i.e. commercial cellulase, crude chitinase and pectinase, KCl (0.6 M) as osmotic stabilizer, pH 6 of the phosphate buffer and an incubation time of 3 hours resulte...

  3. [SapM-induced fusion blocking of autophagosome-lysosome is depended on interaction with Rab7].

    Science.gov (United States)

    Hu, Dong; Wang, Wan; Zhao, Runpeng; Xu, Xuewei; Xing, Yingru; Ni, Shengfa; Xu, Congjing; Tie, Baoxian; Zhang, Rongbo; Wu, Jing

    2016-09-01

    Objective To study the role of Rab7 in the blockage of autophagosome-lysosome fusion induced by secretory acid phosphatase (SapM), a virulence factor of mycobacterium tuberculosis. Methods The Raw264.7 cells were transfected with siRab7, and the P62 was detected using Western blotting. After transfected with mCherry-SapM, the co-localization of SapM and Rab7 in Raw264.7 cells was detected by immunofluorescence cytochemistry and the interaction of SapM with Rab7 was determined by co-immunoprecipitation. SapM mutants including SapM(δ ARCA), SapM(δ FRED) and SapM(δ CT) were used to transfect Raw264.7 cells, and their associations with Rab7 were analyzed. Results The treatment of siRab7 induced a significant increase of P62 in these cells. Immunofluorescence cytochemistry showed the intracellular co-localization of SapM and Rab7. Co-immunoprecipitation showed that SapM and Rab7 were precipitated by each other. Only SapM(δ CT) failed to interact with Rab7 among the three SapM mutants. Conclusion The inhibition of autophagosome-lysosome fusion induced by SapM is dependent on the interaction between SapM and Rab7. PMID:27609571

  4. Prm3p Is a Pheromone-induced Peripheral Nuclear Envelope Protein Required for Yeast Nuclear Fusion

    OpenAIRE

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

    2009-01-01

    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 pheromon...

  5. 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

    genetic and epigenetic (DNA methylation and chromatin) profiling. We found that the basal-like trait is generally dominant and is largely defined by epigenetic repression of luminal transcription factors. Definition of super-enhancers highlighted a core program common in luminal cells but a high degree......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...... 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...

  6. Nuclear fusion induced by X-rays in a crystal

    CERN Document Server

    Belyaev, V B; Otto, J; Rakityansky, S A

    2016-01-01

    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 the synchrotron radiation). Exposing to the X-rays the solid compound LiD (lithium-deuteride) for the duration of 111 hours, we have detected 88 events of the nuclear fusion d+Li6 ---> Be8*. Our theoretical estimate agrees with what we observed. One of possible applications of the phenomenon we found, could be the measurements of the rates of various nuclear reactions (not necessarily fusion) at extremely low energies inaccessible in accelerator experiments.

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

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Kyoungmi; Kim, Hwain; Lee, Daekee, E-mail: daekee@ewha.ac.kr

    2009-10-09

    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{sup tm1Sor}, revealed that treatment of 1-cell or 2-cell embryos with 3 {mu}M 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.

  8. Using ApoE Nanolipoprotein Particles To Analyze SNARE-Induced Fusion Pores.

    Science.gov (United States)

    Bello, Oscar D; Auclair, Sarah M; Rothman, James E; Krishnakumar, Shyam S

    2016-03-29

    Here we introduce ApoE-based nanolipoprotein particle (NLP)-a soluble, discoidal bilayer mimetic of ∼23 nm in diameter, as fusion partners to study the dynamics of fusion pores induced by SNARE proteins. Using in vitro lipid mixing and content release assays, we report that NLPs reconstituted with synaptic v-SNARE VAMP2 (vNLP) fuse with liposomes containing the cognate t-SNARE (Syntaxin1/SNAP25) partner, with the resulting fusion pore opening directly to the external buffer. Efflux of encapsulated fluorescent dextrans of different sizes show that unlike the smaller nanodiscs, these larger NLPs accommodate the expansion of the fusion pore to at least ∼9 nm, and dithionite quenching of fluorescent lipid introduced in vNLP confirms that the NLP fusion pores are short-lived and eventually reseal. The NLPs also have capacity to accommodate larger number of proteins and using vNLPs with defined number of VAMP2 protein, including physiologically relevant copy numbers, we find that 3-4 copies of VAMP2 (minimum 2 per face) are required to keep a nascent fusion pore open, and the SNARE proteins act cooperatively to dilate the nascent fusion pore. PMID:26972604

  9. Microscopic dynamics simulations of heavy-ion fusion reactions induced by neutron-rich nuclei

    CERN Document Server

    Wang, Ning; Zhang, Yingxun; Li, Zhuxia

    2014-01-01

    The heavy-ion fusion reactions induced by neutron-rich nuclei are investigated with the improved quantum molecular dynamics (ImQMD) model. With a subtle consideration of the neutron skin thickness of nuclei and the symmetry potential, the stability of nuclei and the fusion excitation functions of heavy-ion fusion reactions $^{16}$O+$^{76}$Ge, $^{16}$O+$^{154}$Sm, $^{40}$Ca+$^{96}$Zr and $^{132}$Sn+$^{40}$Ca are systematically studied. The fusion cross sections of these reactions at energies around the Coulomb barrier can be well reproduced by using the ImQMD model. The corresponding slope parameter of the symmetry energy adopted in the calculations is $L \\approx 78$ MeV and the surface energy coefficient is $g_{\\rm sur}=18\\pm 1.5$ MeVfm$^2$. In addition, it is found that the surface-symmetry term significantly influences the fusion cross sections of neutron-rich fusion systems. For sub-barrier fusion, the dynamical fluctuations in the densities of the reaction partners and the enhanced surface diffuseness at ...

  10. Horizontal gene transfers and cell fusions in microbiology, immunology and oncology (Review).

    Science.gov (United States)

    Sinkovics, Joseph G

    2009-09-01

    Evolving young genomes of archaea, prokaryota and unicellular eukaryota were wide open for the acceptance of alien genomic sequences, which they often preserved and vertically transferred to their descendants throughout three billion years of evolution. Established complex large genomes, although seeded with ancestral retroelements, have come to regulate strictly their integrity. However, intruding retroelements, especially the descendents of Ty3/Gypsy, the chromoviruses, continue to find their ways into even the most established genomes. The simian and hominoid-Homo genomes preserved and accommodated a large number of endogenous retroviral genomic segments. These retroelements may mature into exogenous retroviruses, or into functional new genes. Phages and viruses have been instrumental in incorporating and transferring host cell genes. These events profoundly influenced and altered the course of evolution. Horizontal (lateral) gene transfers (HGT) overwhelmed the genomes of the ancient protocells and the evolving unicellular microorganisms, actually leading to their Cambrian explosion. While the rigidly organized genomes of multicellular organisms increasingly resist H/LGT, de-differentiated cells assuming the metabolism of their onto- or phylogenetic ancestors, open up widely to the practice of H/LGT by direct transfer, or to transfers mediated by viruses, or by cell fusions. This activity is intensified in malignantly transformed cells, thus rendering these subjects receptive to therapy with oncolytic viruses and with viral vectors of tumor-suppressive or immunogenic genetic materials. Naturally formed hybrids of dendritic and tumor cells are often tolerogenic, whereas laboratory products of these unisons may be immunogenic in the hosts of origin. As human breast cancer stem cells are induced by a treacherous class of CD8+ T cells to undergo epithelial to mesenchymal (ETM) transition and to yield to malignant transformation by the omnipresent proto

  11. In-vitro activation of cytotoxic T lymphocytes by fusion of mouse hepatocellular carcinoma cells and lymphotactin gene-modified dendritic cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To investigate the in-vitro activation of cytotoxic T lymphocytes (CTLs) by fusion of mouse hepatocellular carcinoma (HCC) cells and lymphotactin gene-modified dendritic cells (DCs).METHODS: Lymphotactin gene modified DCs (DCLptn) were prepared by lymphotactin recombinant adenovirus transduction of mature DCs which differentiated from mouse bone marrow cells by stimulation with granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4) and tumor necrosis factor alpha (TNF-α). DCLptn and H22 fusion was prepared using 50% PEG. Lymphotactin gene and protein expression levels were measured by RT-PCR and ELISA, respectively. Lymphotactin chemotactic responses were examined by in-vitro chemotaxis assay. In-vitro activation of CTLs by DCLptn/H22 fusion was measured by detecting CD25 expression and cytokine production after autologous T cell stimulation. Cytotoxic function of activated T lymphocytes stimulated with DCLptn/H22 cells was determined by LDH cytotoxicity assay.RESULTS: Lymphotactin gene could be efficiently transduced to DCs by adenovirus vector and showed an effective biological activity. After fusion, the hybrid DCLptn/H22 cells acquired the phenotypes of both DCLptn and H22 cells. In T cell proliferation assay, flow cytometry showed a very high CD25 expression, and cytokine release assay showed a significantly higher concentration of IFN-γ and IL-2 in DCLptn/H22 group than in DCLptn, DCLptn+H22, DC/H22 or H22 groups. Cytotoxicity assay revealed that T cells derived from DCLptn/H22 group had much higher anti-tumor activity than those derived from DCLptn, H22, DCLptn + H22, DC/H22 groups.CONCLUSION: Lymphotactin gene-modified dendritoma induces T-cell proliferation and strong CTL reaction against allogenic HCC cells. Immunization-engineered fusion hybrid vaccine is an attractive strategy in prevention and treatment of HCC metastases.

  12. 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

    Background: Spinal fusion is indicated in the surgical management of various spinal disorders. To ensure stabile fusion, bone graft materials are essential. Traditionally allo- or autograft has been used, but both are associated with limitations. Synthetic bone graft materials that reassemble today...... 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...... two level uninstrumented PLF at level L2/L3 and L4/L5. Levels were randomised to receive silk fibre enhanced ABM graft with or graft without P15 coating. The sheep were sacrificed after 4.5 months. Levels were harvested and evaluated with Micro-CT 50 scanner and qualitative histology. Fusion rates...

  13. Fusion of phospholipid vesicles induced by muscle glyceraldehyde-3-phosphate dehydrogenase in the absence of calcium.

    Science.gov (United States)

    Morero, R D; Viñals, A L; Bloj, B; Farías, R N

    1985-04-01

    Ca2+-induced fusion of phospholipid vesicles (phosphatidylcholine/phosphatidic acid, 9:1 mol/mol) prepared by ethanolic injection was followed by five different procedures: resonance energy transfer, light scattering, electron microscopy, intermixing of aqueous content, and gel filtration through Sepharose 4-B. The five methods gave concordant results, showing that vesicles containing only 10% phosphatidic acid can be induced to fuse by millimolar concentrations of Ca2+. When the fusing capability of several soluble proteins was assayed, it was found that concanavalin A, bovine serum albumin, ribonuclease, and protease were inactive. On the other hand, lysozyme, L-lactic dehydrogenase, and muscle and yeast glyceraldehyde-3-phosphate dehydrogenase were capable of inducing vesicle fusion. Glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle, the most extensively studied protein, proved to be very effective: 0.1 microM was enough to induce complete intermixing of bilayer phospholipid vesicles. Under conditions used in this work, fusion was accompanied by leakage of internal contents. The fusing capability of glyceraldehyde-3-phosphate dehydrogenase was not affected by 5 mM ethylenediaminetetraacetic acid. The Ca2+ concentration in the medium, as determined by atomic absorption spectroscopy, was 5 ppm. Heat-denatured enzyme was incapable of inducing fusion. We conclude that glyceraldehyde-3-phosphate dehydrogenase is a soluble protein inherently endowed with the capability of fusing phospholipid vesicles.

  14. Intercellular adhesion molecule 1 promotes HIV-1 attachment but not fusion to target cells.

    Directory of Open Access Journals (Sweden)

    Naoyuki Kondo

    Full Text Available Incorporation of intercellular adhesion molecule 1 (ICAM-1 into HIV-1 particles is known to markedly enhance the virus binding and infection of cells expressing lymphocyte function-associated antigen-1 (LFA-1. At the same time, ICAM-1 has been reported to exert a less pronounced effect on HIV-1 fusion with lymphoid cells. Here we examined the role of ICAM-1/LFA-1 interactions in productive HIV-1 entry into lymphoid cells using a direct virus-cell fusion assay. ICAM-1 promoted HIV-1 attachment to cells in a temperature-dependent manner. It exerted a marginal effect on virus binding in the cold, but enhanced binding up to 4-fold at physiological temperature. ICAM-1-independent attachment in the cold was readily reversible upon subsequent incubation at elevated temperature, whereas ICAM-1-bearing particles were largely retained by cells. The better virus retention resulted in a proportional increase in HIV-1 internalization and fusion, suggesting that ICAM-1 did not specifically accelerate endocytosis or fusion steps. We also measured the rates of CD4 engagement, productive endocytosis and HIV-endosome fusion using specific fusion inhibitors. These rates were virtually independent of the presence of ICAM-1 in viral particles. Importantly, irrespective of the presence of ICAM-1, HIV-1 escaped from the low temperature block, which stopped virus endocytosis and fusion, much later than from a membrane-impermeant fusion inhibitor targeting surface-accessible particles. This result, along with the complete inhibition of HIV-1 fusion by a small molecule dynamin inhibitor, implies this virus enters lymphoid cells used in this study via endocytosis and that this pathway is not altered by the viral ICAM-1. Our data highlight the role of ICAM-1 in stabilizing the HIV-1 attachment to LFA-1 expressing cells, which leads to a proportional enhancement of the receptor-mediated uptake and fusion with endosomes.

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

    OpenAIRE

    Wu, Liguo; Lindsey M Hutt-Fletcher

    2007-01-01

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

  16. Bimolecular fluorescence complementation analysis of eukaryotic fusion products

    OpenAIRE

    Lin, Ho-Pi; Vincenz, Claudius; Eliceiri, Kevin W.; Kerppola, Tom K.; Ogle, Brenda M.

    2010-01-01

    Background information. Cell fusion is known to underlie key developmental processes in humans and is postulated to contribute to tissue maintenance and even carcinogenesis. The mechanistic details of cell fusion, especially between different cell types, have been difficult to characterize because of the dynamic nature of the process and inadequate means to track fusion products over time. Here we introduce an inducible system for detecting and tracking live cell fusion products in vitro and ...

  17. LIN-39/Hox triggers cell division and represses EFF-1/fusogen-dependent vulval cell fusion

    OpenAIRE

    Shemer, Gidi; Podbilewicz, Benjamin

    2002-01-01

    General mechanisms by which Hox genes establish cell fates are known. However, a few Hox effectors mediating cell behaviors have been identified. Here we found the first effector of LIN-39/HoxD4/Dfd in Caenorhabditis elegans. In specific vulval precursor cells (VPCs), LIN-39 represses early and late expression of EFF-1, a membrane protein essential for cell fusion. Repression of eff-1 is also achieved by the activity of CEH-20/Exd/Pbx, a known cofactor of Hox proteins. Unfused VPCs in lin-39(...

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

    International Nuclear Information System (INIS)

    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)

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

    DEFF Research Database (Denmark)

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

    2010-01-01

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

  20. INHIBITION OF APOPTOSIS BY bcr-abl FUSION GENE IN K562 CELLS

    Institute of Scientific and Technical Information of China (English)

    WANG Chun-hong; SUN Bing-zhong; YUAN Yue-chuan

    1999-01-01

    Objective: To investigate the effect of bcr-abl fusion gene on CML cell apoptosis. Methods: Apoptosis of exvivo cultured K562 cells were observed after exposure to synthetic 18 mer antisense oligodeoxynucleotide complementary to the bcr-abl junction (b3a2). Results: Apoptosis of K562 cells was significantly increased associated with inhibition of bcr-abl expression. Conclusion: bcr-abl fusion gene formation due to chromosome translocation may be the major mechanism of CML via inhibition of apoptosis.

  1. Enhanced cell-free protein expression by fusion with immunoglobulin Cκ domain

    OpenAIRE

    Palmer, Elizabeth; Liu, Hong; Khan, Farid; Taussig, Michael J; He, Mingyue

    2006-01-01

    While cell-free systems are increasingly used for protein expression in structural and functional studies, several proteins are difficult to express or expressed only at low levels in cell-free lysates. Here, we report that fusion of the human immunoglobulin κ light chain constant domain (Cκ) at the C terminus of four representative proteins dramatically improved their production in the Escherichia coli S30 system, suggesting that enhancement of cell-free protein expression by Cκ fusion will ...

  2. 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

    2016-01-01

    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. PMID:27374619

  3. Influence of adjuvants on protection induced by a recombinant fusion protein against malarial infection.

    OpenAIRE

    Daly, T M; Long, C A

    1996-01-01

    Previously, we described a protective immune response induced by the carboxyl-terminal region of the merozoite surface protein-1 (MSP-1) from the rodent malarial parasite Plasmodium yoelii yoelii 17XL, expressed as a fusion protein and designated glutathione S-transferase (GST)-PYC2. We also demonstrated that the humoral response induced by GST-PYC2 was the primary mechanism by which immunized animals controlled their blood-stage infections. We have now examined the influence of several adjuv...

  4. Fusion of short telomeres in human cells is characterized by extensive deletion and microhomology, and can result in complex rearrangements

    OpenAIRE

    Letsolo, Boitelo T.; Rowson, Jan; Baird, Duncan M.

    2009-01-01

    Telomere fusion is an important mutational event that has the potential to lead to large-scale genomic rearrangements of the types frequently observed in cancer. We have developed single-molecule approaches to detect, isolate and characterize the DNA sequence of telomere fusion events in human cells. Using these assays, we have detected complex fusion events that include fusion with interstitial loci adjacent to fragile sites, intra-molecular rearrangements, and fusion events involving the te...

  5. Kinetic modeling of Sendai virus fusion with PC-12 cells. Effect of pH and temperature on fusion and viral inactivation

    OpenAIRE

    Lima, Maria da Conceição Pedroso de; Ramalho-Santos, João; Martins, Maria de Fátima; Carvalho, Arsélio Pato de; Bairos, Vasco; Nir, Shlomo

    1992-01-01

    We have studied the fusion activity of Sendai virus, a lipid-enveloped paramyxovirus, towards a line of adherent cells designated PC-12. Fusion was monitored by the dequenching of octadecylrhodamine, a fluorescent non-exchangeable probe. The results were analysed with a mass action kinetic model which could explain and predict the kinetics of virus2013cell fusion. When the temperature was lowered from 37°C to 25°C, a sharp inhibition of the fusion process was observed, probably reflecting a c...

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

    International Nuclear Information System (INIS)

    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 3H-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

  7. Bone Marrow Mesenchymal Stem Cells Expressing Baculovirus-Engineered Bone Morphogenetic Protein-7 Enhance Rabbit Posterolateral Fusion.

    Science.gov (United States)

    Liao, Jen-Chung

    2016-01-01

    Previous studies have suggested that bone marrow-derived mesenchymal stem cells (BMDMSCs) genetically modified with baculoviral bone morphogenetic protein-2 (Bac-BMP-2) vectors could achieve successful fusion in a femur defect model or in a spinal fusion model. In this study, BMDMSCs expressing BMP-7 (Bac-BMP-7-BMDMSCs) were generated. We hypothesized that Bac-BMP-7-BMDMSCs could secrete more BMP-7 than untransduced BMDMSCs in vitro and achieve spinal posterolateral fusion in a rabbit model. Eighteen rabbits underwent posterolateral fusion at L4-5. Group I (n = 6) was implanted with collagen-β-tricalcium phosphate (TCP)-hydroxyapatite (HA), Group II (n = 6) was implanted with collagen-β-TCP-HA plus BMDMSCs, and Group III (n = 6) was implanted with collagen-β-TCP-HA plus Bac-BMP-7-BMDMSCs. In vitro production of BMP-7 was quantified with an enzyme-linked immunosorbent assay (ELISA). Spinal fusion was examined using computed tomography (CT), manual palpation, and histological analysis. ELISA demonstrated that Bac-BMP-7-BMDMSCs produced four-fold to five-fold more BMP-7 than did BMDMSCs. In the CT results, 6 fused segments were observed in Group I (50%, 6/12), 8 in Group II (67%, 8/12), and 12 in Group III (100%, 12/12). The fusion rate, determined by manual palpation, was 0% (0/6) in Group I, 0% (0/6) in Group II, and 83% (5/6) in Group III. Histology showed that Group III had more new bone and matured marrow formation. In conclusion, BMDMSCs genetically transduced with the Bac-BMP-7 vector could express more BMP-7 than untransduced BMDMSCs. These Bac-BMP-7-BMDMSCs on collagen-β-TCP-HA scaffolds were able to induce successful spinal fusion in rabbits. PMID:27399674

  8. Bone Marrow Mesenchymal Stem Cells Expressing Baculovirus-Engineered Bone Morphogenetic Protein-7 Enhance Rabbit Posterolateral Fusion

    Directory of Open Access Journals (Sweden)

    Jen-Chung Liao

    2016-07-01

    Full Text Available Previous studies have suggested that bone marrow-derived mesenchymal stem cells (BMDMSCs genetically modified with baculoviral bone morphogenetic protein-2 (Bac-BMP-2 vectors could achieve successful fusion in a femur defect model or in a spinal fusion model. In this study, BMDMSCs expressing BMP-7 (Bac-BMP-7-BMDMSCs were generated. We hypothesized that Bac-BMP-7-BMDMSCs could secrete more BMP-7 than untransduced BMDMSCs in vitro and achieve spinal posterolateral fusion in a rabbit model. Eighteen rabbits underwent posterolateral fusion at L4-5. Group I (n = 6 was implanted with collagen-β-tricalcium phosphate (TCP-hydroxyapatite (HA, Group II (n = 6 was implanted with collagen-β-TCP-HA plus BMDMSCs, and Group III (n = 6 was implanted with collagen-β-TCP-HA plus Bac-BMP-7-BMDMSCs. In vitro production of BMP-7 was quantified with an enzyme-linked immunosorbent assay (ELISA. Spinal fusion was examined using computed tomography (CT, manual palpation, and histological analysis. ELISA demonstrated that Bac-BMP-7-BMDMSCs produced four-fold to five-fold more BMP-7 than did BMDMSCs. In the CT results, 6 fused segments were observed in Group I (50%, 6/12, 8 in Group II (67%, 8/12, and 12 in Group III (100%, 12/12. The fusion rate, determined by manual palpation, was 0% (0/6 in Group I, 0% (0/6 in Group II, and 83% (5/6 in Group III. Histology showed that Group III had more new bone and matured marrow formation. In conclusion, BMDMSCs genetically transduced with the Bac-BMP-7 vector could express more BMP-7 than untransduced BMDMSCs. These Bac-BMP-7-BMDMSCs on collagen-β-TCP-HA scaffolds were able to induce successful spinal fusion in rabbits.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    International Nuclear Information System (INIS)

    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

  11. 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

  12. Genetic complementation of human muscle cells via directed stem cell fusion.

    Science.gov (United States)

    Gonçalves, Manuel A F V; Swildens, Jim; Holkers, Maarten; Narain, Anjali; van Nierop, Gijsbert P; van de Watering, Marloes J M; Knaän-Shanzer, Shoshan; de Vries, Antoine A F

    2008-04-01

    Duchenne muscular dystrophy (DMD) is caused by mutations in the X chromosome-linked DMD gene, which encodes the sarcolemma-stabilizing protein-dystrophin. Initial attempts at DMD therapy deployed muscle progenitor cells from healthy donors. The utilization of these cells is, however, hampered by their immunogenicity, while those from DMD patients are scarce and display limited ex vivo replication. Nonmuscle cells with myogenic capacity may offer valuable alternatives especially if, to allow autologous transplantation, they are amenable to genetic intervention. As a paradigm for therapeutic gene transfer by heterotypic cell fusion we are investigating whether human mesenchymal stem cells (hMSCs) can serve as donors of recombinant DMD genes for recipient human muscle cells. Here, we show that forced MyoD expression in hMSCs greatly increases their tendency to participate in human myotube formation turning them into improved DNA delivery vehicles. Efficient loading of hMSCs with recombinant DMD was achieved through a new tropism-modified high-capacity adenoviral (hcAd) vector directing striated muscle-specific synthesis of full-length dystrophin. This study introduces the principle of genetic complementation of gene-defective cells via directed cell fusion and provides an initial framework to test whether transient MyoD synthesis in autologous, gene-corrected hMSCs increases their potential for treating DMD and, possibly, other muscular dystrophies.

  13. Tritium adsorption and desorption measurement on fusion relevant materials by beta induced spectrometry

    International Nuclear Information System (INIS)

    The use of tritium in future fusion power plants has the potential to make a major contribution to a sustainable and secure energy supply. For economic, licensing and safety reasons, tritium confinement in the fusion fuel system must be absolutely assured. Tritium adsorbed on a surface or diffused into a bulk material can escape from the fuel system and is lost for the process. Therefore, tritium confinement can be optimized by the use of materials with low adsorption and diffusion tendency. The Tritium Adsorption Desorption (TRIADE) Experiment is dedicated to investigate the tritium adsorption/desorption on fusion relevant materials by Beta Induced X-ray Spectrometry (BIXS) and mass spectrometry. The experimental setup and first results of the tritium measurements are presented.

  14. Neutron-induced dpa, transmutations, gas production, and helium embrittlement of fusion materials

    CERN Document Server

    Gilbert, M R; Nguyen-Manh, D; Zheng, S; Packer, L W; Sublet, J -Ch

    2013-01-01

    In a fusion reactor materials will be subjected to significant fluxes of high-energy neutrons. As well as causing radiation damage, the neutrons also initiate nuclear reactions leading to changes in the chemical composition of materials (transmutation). Many of these reactions produce gases, particularly helium, which cause additional swelling and embrittlement of materials. This paper investigates, using a combination of neutron-transport and inventory calculations, the variation in displacements per atom (dpa) and helium production levels as a function of position within the high flux regions of a recent conceptual model for the "next-step" fusion device DEMO. Subsequently, the gas production rates are used to provide revised estimates, based on new density-functional-theory results, for the critical component lifetimes associated with the helium-induced grain-boundary embrittlement of materials. The revised estimates give more optimistic projections for the lifetimes of materials in a fusion power plant co...

  15. Recent progress on MHD-induced loss of D-D fusion products in TFTR

    International Nuclear Information System (INIS)

    This paper reviews the recent progress made toward understanding the MHD-induced loss of D-D fusion products which has been seen on TFTR since 1988. These measurements have been made using the ''lost alpha'' diagnostic, which is described briefly. The largest MHD- induced loss occurs with coherent 3/2 or 2/1 MHD activity (kink/tearing modes), which can cause up to ∼3--5 times the first-orbit loss at I∼1.6--1.8 MA, roughly a ∼20--30% global los of D-D fusion products. Modeling of these MHD-induced losses has progressed to the point where the basic loss mechanism can be accounted for qualitatively, but the experimental results can not yet be understood quantitatively. Several alpha loss codes are being developed to improve the quantitative comparison between experiment and theory

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

    International Nuclear Information System (INIS)

    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.

  17. Horizontal Transmission and Retention of Malignancy, as well as Functional Human Genes, After Spontaneous Fusion of Human Glioblastoma and Hamster Host Cells In Vivo

    Science.gov (United States)

    Goldenberg, David M.; Zagzag, David; Heselmeyer-Haddad, Kerstin M.; Berroa Garcia, Lissa Y; Ried, Thomas; Loo, Meiyu; Chang, Chien-Hsing; Gold, David V.

    2011-01-01

    Cell fusion in vitro has been used to study cancer, gene mapping and regulation, and the production of antibodies via hybridomas. However, in-vivo heterosynkaryon formation by cell-cell fusion has received less attention. This investigation describes the spontaneous fusion of a human glioblastoma with normal hamster cells after xenogeneic transplantation, resulting in malignant cells that express both human and hamster genes and gene products, and retention of glioblastoma traits with an enhanced ability to metastasize. Three of 7 human genes found showed translation of their proteins during serial propagation in vivo or in vitro for years; namely, CD74, CXCR4, and PLAGL2, each implicated with malignancy or glioblastoma. This supports the thesis that genetic hybridization of cancer and normal cells can transmit malignancy and also, as first described herein, regulatory genes involved in the tumor’s organotypic morphology. Evidence also is increasing that even cell-free human cancer DNA can induce malignancy and transfer genetic information to normal cells. Hence, we posit that the transfer of genetic information between tumor and stromal cells, whether by cell-cell fusion or other mechanisms, is implicated in the progression of malignancy, and may further define the crosstalk between cancer cells and their stromal neighbors. PMID:21796629

  18. Geometry of the Contact Zone between Fused Membrane-Coated Beads Mimicking Cell-Cell Fusion.

    Science.gov (United States)

    Savić, Filip; Kliesch, Torben-Tobias; Verbeek, Sarah; Bao, Chunxiao; Thiart, Jan; Kros, Alexander; Geil, Burkhard; Janshoff, Andreas

    2016-05-24

    The fusion of lipid membranes is a key process in biology. It enables cells and organelles to exchange molecules with their surroundings, which otherwise could not cross the membrane barrier. To study such complex processes we use simplified artificial model systems, i.e., an optical fusion assay based on membrane-coated glass spheres. We present a technique to analyze membrane-membrane interactions in a large ensemble of particles. Detailed information on the geometry of the fusion stalk of fully fused membranes is obtained by studying the diffusional lipid dynamics with fluorescence recovery after photobleaching experiments. A small contact zone is a strong obstruction for the particle exchange across the fusion spot. With the aid of computer simulations, fluorescence-recovery-after-photobleaching recovery times of both fused and single-membrane-coated beads allow us to estimate the size of the contact zones between two membrane-coated beads. Minimizing delamination and bending energy leads to minimal angles close to those geometrically allowed. PMID:27224487

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

    OpenAIRE

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

    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...

  20. Cdc42p and Fus2p act together late in yeast cell fusion

    OpenAIRE

    Ydenberg, Casey A.; Stein, Richard A; Rose, Mark D.

    2012-01-01

    Cell fusion is the key event of fertilization that gives rise to the diploid zygote and is a nearly universal aspect of eukaryotic biology. In the yeast Saccharomyces cerevisiae, several mutants have been identified that are defective for cell fusion, and yet the molecular mechanism of this process remains obscure. One obstacle has been that genetic screens have mainly focused on mating-specific factors, whereas the process likely involves housekeeping proteins as well. Here we implicate Cdc4...

  1. 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.

  2. Isolation, Regeneration and PEG-Induced Fusion of Protoplasts of Pleurotus pulmonarius and Pleurotus florida.

    Science.gov (United States)

    Eyini, M; Rajkumar, K; Balaji, P

    2006-06-01

    Inter-specific hybridization between Pleurotus pulmonarius and P. florida was attempted through PEG-induced protoplast fusion to select a fusant. The protocol for protoplast release, regeneration and fusion in these two Pleurotus species was standardized using the variables controlling the process. The mixture of mycolytic enzymes, i.e. commercial cellulase, crude chitinase and pectinase, KCl (0.6 M) as osmotic stabilizer, pH 6 of the phosphate buffer and an incubation time of 3 hours resulted in the maximum release of protoplasts from 3-day-old mycelia of P. florida (5.3~5.75 × 10(7) protoplasts/g) and P. pulmonarius (5.6~6 × 10(7) protoplasts/g). The isolated protoplasts of P. florida regenerated mycelium with 3.3% regeneration efficiency while P. pulmonarius showed 4.1% efficiency of regeneration. Polyethyleneglycol (PEG) - induced fusion of protoplasts of these two species resulted in 0.28% fusion frequency. The fusant produced fruiting bodies on paddy straw but required a lower temperature of crop running (24 ± 2℃) than its parents which could fruit at 28 ± 2℃. The stable fusant strain was selected by testing for the selected biochemical markers i.e. Carbendazim tolerance and utilization of the lignin degradation product, vanillin. PMID:24039474

  3. Study of the role of breakup following neutron transfer in fusion induced by 9Be at near barrier energies

    International Nuclear Information System (INIS)

    The studies on the breakup effects of weakly bound nuclei, both stable and radioactive, on fusion cross section is a subject of contemporary interest. These weakly bound nuclei have low breakup threshold (binding energy) that makes the fusion induced by these nuclei fundamentally different from that induced by tightly bound nuclei. Among these nuclei, the nucleus 9Be is very interesting because it has low break up threshold of 1.67 MeV and has a possible three body n+α+α Borromean structure. So far, numbers of experiments have been carried out to study the fusion reactions induced by 9Be on various targets at near barrier energies

  4. Truncated SSX protein suppresses synovial sarcoma cell proliferation by inhibiting the localization of SS18-SSX fusion protein.

    Directory of Open Access Journals (Sweden)

    Yasushi Yoneda

    Full Text Available Synovial sarcoma is a relatively rare high-grade soft tissue sarcoma that often develops in the limbs of young people and induces the lung and the lymph node metastasis resulting in poor prognosis. In patients with synovial sarcoma, specific chromosomal translocation of t(X; 18 (p11.2;q11.2 is observed, and SS18-SSX fusion protein expressed by this translocation is reported to be associated with pathogenesis. However, role of the fusion protein in the pathogenesis of synovial sarcoma has not yet been completely clarified. In this study, we focused on the localization patterns of SS18-SSX fusion protein. We constructed expression plasmids coding for the full length SS18-SSX, the truncated SS18 moiety (tSS18 and the truncated SSX moiety (tSSX of SS18-SSX, tagged with fluorescent proteins. These plasmids were transfected in synovial sarcoma SYO-1 cells and we observed the expression of these proteins using a fluorescence microscope. The SS18-SSX fusion protein showed a characteristic speckle pattern in the nucleus. However, when SS18-SSX was co-expressed with tSSX, localization of SS18-SSX changed from speckle patterns to the diffused pattern similar to the localization pattern of tSSX and SSX. Furthermore, cell proliferation and colony formation of synovial sarcoma SYO-1 and YaFuSS cells were suppressed by exogenous tSSX expression. Our results suggest that the characteristic speckle localization pattern of SS18-SSX is strongly involved in the tumorigenesis through the SSX moiety of the SS18-SSX fusion protein. These findings could be applied to further understand the pathogenic mechanisms, and towards the development of molecular targeting approach for synovial sarcoma.

  5. Surface apposition and multiple cell contacts promote myoblast fusion in Drosophila flight muscles.

    Science.gov (United States)

    Dhanyasi, Nagaraju; Segal, Dagan; Shimoni, Eyal; Shinder, Vera; Shilo, Ben-Zion; VijayRaghavan, K; Schejter, Eyal D

    2015-10-12

    Fusion of individual myoblasts to form multinucleated myofibers constitutes a widely conserved program for growth of the somatic musculature. We have used electron microscopy methods to study this key form of cell-cell fusion during development of the indirect flight muscles (IFMs) of Drosophila melanogaster. We find that IFM myoblast-myotube fusion proceeds in a stepwise fashion and is governed by apparent cross talk between transmembrane and cytoskeletal elements. Our analysis suggests that cell adhesion is necessary for bringing myoblasts to within a minimal distance from the myotubes. The branched actin polymerization machinery acts subsequently to promote tight apposition between the surfaces of the two cell types and formation of multiple sites of cell-cell contact, giving rise to nascent fusion pores whose expansion establishes full cytoplasmic continuity. Given the conserved features of IFM myogenesis, this sequence of cell interactions and membrane events and the mechanistic significance of cell adhesion elements and the actin-based cytoskeleton are likely to represent general principles of the myoblast fusion process.

  6. Lack of lysosomal fusion with phagosomes containing Ehrlichia risticii in P388D1 cells: abrogation of inhibition with oxytetracycline.

    OpenAIRE

    Wells, M Y; Rikihisa, Y

    1988-01-01

    Fusion of lysosomes with phagosomes containing Ehrlichia risticii, an obligate intracellular parasite, was evaluated in P388D1 murine macrophagelike cells. Lysosomes in cells ranging in infectivity from 30 to 70% were labeled cytochemically with acid phosphatase or via endocytosis of thorium dioxide or cationized ferritin to document phagosome-lysosome (P-L) fusion in untreated cells and cells treated with oxytetracycline. Regardless of the marker used, P-L fusion was generally not observed i...

  7. Cluster Model for Near-barrier Fusion Induced by Weakly Bound and Halo Nuclei

    OpenAIRE

    Beck, C; Keeley, N.; Diaz-Torres, A.

    2007-01-01

    The influence on the fusion process of coupling transfer/breakup channels is investigated for the medium weight $^{6,7}$Li+$^{59}$Co systems in the vicinity of the Coulomb barrier. Coupling effects are discussed within a comparison of predictions of the Continuum Discretized Coupled-Channels model. Applications to $^{6}$He+$^{59}$Co induced by the borromean halo nucleus $^{6}$He are also proposed.

  8. Finite element analysis of an eddy current induced in thin structures of a magnetic fusion reactor

    International Nuclear Information System (INIS)

    A numerical method, using finite element analysis, has been developed to investigate the eddy currents induced in metallic components such as the vacuum vessel, the first wall and the limiters, of a magnetic fusion reactor. The devices are treated as thin shells thus reducing the three dimensional problem to a two-dimensional one due to the shallow skin depth of the eddy current. (U.K.)

  9. An induced junction photovoltaic cell

    Science.gov (United States)

    Call, R. L.

    1974-01-01

    Silicon solar cells operating with induced junctions rather than diffused junctions have been fabricated and tested. Induced junctions were created by forming an inversion layer near the surface of the silicon by supplying a sheet of positive charge above the surface. Measurements of the response of the inversion layer cell to light of different wavelengths indicated it to be more sensitive to the shorter wavelengths of the sun's spectrum than conventional cells. The greater sensitivity occurs because of the shallow junction and the strong electric field at the surface.

  10. Red fluorescent protein-aequorin fusions as improved bioluminescent Ca2+ reporters in single cells and mice.

    Directory of Open Access Journals (Sweden)

    Adil Bakayan

    Full Text Available Bioluminescence recording of Ca(2+ signals with the photoprotein aequorin does not require radiative energy input and can be measured with a low background and good temporal resolution. Shifting aequorin emission to longer wavelengths occurs naturally in the jellyfish Aequorea victoria by bioluminescence resonance energy transfer (BRET to the green fluorescent protein (GFP. This process has been reproduced in the molecular fusions GFP-aequorin and monomeric red fluorescent protein (mRFP-aequorin, but the latter showed limited transfer efficiency. Fusions with strong red emission would facilitate the simultaneous imaging of Ca(2+ in various cell compartments. In addition, they would also serve to monitor Ca(2+ in living organisms since red light is able to cross animal tissues with less scattering. In this study, aequorin was fused to orange and various red fluorescent proteins to identify the best acceptor in red emission bands. Tandem-dimer Tomato-aequorin (tdTA showed the highest BRET efficiency (largest energy transfer critical distance R(0 and percentage of counts in the red band of all the fusions studied. In addition, red fluorophore maturation of tdTA within cells was faster than that of other fusions. Light output was sufficient to image ATP-induced Ca(2+ oscillations in single HeLa cells expressing tdTA. Ca(2+ rises caused by depolarization of mouse neuronal cells in primary culture were also recorded, and changes in fine neuronal projections were spatially resolved. Finally, it was also possible to visualize the Ca(2+ activity of HeLa cells injected subcutaneously into mice, and Ca(2+ signals after depositing recombinant tdTA in muscle or the peritoneal cavity. Here we report that tdTA is the brightest red bioluminescent Ca(2+ sensor reported to date and is, therefore, a promising probe to study Ca(2+ dynamics in whole organisms or tissues expressing the transgene.

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

    International Nuclear Information System (INIS)

    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

  13. Intercellular Adhesion Molecule 1 Promotes HIV-1 Attachment but Not Fusion to Target Cells

    OpenAIRE

    Naoyuki Kondo; Melikyan, Gregory B.

    2012-01-01

    Incorporation of intercellular adhesion molecule 1 (ICAM-1) into HIV-1 particles is known to markedly enhance the virus binding and infection of cells expressing lymphocyte function-associated antigen-1 (LFA-1). At the same time, ICAM-1 has been reported to exert a less pronounced effect on HIV-1 fusion with lymphoid cells. Here we examined the role of ICAM-1/LFA-1 interactions in productive HIV-1 entry into lymphoid cells using a direct virus-cell fusion assay. ICAM-1 promoted HIV-1 attachme...

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

    Science.gov (United States)

    Prada, Ilaria; Meldolesi, Jacopo

    2016-01-01

    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. PMID:27517914

  15. Aqueous extract from a Chaga medicinal mushroom, Inonotus obliquus (higher Basidiomycetes), prevents herpes simplex virus entry through inhibition of viral-induced membrane fusion.

    Science.gov (United States)

    Pan, Hong-Hui; Yu, Xiong-Tao; Li, Ting; Wu, Hong-Ling; Jiao, Chun-Wei; Cai, Mian-Hua; Li, Xiang-Min; Xie, Yi-Zhen; Wang, Yi; Peng, Tao

    2013-01-01

    Chaga medicinal mushroom, Inonotus obliquus, a popular prescription in traditional medicine in Europe and Asia, was used to reduce inflammation in the nasopharynx and to facilitate breathing. The aqueous extract from I. obliquus (AEIO) exhibited marked decrease in herpes simplex virus (HSV) infection (the 50% inhibitory concentration was 3.82 μg/mL in the plaque reduction assay and 12.29 μg/mL in the HSV-1/blue assay) as well as safety in Vero cells (the 50% cellular cytotoxicity was > 1 mg/mL, and selection index was > 80). Using a time course assay, effective stage analysis, and fusion inhibition assay, the mechanism of anti-HSV activity was found against the early stage of viral infection through inhibition of viral-induced membrane fusion. Therefore, AEIO could effectively prevent HSV-1 entry by acting on viral glycoproteins, leading to the prevention of membrane fusion, which is different from nucleoside analog antiherpetics. PMID:23510282

  16. 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

  17. A mechanistic paradigm for broad-spectrum antivirals that target virus-cell fusion.

    Directory of Open Access Journals (Sweden)

    Frederic Vigant

    Full Text Available LJ001 is a lipophilic thiazolidine derivative that inhibits the entry of numerous enveloped viruses at non-cytotoxic concentrations (IC50 ≤ 0.5 µM, and was posited to exploit the physiological difference between static viral membranes and biogenic cellular membranes. We now report on the molecular mechanism that results in LJ001's specific inhibition of virus-cell fusion. The antiviral activity of LJ001 was light-dependent, required the presence of molecular oxygen, and was reversed by singlet oxygen ((1O2 quenchers, qualifying LJ001 as a type II photosensitizer. Unsaturated phospholipids were the main target modified by LJ001-generated (1O2. Hydroxylated fatty acid species were detected in model and viral membranes treated with LJ001, but not its inactive molecular analog, LJ025. (1O2-mediated allylic hydroxylation of unsaturated phospholipids leads to a trans-isomerization of the double bond and concurrent formation of a hydroxyl group in the middle of the hydrophobic lipid bilayer. LJ001-induced (1O2-mediated lipid oxidation negatively impacts on the biophysical properties of viral membranes (membrane curvature and fluidity critical for productive virus-cell membrane fusion. LJ001 did not mediate any apparent damage on biogenic cellular membranes, likely due to multiple endogenous cytoprotection mechanisms against phospholipid hydroperoxides. Based on our understanding of LJ001's mechanism of action, we designed a new class of membrane-intercalating photosensitizers to overcome LJ001's limitations for use as an in vivo antiviral agent. Structure activity relationship (SAR studies led to a novel class of compounds (oxazolidine-2,4-dithiones with (1 100-fold improved in vitro potency (IC50<10 nM, (2 red-shifted absorption spectra (for better tissue penetration, (3 increased quantum yield (efficiency of (1O2 generation, and (4 10-100-fold improved bioavailability. Candidate compounds in our new series moderately but significantly (p≤0

  18. Acute inhibition of PI3K-PDK1-Akt pathway potentiates insulin secretion through upregulation of newcomer granule fusions in pancreatic β-cells.

    Directory of Open Access Journals (Sweden)

    Kyota Aoyagi

    Full Text Available In glucose-induced insulin secretion from pancreatic β-cells, a population of insulin granules fuses with the plasma membrane without the typical docking process (newcomer granule fusions, however, its mechanism is unclear. In this study, we investigated the PI3K signaling pathways involved in the upregulation of newcomer granule fusions. Acute treatment with the class IA-selective PI3K inhibitors, PIK-75 and PI-103, enhanced the glucose-induced insulin secretion. Total internal reflection fluorescent microscopy revealed that the PI3K inhibitors increased the fusion events from newcomer granules. We developed a new system for transfection into pancreatic islets and demonstrated the usefulness of this system in order for evaluating the effect of transfected genes on the glucose-induced secretion in primary cultured pancreatic islets. Using this transfection system together with a series of constitutive active mutants, we showed that the PI3K-3-phosphoinositide dependent kinase-1 (PDK1-Akt pathway mediated the potentiation of insulin secretion. The Akt inhibitor also enhanced the glucose-induced insulin secretion in parallel with the upregulation of newcomer granule fusions, probably via increased motility of intracellular insulin granules. These data suggest that the PI3K-PDK1-Akt pathway plays a significant role in newcomer granule fusions, probably through an alteration of the dynamics of the intracellular insulin granules.

  19. 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

    2016-07-01

    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

  20. Role of projectile breakup in {sup 6}He and {sup 6}Li induced fusion reactions around barrier energies

    Energy Technology Data Exchange (ETDEWEB)

    Kumari, Anju; Kharab, Rajesh, E-mail: kharabrajesh@rediffmail.com

    2015-09-15

    The influence of projectile breakup on fusion cross section for {sup 6}He + {sup 209}Bi, {sup 6}He + {sup 64}Zn, {sup 6}Li + {sup 209}Bi and {sup 6}Li + {sup 64}Zn reactions at near barrier energies is studied within the framework of quantum diffusion approach. The breakup does not affect the fusion induced by {sup 6}He, whereas a significant suppression for {sup 6}Li induced reaction is observed in below barrier energy region.

  1. 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.

  2. Significance of neck length parameter in 32S induced fusion reactions

    International Nuclear Information System (INIS)

    The dynamics of heavy ion induced reactions give a pathway to number of interesting characteristics of atomic nuclei. The heavy ion fusion reactions are observed to be strongly influenced by the nuclear structure of the interacting nuclei and the intrinsic properties associated with them. The exclusive studies have been made to study the effects of nuclear shape, structure and angular momentum etc. Present work is a further attempt to establish the predictability of the DCM, while studying the reactions induced by the stable projectile 32S at fixed incident energy Elab ∼142 MeV on different targets

  3. Polarized exocyst-mediated vesicle fusion directs intracellular lumenogenesis within the C. elegans excretory cell

    OpenAIRE

    Armenti, Stephen T.; Chan, Emily; Nance, Jeremy

    2014-01-01

    Lumenogenesis of small seamless tubes occurs through intracellular membrane growth and directed vesicle fusion events. Within the C. elegans excretory cell, which forms seamless intracellular tubes (canals) that mediate osmoregulation, lumens grow in length and diameter when vesicles fuse with the expanding lumenal surface. Here, we show that lumenal vesicle fusion depends on the small GTPase RAL-1, which localizes to vesicles and acts through the exocyst vesicle-tethering complex. Loss of ei...

  4. Long-term live cell microscopy studies of lipid droplet fusion dynamics in adipocytes[S

    OpenAIRE

    Jüngst, Christian; Klein, Matthias; Zumbusch, Andreas

    2013-01-01

    During the adipogenic differentiation process of mesenchymal stem cells, lipid droplets (LDs) grow slowly by transferring lipids between each other. Recent findings hint at the possibility that a fusion pore is involved. In this study, we analyze lipid transfer data obtained in long-term label-free microscopy studies in the framework of a Hagen-Poiseuille model. The data obtained show a LD fusion process in which the lipid transfer directionality depends on the size difference between LDs, wh...

  5. Nanoscale organization of β2-adrenergic receptor-Venus fusion protein domains on the surface of mammalian cells

    International Nuclear Information System (INIS)

    Adrenergic receptors are a key component of nanoscale multiprotein complexes that are responsible for controlling the beat rate in a mammalian heart. We demonstrate the ability of near-field scanning optical microscopy (NSOM) to visualize β2-adrenergic receptors (β2AR) fused to the GFP analogue Venus at the nanoscale on HEK293 cells. The expression of the β2AR-Venus fusion protein was tightly controlled using a tetracycline-induced promoter. Both the size and density of the observed nanoscale domains are dependent on the level of induction and thus the level of protein expression. At concentrations between 100 and 700 ng/ml of inducer doxycycline, the size of domains containing the β2AR-Venus fusion protein appears to remain roughly constant, but the number of domains per cell increase. At 700 ng/ml doxycycline the functional receptors are organized into domains with an average diameter of 150 nm with a density similar to that observed for the native protein on primary murine cells. By contrast, larger micron-sized domains of β2AR are observed in the membrane of the HEK293 cells that stably overexpress β2AR-GFP and β2AR-eYFP. We conclude that precise chemical control of gene expression is highly advantageous for the use β2AR-Venus fusion proteins as models for β2AR function. These observations are critical for designing future cell models and assays based on β2AR, since the receptor biology is consistent with a relatively low density of nanoscale receptor domains.

  6. Nanoscale organization of {beta}{sub 2}-adrenergic receptor-Venus fusion protein domains on the surface of mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Vobornik, Dusan; Rouleau, Yanouchka; Haley, Jennifer [Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa, ON, Canada K1A 0R6 (Canada); Bani-Yaghoub, Mahmud [Institute for Biological Sciences, National Research Council Canada, Ottawa, ON, Canada K1A 0R6 (Canada); Taylor, Rod [Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa, ON, Canada K1A 0R6 (Canada); Johnston, Linda J., E-mail: Linda.Johnston@nrc-cnrc.gc.ca [Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa, ON, Canada K1A 0R6 (Canada); Pezacki, John Paul, E-mail: John.Pezacki@nrc-cnrc.gc.ca [Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa, ON, Canada K1A 0R6 (Canada)

    2009-04-24

    Adrenergic receptors are a key component of nanoscale multiprotein complexes that are responsible for controlling the beat rate in a mammalian heart. We demonstrate the ability of near-field scanning optical microscopy (NSOM) to visualize {beta}{sub 2}-adrenergic receptors ({beta}{sub 2}AR) fused to the GFP analogue Venus at the nanoscale on HEK293 cells. The expression of the {beta}{sub 2}AR-Venus fusion protein was tightly controlled using a tetracycline-induced promoter. Both the size and density of the observed nanoscale domains are dependent on the level of induction and thus the level of protein expression. At concentrations between 100 and 700 ng/ml of inducer doxycycline, the size of domains containing the {beta}{sub 2}AR-Venus fusion protein appears to remain roughly constant, but the number of domains per cell increase. At 700 ng/ml doxycycline the functional receptors are organized into domains with an average diameter of 150 nm with a density similar to that observed for the native protein on primary murine cells. By contrast, larger micron-sized domains of {beta}{sub 2}AR are observed in the membrane of the HEK293 cells that stably overexpress {beta}{sub 2}AR-GFP and {beta}{sub 2}AR-eYFP. We conclude that precise chemical control of gene expression is highly advantageous for the use {beta}{sub 2}AR-Venus fusion proteins as models for {beta}{sub 2}AR function. These observations are critical for designing future cell models and assays based on {beta}{sub 2}AR, since the receptor biology is consistent with a relatively low density of nanoscale receptor domains.

  7. Melanoma Cells Can Adopt the Phenotype of Stromal Fibroblasts and Macrophages by Spontaneous Cell Fusion in Vitro

    Science.gov (United States)

    Kemény, Lajos V.; Kurgyis, Zsuzsanna; Buknicz, Tünde; Groma, Gergely; Jakab, Ádám; Zänker, Kurt; Dittmar, Thomas; Kemény, Lajos; Németh, István B.

    2016-01-01

    After the removal of primary cutaneous melanoma some patients develop local recurrences, even after having histologically tumor-free re-excision. A potential explanation behind this phenomenon is that tumor cells switch their phenotype, making their recognition via standard histopathological assessments extremely difficult. Tumor-stromal cell fusion has been proposed as a potential mechanism for tumor cells to acquire mesenchymal traits; therefore, we hypothesized that melanoma cells could acquire fibroblast- and macrophage-like phenotypes via cell fusion. We show that melanoma cells spontaneously fuse with human dermal fibroblasts and human peripheral blood monocytes in vitro. The hybrid cells’ nuclei contain chromosomes from both parental cells and are indistinguishable from the parental fibroblasts or macrophages based on their morphology and immunophenotype, as they could lose the melanoma specific MART1 marker, but express the fibroblast marker smooth muscle actin or the macrophage marker CD68. Our results suggest that, by spontaneous cell fusion in vitro, tumor cells can adopt the morphology and immunophenotype of stromal cells while still carrying oncogenic, tumor-derived genetic information. Therefore, melanoma–stromal cell fusion might play a role in missing tumor cells by routine histopathological assessments. PMID:27271591

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

    International Nuclear Information System (INIS)

    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

  9. 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.

  10. Induction of apoptosis in MCF‑7 human breast cancer cells by Khz (fusion of Ganoderma lucidum and Polyporus umbellatus mycelium).

    Science.gov (United States)

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

    2016-02-01

    Khz (fusion of Ganoderma lucidum and Polyporus umbellatus), isolated from the mycelia of G. lucidum and P. umbellatus, exerts anti‑proliferative effects against malignant cells; however, its activity against human breast cancer cells remains to be elucidated. In the present study, cell proliferation was assessed using a 3-(4,5‑dimethylthiazol‑2‑yl)-2,5‑diphenyltetrazolium bromide assay, and poptosis was examined using annexin V‑propidium iodide staining and flow cytometry. The activation of caspases 7, 8 and 9 were detected in the Khz‑treated cells using western blotting. The results demonstrated that Khz increased the intracellular calcium concentration and induced the production of reactive oxygen species in MCF‑7 breast cancer cells, as determined using flow cytometry. The results also demonstrated that Khz inhibited cell proliferation and induced apoptosis in the MCF‑7 cells. In addition, the mechanism by which Khz induces apoptosis in cancer cells was investigated. Khz induced apoptosis preferentially in transformed cells, with a minimal effect on non‑transformed cells, suggesting its potential as an anticancer therapeutic agent. Oxidative stress is associated with apoptotic and non‑apoptotic cell death, although pro‑oxidative conditions are not a pre‑requisite for apoptosis. Assessment of the activation status of caspases 7, 8 and 9 revealed that the levels of cleaved caspases were significantly increased in the cells treated with Khz. It is widely accepted that calcium signaling is important in apoptosis, and the present study observed an increase in [Ca2+]i in response to Khz treatment. The anti‑proliferative and pro‑apoptotic effects of Khz suggest that this extract may be developed as a potential anticancer agent. PMID:26648109

  11. [Expression of human IL-35-IgG4 (Fc) fusion protein in CHO/DG44 cells].

    Science.gov (United States)

    Tang, Jing; Gao, Wenda; Zhang, Qing; Zhang, Dawei; Chen, Yang; He, Bo; Liu, Quansheng

    2009-01-01

    We constructed the eukaryotic expression vector of human IL-35-IgG4 (Fc)-pOptiVEC-TOPO by gene recombination technique and expressed the fusion protein human IL-35-IgG4 (Fc) in CHO/DG44 cells. The two components of the newly discovered cytokine human IL-35, EBI3 and IL-12p35, were amplified by PCR from the cDNA library derived from the KG-I cells after LPS induction. The two PCR-amplified cDNA fragments of human IL-35 were linked by over-lapping PCR and then cloned into the IgG4 (Fc)-pOptiVEC-TOPO vector. The constructed plasmid with the recombinant cDNA IL-35-IgG4 (Fc) was verified by restriction enzyme digestion analysis, PCR and DNA sequencing. The verified plasmid with the recombinant cDNA was transfected into CHO/DG44 cells using Lipofectamine 2000. The success of the transfection was examined and confirmed by RT-PCR. After selection in alpha-MEM (-) medium, the IL-35-Ig G4 (Fc) positive CHO/DG44 clones were chosen and the media from these positive clones were collected to be used to purify the fusion protein. The positive CHO/DG44 clones were further cultured in increasing concentrations of MTX and the expression levels of the fusion protein IL-35-Ig G4 (Fc) were repetitively induced by MTX-induced gene amplification. The IL-35-IgG4 (Fc) fusion protein was purified from the media collected from the positive CHO/DG44 clones by protein G affinity chromatography and then identified by SDS-PAGE and Western blotting. The results showed that one protein band was found to match well with the predicted relative molecular mass of human IL-35-IgG4 (Fc) and this protein could specifically bind to anti-human IgG4 (Fc) monoclonal antibody. In conclusion, our study successfully established an IL-35-IgG4 (Fc) positive DG44 cell line which could stably express IL-35-IgG4 (Fc) fusion protein.

  12. 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. PMID:21556691

  13. Antitumor immunopreventive effect in mice induced by DNA vaccine encoding a fusion protein of α-fetoprotein and CTLA4

    Institute of Scientific and Technical Information of China (English)

    Geng Tian; Ji-Lin Yi; Ping Xiong

    2004-01-01

    AIM: To develop a tumor DNA vaccine encoding a fusion protein of murine AFP and CTLA4, and to study its ability to induce specific CTL response and its protective effect against AFP-producing tumor.METHODS: Murine α-fetoprotein (mAFP) gene was cloned from total RNA of Hepa1-6 cells by RT-PCR. A DNA vaccine was constructed by fusion murine α-fetoprotein gene and extramembrane domain of murine CTLA4 gene. The DNA vaccine was identified by restriction enzyme analysis,sequencing and expression. EL-4 (mAFP) was developed by stable transfection of EL-4 cells with pmAFP. The frequency of cells produdng IFN-γ in splenocytes harvested from the immunized mice was measured by ELISPOT. Mice immunized with DNA vaccine were inoculated with EL-4 (mAFP) cells in back to observe the protective effect of immunization on tumor. On the other hand, blood samples were collected from the immunized mice to check the functions of liver and kidney.RESULTS: 1.8 kb mAFP cDNA was cloned from total RNA of Hepa1-6 cells by RT-PCR. The DNA vaccine encoding a fusion protein of mAFP-CTLA4 was constructed and confirmed by restriction enzyme analysis, sequencing and expression. The expression of mAFP mRNA in EL-4 (mAFP) was confirmed by RT-PCR. The ELISPOT results showed that the number of IFN-γ-producing cells in pmAFP-CTLA4 group was significantly higher than that in pmAFP, pcDNA3.1 and PBS group. The tumor volume in pmAFP-CTLA4 group was significantly smaller than that in pmAFP, pcDNA3.1 and PBS group, respectively. The hepatic and kidney functions in each group were not altered.CONCLUSION: AFP-CTLA4 DNA vaccine can stimulate potent specific CTL responses and has distinctive antitumor effect on AFP-producing tumor. The vaccine has no impact on the function of mouse liver and kidney.

  14. Fusion Pore Size Limits 5-HT Release From Single Enterochromaffin Cell Vesicles.

    Science.gov (United States)

    Raghupathi, Ravinarayan; Jessup, Claire F; Lumsden, Amanda L; Keating, Damien J

    2016-07-01

    Enterochromaffin cells are the major site of serotonin (5-HT) synthesis and secretion providing ∼95% of the body's total 5-HT. 5-HT can act as a neurotransmitter or hormone and has several important endocrine and paracrine roles. We have previously demonstrated that EC cells release small amounts of 5-HT per exocytosis event compared to other endocrine cells. We utilized a recently developed method to purify EC cells to demonstrate the mechanisms underlying 5-HT packaging and release. Using the fluorescent probe FFN511, we demonstrate that EC cells express VMAT and that VMAT plays a functional role in 5-HT loading into vesicles. Carbon fiber amperometry studies illustrate that the amount of 5-HT released per exocytosis event from EC cells is dependent on both VMAT and the H(+)-ATPase pump, as demonstrated with reserpine or bafilomycin, respectively. We also demonstrate that increasing the amount of 5-HT loaded into EC cell vesicles does not result in an increase in quantal release. As this indicates that fusion pore size may be a limiting factor involved, we compared pore diameter in EC and chromaffin cells by assessing the vesicle capture of different-sized fluorescent probes to measure the extent of fusion pore dilation. This identified that EC cells have a reduced fusion pore expansion that does not exceed 9 nm in diameter. These results demonstrate that the small amounts of 5-HT released per fusion event in EC cells can be explained by a smaller fusion pore that limits 5-HT release capacity from individual vesicles. PMID:26574734

  15. 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

  16. Model-independent determination of the astrophysical S-factor in laser-induced fusion plasmas

    CERN Document Server

    Lattuada, D; Bonasera, A; Bang, W; Quevedo, H J; Warren, M; Consoli, F; De Angelis, R; Andreoli, P; Kimura, S; Dyer, G; Bernstein, A C; Hagel, K; Barbui, M; Schmidt, K; Gaul, E; Donovan, M E; Natowitz, J B; Ditmire, T

    2016-01-01

    In this work, we present a new and general method for measuring the astrophysical S-factor of nuclear reactions in laser-induced plasmas and we apply it to d(d,n)$^{3}$He. The experiment was performed with the Texas Petawatt laser, which delivered 150-270 fs pulses of energy ranging from 90 to 180 J to D$_{2}$ or CD$_{4}$ molecular clusters. After removing the background noise, we used the measured time-of-flight data of energetic deuterium ions to obtain their energy distribution. We derive the S-factor using the measured energy distribution of the ions, the measured volume of the fusion plasma and the measured fusion yields. This method is model-independent in the sense that no assumption on the state of the system is required, but it requires an accurate measurement of the ion energy distribution especially at high energies and of the relevant fusion yields. In the d(d,n)$^{3}$He and $^{3}$He(d,p)$^{4}$He cases discussed here, it is very important to apply the background subtraction for the energetic ions ...

  17. Parameterization of fusion barriers for light-projectiles-induced reactions using the proximity approach

    Science.gov (United States)

    Gharaei, R.; Sheibani, J.

    2016-05-01

    In this article we propose a pocket formula for fusion barriers calculated by three versions of the proximity formalism, namely AW 95, Bass 80 and Prox. 2010 potentials, for fusion reactions involving the collisions of the proton and helium projectiles with different targets in mass ranges 51≤ AT ≤ 130 and 40≤ AT ≤ 233 , respectively. For the first type of the colliding systems, it is shown that the proposed pocket formulas are able to predict the actual values of RB and VB within accuracies of ±0.4% and ±0.45% , respectively. Moreover, for the second type of the selected reactions, these accuracies are obtained ±0.24% and ±0.36% , respectively. In this study, the ability of the present pocket formulas is also demonstrated to predict the exact values of the fusion cross sections for our selected mass ranges. A comparison with the results of the previous pocket formulas reveals that our parameterized forms are more successful to reproduce the empirical data of the barrier height and position in the proton- and helium-induced reactions.

  18. Parameterization of fusion barriers for light-projectiles-induced reactions using the proximity approach

    Energy Technology Data Exchange (ETDEWEB)

    Gharaei, R. [Hakim Sabzevari University, Department of Physics, Sciences Faculty, P. O. Box 397, Sabzevar (Iran, Islamic Republic of); Sheibani, J. [University of Birjand, Department of Physics, Ferdows Faculty of Engineering, Ferdows (Iran, Islamic Republic of)

    2016-05-15

    In this article we propose a pocket formula for fusion barriers calculated by three versions of the proximity formalism, namely AW 95, Bass 80 and Prox. 2010 potentials, for fusion reactions involving the collisions of the proton and helium projectiles with different targets in mass ranges 51≤ A{sub T}≤130 and 40≤A{sub T}≤233, respectively. For the first type of the colliding systems, it is shown that the proposed pocket formulas are able to predict the actual values of R{sub B} and V{sub B} within accuracies of ±0.4% and ±0.45%, respectively. Moreover, for the second type of the selected reactions, these accuracies are obtained ±0.24% and ±0.36%, respectively. In this study, the ability of the present pocket formulas is also demonstrated to predict the exact values of the fusion cross sections for our selected mass ranges. A comparison with the results of the previous pocket formulas reveals that our parameterized forms are more successful to reproduce the empirical data of the barrier height and position in the proton- and helium-induced reactions. (orig.)

  19. Diphtheria Toxin/Human B-Cell Activating Factor Fusion Protein Kills Human Acute Lymphoblastic Leukemia BALL-1 Cells: An Experimental Study

    Institute of Scientific and Technical Information of China (English)

    Xin-pu Gao; Zheng-min Liu; Yu-lian Jiao; Bin Cui; Yue-ting Zhu; Jie Zhang; Lai-cheng Wang; Yue-ran Zhao

    2012-01-01

    Objective:This study aimed to express a fusion protein of diphtheria toxin and human B ceil-activating factor (DT388sBAFF) in Escherichia coli (E.coli) and investigate its activity in human B-lineage acute lymphoblastic leukemia 1 cells (BALL-1).Methods:A fragment of DT388sBAFF fusion gene was separated from plasmid pUC57-DT388sBAFF digested with Nde Ⅰ and Xho Ⅰ,and inserted into the expression vector pcold Ⅱ digested with the same enzymes.Recombinants were screened by the colony polymerase chain reaction (PCR) and restriction map.The recombinant expression vector was transformed into BL21 and its expression was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG).The recombinant protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot,and then purified by Ni2+-NTA affinity chromatography.The expression level of B cell-activating factor receptor (BAFF-R) on BALL-1 cells was assessed by real-time PCR.The receptor binding capacity of recombinant protein was determined by cell fluorescent assay.The specific cytotoxicity of recombinant protein on BALL-1 cells was detected by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay.Results:The expression level of recombinant protein was 50% of total bacterial proteins in E.coli,and the recombinant protein could bind to BAFF-R-positive BALL-1 cells and thereby produce a cytotoxic effect on the cells.Conclusion:The fusion protein expression vector DT388sBAFF was successfully constructed and the recombinant protein with selective cytotoxicity against BALL-1 cells was obtained,providing foundation for further study of the therapy of human B-lineage acute lymphoblastic leukemia.

  20. The apoptotic response in HCT116BAX-/- cancer cells becomes rapidly saturated with increasing expression of a GFP-BAX fusion protein

    International Nuclear Information System (INIS)

    Many chemotherapeutic agents promote tumor cell death by activating the intrinsic pathway of apoptosis. Intrinsic apoptosis involves permeabilization of the mitochondrial outer membrane and the release of cytochrome c, a process that is controlled by proteins of the BCL2 gene family. Chemoresistance is often associated with abnormalities in concentrations of BCL2 family proteins. Although stoichiometirc interactions between anti-apoptotic and BH3-only BCL2 family proteins have been well documented as affecting cell death, the association between changes in BAX concentration and intrinsic apoptosis are poorly understood. Exogenous GFP-murine Bax fusion constructs were transfected into BAX-deficient HCT116 cells. To titrate the expression of the fusion protein, GFP-BAX was cloned into a tetracycline sensitive expression cassette and cotransfected with a plasmid expressing the rtTA transcription factor into HCT116BAX-/- cells. Linear expression of the fusion gene was induced with doxycycline and monitored by quantitative PCR and immunoblotting. Cell death was assayed by DAPI staining cells after exposure to indomethacin, and scoring nuclei for condensed chromatin and fragmented nuclei. HCT116BAX-/- cells were resistant to indomethacin, but susceptibility could be recovered in cells expressing a GFP-BAX fusion protein. Titration of GFP-BAX expression revealed that the concentration of BAX required to induce a saturating apoptosis response from baseline, was rapidly achieved. Increased levels of GFP-BAX were unable to stimulate higher levels of cell death. Examination of GFP-BAX distribution before and after indomethacin treatment indicated that BAX protein did not form aggregates when present at sub-lethal concentrations. Within the limitations of this experimental system, BAX-dependent apoptosis in HCT116 cells exhibits an all-or-none response depending on the level of BAX protein present. The lack of BAX aggregation at sub-saturation levels suggests that the

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

    Science.gov (United States)

    Klix, Axel; Fischer, Ulrich; Gehre, Daniel

    2016-02-01

    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. The Flocculating Cationic Polypetide from Moringa oleifera Seeds Damages Bacterial Cell Membranes by Causing Membrane Fusion.

    Science.gov (United States)

    Shebek, Kevin; Schantz, Allen B; Sines, Ian; Lauser, Kathleen; Velegol, Stephanie; Kumar, Manish

    2015-04-21

    A cationic protein isolated from the seeds of the Moringa oleifera tree has been extensively studied for use in water treatment in developing countries and has been proposed for use in antimicrobial and therapeutic applications. However, the molecular basis for the antimicrobial action of this peptide, Moringa oleifera cationic protein (MOCP), has not been previously elucidated. We demonstrate here that a dominant mechanism of MOCP antimicrobial activity is membrane fusion. We used a combination of cryogenic electron microscopy (cryo-EM) and fluorescence assays to observe and study the kinetics of fusion of membranes in liposomes representing model microbial cells. We also conducted cryo-EM experiments on E. coli cells where MOCP was seen to fuse the inner and outer membranes. Coarse-grained molecular dynamics simulations of membrane vesicles with MOCP molecules were used to elucidate steps in peptide adsorption, stalk formation, and fusion between membranes.

  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. Neutron Induced D Breakup in Inertial Confinement Fusion at the Omega Laser Facility

    Science.gov (United States)

    Forrest, C. J.; Glebov, V. Yu.; Knauer, J. P.; Radha, P. B.; Regan, S. P.; Sangster, T. C.; Stoeckl, C.; Schroder, W. U.; Frenje, J. A.; Gatu Johnson, M.

    2015-11-01

    High-resolution neutron spectroscopy is used to study the deuteron breakup reaction D(n,n ') np in the thermonuclear environment created in inertial confinement fusion experiments at the Omega Laser Facility. Neutrons with an energy of 14.1 MeV generated in the primary D-T fusion reactions scatter elastically and inelastically off the dense (cryogenic) D-T fuel assembly surrounding the central hot spot at peak fuel compression. These neutrons also induce a breakup of the fuel deuterons. The corresponding breakup cross section is measured relative to elastic n -D and n -T scattering, i.e., simultaneously in the same environment. Apart from astrophysical and technological interest, the neutron-induced deuteron breakup reaction is of interest to the physics of nucleon -nucleon forces. For example, theoretical calculations predict a noticeable influence of nucleonic three-body forces on the magnitude of the breakup cross section. Preliminary results from measurements of the neutron contribution in the 2- to 6-MeV range show reasonable agreement with the published ENDL 2008.2 semi-empirical cross-section. This material is based upon work supported by the Department of Energy National Nuclear Security Administration under Award Number DE-NA0001944.

  5. Application of remote-sensing-image fusion to the monitoring of mining induced subsidence

    Institute of Scientific and Technical Information of China (English)

    LI Liang-jun; WU Yan-bin

    2008-01-01

    We discuss remote-sensing-image fusion based on a multi-band wavelet and RGB feature fusion method. The fused data can be used to monitor the dynamic evolution of mining induced subsidence. High resolution panchromatic image data and multi-spectral image data were first decomposed with a multi-ary wavelet method. Then the high frequency components of the high resolution image were fused with the features from the R, G, B bands of the multi-spectral image to form a new high frequency component. Then the newly formed high frequency component and the low frequency component were inversely transformed using a multi-ary wavelet method. Finally, color images were formed from the newly formed R, G, B bands. In our experiment we used images with a resolution of 10 m (SPOT), and TM30 images, of the Huainan mining area. These images were fused with a trinary wavelet method. In addition, we used four indexes-entropy, average gradient, wavelet energy and spectral distortion-to assess the new method. The result indicates that this new method can improve the clarity and resolution of the images and also preserves the information from the original images. Using the fused images for monitoring mining induced subsidence achieves a good effect.

  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 syst

  7. Introduction of transformed chloroplasts from tobacco into petunia by asymmetric cell fusion.

    Science.gov (United States)

    Sigeno, Asako; Hayashi, Sugane; Terachi, Toru; Yamagishi, Hiroshi

    2009-11-01

    Plastid engineering technique has been established only in Nicotiana tabacum, and the widespread application is severely limited so far. In order to exploit a method to transfer the genetically transformed plastomes already obtained in tobacco into other plant species, somatic cell fusion was conducted between a plastome transformant of tobacco and a cultivar of petunia (Petunia hybrida). A tobacco strain whose plastids had been transformed with aadA (a streptomycin/spectinomycin adenylyltransferase gene) and mdar [a gene for monodehydroascorbate reductase (MDAR)] and a petunia variety, 'Telstar', were used as cell fusion partners. An efficient regeneration system from the protoplasts of both the parents, and effectiveness of selection for the aadA gene with spectinomycin were established before the cell fusion. In addition, the influence of UV irradiation on the callus development from the protoplasts and shoot regeneration of tobacco was investigated. Protoplasts were cultured after cell fusion treatment with polyethylene glycol, and asymmetric somatic cybrids were selected using the aadA gene as a marker. Although many shoots of tobacco that had escaped the UV irradiation regenerated, several shoots possessing the morphology of petunia and the resistance to spectinomycin were obtained. Molecular analyses of the petunia type regenerants demonstrated that they had the nuclear and mitochondrial genomes derived from petunia besides the chloroplasts of tobacco transformed with aadA and mdar. Furthermore, it was ascertained that mdar was transcribed in the somatic cybrids. The results indicate the success in intergeneric transfer of transformed plastids of tobacco into petunia. PMID:19727738

  8. Multicharged ion-induced emission from metal- and insulator surfaces related to magnetic fusion research

    Energy Technology Data Exchange (ETDEWEB)

    Winter, H.P. [Technische Univ., Vienna (Austria). Inst. fuer Allgemeine Physik

    1997-01-01

    The edge region of magnetically confined plasmas in thermonuclear fusion experiments couples the hot plasma core with the cold first wall. We consider the dependence of plasma-wall interaction processes on edge plasma properties, with particular emphasis on the role of slow multicharged ions (MCI). After a short survey on the physics of slow MCI-surface interaction we discuss recent extensive studies on MCI-induced electron emission from clean metal surfaces conducted at impact velocities << 1 a.u., from which generally reliable total electron yields can be obtained. We then demonstrate the essentially different role of the MCI charge for electron emission from metallic and insulator surfaces, respectively. Furthermore, we present recent results on slow MCI-induced `potential sputtering` of insulators which, in contrast to the well established kinetic sputtering, already occurs at very low ion impact energy and strongly increases with the MCI charge state. (J.P.N.). 55 refs.

  9. Measurement of DT fusion and neutron-induced gamma-rays using gas Cherenkov Detector

    Science.gov (United States)

    Kim, Y.; Herrmann, H. W.; Evans, S.; Sedillo, T.; Langenbrunner, J. R.; Young, C. S.; Mack, J. M.; McEvoy, A.; Horsfield, C. J.; Rubery, M.; Ali, Z.; Stoeffl, W.

    2010-08-01

    A secondary gamma experiment was carried out using a Gas Cherenkov Detector (GCD) at the OMEGA laser facility. The primary experimental objective was to simulate neutron-induced secondary gamma production (n-γ) from a NIF implosion capsule, hohlraum, and thermo-mechanical package. The high-band width of the GCD enabled us to detect time delayed and Doppler broadened n-γ signals from five different puck materials (Si, SiO2, Al, Al2O3, Cu) placed near target chamber center. These measurements were used for MCNP & ITS ACCEPT code validation purposes. By a simple change of the GCD CO2 gas pressure the system can effectively eliminate signals induced by n-γ reactions and thereby allow quality measurements of DT fusion γ-rays that are produced at NIF (National Ignition Facility).

  10. Chlorotoxin-Fc Fusion Inhibits Release of MMP-2 from Pancreatic Cancer Cells

    Directory of Open Access Journals (Sweden)

    Samah El-Ghlban

    2014-01-01

    Full Text Available Chlorotoxin (CTX is a 36-amino acid peptide derived from Leiurus quinquestriatus (scorpion venom, which inhibits low-conductance chloride channels in colonic epithelial cells. It has been reported that CTX also binds to matrix metalloproteinase-2 (MMP-2, membrane type-1 MMP, and tissue inhibitor of metalloproteinase-2, as well as CLC-3 chloride ion channels and other proteins. Pancreatic cancer cells require the activation of MMP-2 during invasion and migration. In this study, the fusion protein was generated by joining the CTX peptide to the amino terminus of the human IgG-Fc domain without a hinge domain, the monomeric form of chlorotoxin (M-CTX-Fc. The resulting fusion protein was then used to target pancreatic cancer cells (PANC-1 in vitro. M-CTX-Fc decreased MMP-2 release into the media of PANC-1 cells in a dose-dependent manner. M-CTX-Fc internalization into PANC-1 cells was observed. When the cells were treated with chlorpromazine (CPZ, the internalization of the fusion protein was reduced, implicating a clathrin-dependent internalization mechanism of M-CTX-Fc in PANC-1 cells. Furthermore, M-CTX-Fc clearly exhibited the inhibition of the migration depending on the concentration, but human IgG, as negative control of Fc, was not affected. The M-CTX-Fc may be an effective instrument for targeting pancreatic cancer.

  11. Fusion of bacterial spheroplasts by electric fields.

    Science.gov (United States)

    Ruthe, H J; Adler, J

    1985-09-25

    Spheroplasts of Escherichia coli or Salmonella typhimurium were found to fuse in an electric field. We employed the fusion method developed by Zimmermann and Scheurich (1981): Close membrane contact between cells is established by dielectrophoresis (formation of chains of cells by an a.c. field), then membrane fusion is induced by the application of short pulses of direct current. Under optimum conditions the fusion yield was routinely 90%. Fusable spheroplasts were obtained by first growing filamentous bacteria in the presence of cephalexin, then converting these to spheroplasts by the use of lysozyme. The fusion products were viable and regenerated to the regular bacterial form. Fusion of genetically different spheroplasts resulted in strains of bacteria possessing a combination of genetic markers. Fusion could not be achieved with spheroplasts obtained by growing the cells in the presence of penicillin or by using lysozyme on bacteria of usual size. PMID:3899175

  12. An experimental system for determining the influence of microgravity on B lymphocyte activation and cell fusion

    Science.gov (United States)

    Sammons, D. W.; Zimmermann, U.; Klinman, N. R.; Gessner, P.; Humphreys, R. C.; Emmons, S. P.; Neil, G. A.

    The influence of microgravity on lymphocyte activation is central to the understanding of immunological function in space. Moreover, the adaptation of groundbased technologies to microgravity conditions presents opportunities for biotechnological applications including high efficiency production of antibody forming hybridomas. Because the emerging technology of microgravity hybridoma generation is dependent upon activation and cultivation of B lymphocytes during flight, we have adapted mitogen-driven B lymphocyte stimulation and culture that allows for the in vitro generation of large numbers of antibody forming cells suitable for cell fusion over a period of 1-2 weeks. We believe that this activation and cultivation system can be flown on near-term space flights to test fundamental hypotheses about mammalian cell activation, cell fusion, metabolism, secretion, growth, and bio-separation.

  13. Humoral immune responses induced by anti-idiotypic antibody fusion protein of 6B11scFv/hGM-CSF in BALB/c mice

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Background We have previously developed and characterized a monoclonal anti-idiotype antibody, designated 6B11, which mimics an ovarian carcinoma associated antigen OC166-9 and whose corresponding monoclonal antibody is COC166-9 (Ab1). In this study, we evaluate the humoral immune responses induced by the fusion protein 6B11 single-chain variable fragment (scFv)/human granulocyte macrophage colony-stimulating factor (hGM-CSF) and 6B11scFv in BALB/c mice. Methods The fusion protein 6B11scFv/hGM-CSF was constructed by fusing a recombinant single-chain variable fragment of 6B11scFv to GM-CSF. BALB/c mice were administrated by 6B11scFv/hGM-CSF and 6B11scFv, respectively. Results The fusion protein 6B11scFv/hGM-CSF retained binding to the anti-mouse F(ab)2' and was also biologically active as measured by proliferation of human GM-CSF dependent cell TF1 in vitro. After immunization with the 6B11scFv/hGM-CSF and 6B11ScFv, BALB/c mice showed significantly enhanced Ab3 antibody responses to 6B11scFv/hGM-CSF compared with the 6B11scFv alone. The level of Ab3 was the highest after the first week and maintained for five weeks after the last immunization. Another booster was given when the Ab3 titer descended, and it would reach to the high level in a week. Conclusion The fusion protein 6B11scFv/hGM-CSF can induce humoral immunity against ovarian carcinoma in vivo. We also provide the theoretical foundation for the application of the fusion protein 6B11scFv/hGM-CSF for active immunotherapy of ovarian cancer.

  14. Cloning of murine BRI3 gene and study on its function for inducing cell death

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    To understand the molecular mechanism of TNFα effects, the cDNA of murine BRI3 gene was cloned from the total RNA of murine brain endothelial cells (bEnd.3)treated with hTNFα by using the suppression subtractive hybridization (SSH) and the RT-PCR method. The fusion expression vector harbouring BRI3 gene and enhanced green fluorescence protein (EGFP) thus obtained were designated as pEGFP/I3. Then pEGFP/I3 was transiently transfected into L929 cells and the fusion protein EGFP/I3 was localized in cytoplasm. It is found that the expression of EGFP/I3 could induce cell death in L929 cells detected by TUNEL method and flow cytometry. And the overexpression of Bci-2 in L929 cells can block cell death induced by EGFP/I3, indicating that murine BRI3 gene might related to the TNFα mediated cytotoxicity.

  15. A cell penetrating peptide-integrated and enediyne-energized fusion protein shows potent antitumor activity.

    Science.gov (United States)

    Ru, Qin; Shang, Bo-Yang; Miao, Qing-Fang; Li, Liang; Wu, Shu-Ying; Gao, Rui-Juan; Zhen, Yong-Su

    2012-11-20

    Arginine-rich peptides belong to a subclass of cell penetrating peptides that are taken up by living cells and can be detected freely diffusing inside the cytoplasm and nucleoplasm. This phenomenon has been attributed to either an endocytotic mode of uptake and a subsequent release from vesicles or a direct membrane penetration. Lidamycin is an antitumor antibiotic, which consists of an active enediyne chromophore (AE) and a noncovalently bound apoprotein (LDP). In the present study, a fusion protein (Arg)(9)-LDP composed of cell penetrating peptide (Arg)(9) and LDP was prepared by DNA recombination, and the enediyne-energized fusion protein (Arg)(9)-LDP-AE was prepared by molecular reconstitution. The data in fixed cells demonstrated that (Arg)(9)-LDP could rapidly enter cells, and the results based on fluorescence activated cell sorting indicated that the major route for (Arg)(9)-mediated cellular uptake of protein molecules was endocytosis. (Arg)(9)-LDP-AE demonstrated more potent cytotoxicity against different carcinoma cell lines than lidamycin in vitro. In the mouse hepatoma 22 model, (Arg)(9)-LDP-AE (0.3mg/kg) suppressed the tumor growth by 89.2%, whereas lidamycin (0.05 mg/kg) by 74.6%. Furthermore, in the glioma U87 xenograft model in nude mice, (Arg)(9)-LDP-AE at 0.2mg/kg suppressed tumor growth by 88.8%, compared with that of lidamycin by 62.9% at 0.05 mg/kg. No obvious toxic effects were observed in all groups during treatments. The results showed that energized fusion protein (Arg)(9)-LDP-AE was more effective than lidamycin and would be a promising candidate for glioma therapy. In addition, this approach to manufacturing fusion proteins might serve as a technology platform for the development of new cell penetrating peptides-based drugs. PMID:22982402

  16. Fusion pore expansion is a slow, discontinuous, and Ca2+ -dependent process regulating secretion from alveolar type II cells

    OpenAIRE

    Haller, Thomas; Dietl, Paul; Pfaller, Kristian; Frick, Manfred; Mair, Norbert; Paulmichl, Markus; Hess, Michael W.; Fürst, Johannes; Maly, Karl

    2001-01-01

    In alveolar type II cells, the release of surfactant is considerably delayed after the formation of exocytotic fusion pores, suggesting that content dispersal may be limited by fusion pore diameter and subject to regulation at a postfusion level. To address this issue, we used confocal FRAP and N-(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl) pyridinium dibromide (FM 1-43), a dye yielding intense localized fluorescence of surfactant when entering the vesicle lumen through the fusion po...

  17. Continuous Flow Separation of Hydrophobin Fusion Proteins from Plant Cell Culture Extract.

    Science.gov (United States)

    Reuter, Lauri J; Conley, Andrew J; Joensuu, Jussi J

    2016-01-01

    Fusion to fungal hydrophobins has proven to be a useful tool to enhance accumulation and recovery of recombinant proteins in plants. Aqueous two-phase separation (ATPS) is an attractive system to capture hydrophobin fusion proteins from plant extracts. The process can simultaneously purify and concentrate target protein with minimal background. ATPS avoids the use of chromatographic column steps, can be carried out in a short time frame, and is amenable to industrial-scale protein purification. A drawback of performing ATPS in large volumes is the lengthy time required for phase separation; however, this can be avoided by incorporating continuous systems, which are often preferred by the processing industry. This method chapter illustrates the capture of GFP-HFBI hydrophobin fusion protein from BY-2 plant cell suspension extract using a semi-continuous ATPS method. PMID:26614291

  18. 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.

  19. Critical factors in transitioning from fuel cell to cold fusion technology

    International Nuclear Information System (INIS)

    The fuel cell industry possesses much of the required manufacturing equipment and knowledge-base (e.g., proton conduction and hydrogen safety) necessary to develop cold fusion systems. Key factors in making a transition to cold fusion technology are discussed. Loading of reaction material can be provided by electrolytic charging and high gas over-pressure. Effective pressures over 10,000 atmospheres are required in cold fusion systems, giving a loading of H/M = 1; and a combination of loading methods is highly desirable. Systems must be designed to provide continuous flow of hydrogen ions (muchgt1017/sec for ten kilowatts), with an input power source of 50 watts (est.). Cold fusion experiments have shown that helium is formed during the reaction, and physical changes occur in the reaction material. These revelations impact design and operation of cold fusion systems, as the reaction material must be replaced periodically, while the systems must maintain integrity during operation. Safety and cost are also highly important considerations

  20. Enhanced binding and killing of target tumor cells by drug-loaded liposomes modified with tumor-specific phage fusion coat protein

    Science.gov (United States)

    Wang, Tao; D’Souza, Gerard GM; Bedi, Deepa; Fagbohun, Olusegun A; Potturi, L Prasanna; Papahadjopoulos-Sternberg, Brigitte; Petrenko, Valery A; Torchilin, Vladimir P

    2010-01-01

    Aim To explore cancer cell-specific phage fusion pVIII coat protein, identified using phage display, for targeted delivery of drug-loaded liposomes to MCF-7 breast cancer cells. Material & methods An 8-mer landscape library f8/8 and a biopanning protocol against MCF-7 cells were used to select a landscape phage protein bearing MCF-7-specific peptide. Size and morphology of doxorubicin-loaded liposomes modified with the tumor-specific phage fusion coat protein (phage–Doxil) were determined by dynamic light scattering and freeze-fraction electron microscopy. Topology of the phage protein in liposomes was examined by western blot. Association of phage–Doxil with MCF-7 cells was evaluated by fluorescence microscopy and fluorescence spectrometry. Selective targeting to MCF-7 was shown by FACS using a coculture model with target and nontarget cells. Phage–Doxil-induced tumor cell killing and apoptosis were confirmed by CellTiter-Blue® Assay and caspase-3/CPP32 fluorometric assay. Results A chimeric phage fusion coat protein specific towards MCF-7 cells, identified from a phage landscape library, was directly incorporated into the liposomal bilayer of doxorubicin-loaded PEGylated liposomes (Doxil®) without additional conjugation with lipophilic moieties. Western blotting confirmed the presence of both targeting peptide and pVIII coat protein in the phage–Doxil, which maintained the liposomal morphology and retained a substantial part of the incorporated drug after phage protein incorporation. The binding activity of the phage fusion pVIII coat protein was retained after incorporation into liposomes, and phage–Doxil strongly and specifically targeted MCF-7 cells, demonstrating significantly increased cytotoxicity towards target cells in vitro. Conclusion We present a novel and straightforward method for making tumor-targeted nanomedicines by anchoring specific phage proteins (substitute antibodies) on their surface. PMID:20528452

  1. 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.

  2. Events leading to the opening and closing of the exocytotic fusion pore have markedly different temperature dependencies. Kinetic analysis of single fusion events in patch-clamped mouse mast cells.

    OpenAIRE

    Oberhauser, A F; Monck, J R; Fernandez, J.M.

    1992-01-01

    The earliest event in exocytosis is the formation of a fusion pore, an aqueous channel that connects the lumen of a secretory granule with the extracellular space. We can observe the formation of individual fusion pores and their subsequent dilation or closure by measuring the changes in the admittance of patch-clamped mast cells during GTP gamma S-stimulated exocytotic fusion. To investigate the molecular structure of the fusion pore, we have studied the temperature dependency of the rate co...

  3. DNA Triplex-Based Complexes Display Anti-HIV-1-Cell Fusion Activity.

    Science.gov (United States)

    Xu, Liang; Zhang, Tao; Xu, Xiaoyu; Chong, Huihui; Lai, Wenqing; Jiang, Xifeng; Wang, Chao; He, Yuxian; Liu, Keliang

    2015-08-01

    DNA triplexes with hydrophobic modifications were designed and evaluated for their activity as inhibitors of the cell fusion of human immunodeficiency virus type 1 (HIV-1). Triplex inhibitors displayed low micromolar activities in the cell-cell fusion assay and nanomolar activities in the anti-HIV-1 pseudovirus test. Helix structure and the presence of sufficient numbers of hydrophobic regions were essential for the antifusion activity. Results from native polyacrylamide gel electrophoresis and a fluorescent resonance energy transfer-based inhibitory assay indicated that these triplexes may interact with the primary pocket at the glycoprotein 41 (gp41) N-heptad repeat, thereby inhibiting formation of the HIV-1 gp41 6-helical bundle. Triplex-based complexes may represent a novel category of HIV-1 inhibitors in anti-HIV-1 drug discovery. PMID:26192705

  4. Enhanced protein expression in the baculovirus/insect cell system using engineered SUMO fusions.

    Science.gov (United States)

    Liu, Li; Spurrier, Joshua; Butt, Tauseef R; Strickler, James E

    2008-11-01

    Recombinant protein expression in insect cells varies greatly from protein to protein. A fusion tag that is not only a tool for detection and purification, but also enhances expression and/or solubility would greatly facilitate both structure/function studies and therapeutic protein production. We have shown that fusion of SUMO (small ubiquitin-related modifier) to several test proteins leads to enhanced expression levels in Escherichia coli. In eukaryotic expression systems, however, the SUMO tag could be cleaved by endogenous desumoylase. In order to adapt SUMO-fusion technology to these systems, we have developed an alternative SUMO-derived tag, designated SUMOstar, which is not processed by native SUMO proteases. In the present study, we tested the SUMOstar tag in a baculovirus/insect cell system with several proteins, i.e. mouse UBP43, human tryptase beta II, USP4, USP15, and GFP. Our results demonstrate that fusion to SUMOstar enhanced protein expression levels at least 4-fold compared to either the native or His(6)-tagged proteins. We isolated active SUMOstar tagged UBP43, USP4, USP15, and GFP. Tryptase was active following cleavage with a SUMOstar specific protease. The SUMOstar system will make significant impact in difficult-to-express proteins and especially to those proteins that require the native N-terminal residue for function.

  5. Anticancer Effects of Fusion Protein CAtin on DMBA-induced Carcinogenesis in Buccal Pouch of Chinese Hamster

    Institute of Scientific and Technical Information of China (English)

    BAI Jie-ying; LI Xiao; LI Chang; ZHANG Xiao-fei; LI Zhi-xin; ZHAO Shuang; LIU Xiao; ZENG Lin; CHI Bao-rong

    2012-01-01

    Aberrant expression ofcarcinoembryonic antigen(CEA)is a common feature for multiple types of cancer,which makes it an attractive target for anticancer therapy.CAtin is a novel dual cancer-specific fusion protein,composed of an anti-CEA single-chain disulfide-stabilized Fv antibody(scdsFv)and Apoptin,a tumor-specific apoptosis-inducing protein.Oral squamous cell carcinoma(OSCC)is an important healthcare problem in the clinic.To evaluate the anticancer effects of CAtin on OSCC,7,12-dimethylbenz[a]anthracene(DMBA)was used to induce oral carcinogenesis and premalignant lesions in the buccal pouch of Chinese hamster,and the antitumor effects of CAtin were determined in pre-cancer,cancer and post-operatative cancer models,respectively.The results show that the administration of CAtin delayed the malignant transformation of early stage cancerous lesions,inhibited the growth of established solid oral tumors and reduced the post-operatative relapse of lesions,with no significant systemic toxicity.This study demonstrates that CAtin may have potential for the treatment of OSCC,and the development of preventive strategies based on CAtin may offer a practical approach for the treatment of human oral tumors.

  6. Melanoma-Derived BRAFV600E Mutation in Peritumoral Stromal Cells: Implications for in Vivo Cell Fusion

    Science.gov (United States)

    Kurgyis, Zsuzsanna; Kemény, Lajos V.; Buknicz, Tünde; Groma, Gergely; Oláh, Judit; Jakab, Ádám; Polyánka, Hilda; Zänker, Kurt; Dittmar, Thomas; Kemény, Lajos; Németh, István B.

    2016-01-01

    Melanoma often recurs in patients after the removal of the primary tumor, suggesting the presence of recurrent tumor-initiating cells that are undetectable using standard diagnostic methods. As cell fusion has been implicated to facilitate the alteration of a cell’s phenotype, we hypothesized that cells in the peritumoral stroma having a stromal phenotype that initiate recurrent tumors might originate from the fusion of tumor and stromal cells. Here, we show that in patients with BRAFV600E melanoma, melanoma antigen recognized by T-cells (MART1)-negative peritumoral stromal cells express BRAFV600E protein. To confirm the presence of the oncogene at the genetic level, peritumoral stromal cells were microdissected and screened for the presence of BRAFV600E with a mutation-specific polymerase chain reaction. Interestingly, cells carrying the BRAFV600E mutation were not only found among cells surrounding the primary tumor but were also present in the stroma of melanoma metastases as well as in a histologically tumor-free re-excision sample from a patient who subsequently developed a local recurrence. We did not detect any BRAFV600E mutation or protein in the peritumoral stroma of BRAFWT melanoma. Therefore, our results suggest that peritumoral stromal cells contain melanoma-derived oncogenic information, potentially as a result of cell fusion. These hybrid cells display the phenotype of stromal cells and are therefore undetectable using routine histological assessments. Our results highlight the importance of genetic analyses and the application of mutation-specific antibodies in the identification of potentially recurrent-tumor-initiating cells, which may help better predict patient survival and disease outcome. PMID:27338362

  7. Melanoma-Derived BRAFV600E Mutation in Peritumoral Stromal Cells: Implications for in Vivo Cell Fusion

    Directory of Open Access Journals (Sweden)

    Zsuzsanna Kurgyis

    2016-06-01

    Full Text Available Melanoma often recurs in patients after the removal of the primary tumor, suggesting the presence of recurrent tumor-initiating cells that are undetectable using standard diagnostic methods. As cell fusion has been implicated to facilitate the alteration of a cell’s phenotype, we hypothesized that cells in the peritumoral stroma having a stromal phenotype that initiate recurrent tumors might originate from the fusion of tumor and stromal cells. Here, we show that in patients with BRAFV600E melanoma, melanoma antigen recognized by T-cells (MART1-negative peritumoral stromal cells express BRAFV600E protein. To confirm the presence of the oncogene at the genetic level, peritumoral stromal cells were microdissected and screened for the presence of BRAFV600E with a mutation-specific polymerase chain reaction. Interestingly, cells carrying the BRAFV600E mutation were not only found among cells surrounding the primary tumor but were also present in the stroma of melanoma metastases as well as in a histologically tumor-free re-excision sample from a patient who subsequently developed a local recurrence. We did not detect any BRAFV600E mutation or protein in the peritumoral stroma of BRAFWT melanoma. Therefore, our results suggest that peritumoral stromal cells contain melanoma-derived oncogenic information, potentially as a result of cell fusion. These hybrid cells display the phenotype of stromal cells and are therefore undetectable using routine histological assessments. Our results highlight the importance of genetic analyses and the application of mutation-specific antibodies in the identification of potentially recurrent-tumor-initiating cells, which may help better predict patient survival and disease outcome.

  8. Residue cross sections of 50Ti-induced fusion reactions based on the two-step model

    International Nuclear Information System (INIS)

    50Ti-induced fusion reactions to synthesize superheavy elements are studied systematically with the two-step model developed recently, where fusion process is divided into approaching phase and formation phase. Furthermore, the residue cross sections for different neutron evaporation channels are evaluated with the statistical evaporation model. In general, the calculated cross sections are much smaller than that of 48Ca-induced fusion reactions, but the results are within the detection capability of experimental facilities nowadays. The maximum calculated residue cross section for producing superheavy element Z=119 is in the reaction 50Ti + 247Bk in 3n channels with σres(3n) = 0.043 pb at E* = 37.0 MeV. (orig.)

  9. Residue cross sections of $^{50}$Ti-induced fusion reactions based on the two-step model

    CERN Document Server

    Liu, Ling; Li, Qingfeng; Tu, Ya; Wang, Xiaobao; Wang, Yongjia

    2015-01-01

    $^{50}$Ti-induced fusion reactions to synthesize superheavy elements are studied systematically with the two-step model developed recently, where fusion process is divided into approaching phase and formation phase. Furthermore, the residue cross sections for different neutron evaporation channels are evaluated with the statistical evaporation model. In general, the calculated cross sections are much smaller than that of $^{48}$Ca-induced fusion reactions, but the results are within the detection capability of experimental facilities nowadays. The maximum calculated residue cross section for producing superheavy element $Z=119$ is in the reaction $^{50}$Ti+$^{247}$Bk in $3n$ channels with $\\sigma_{\\rm res}(3n)=0.043$ pb at $E^{*}$ = 37.0 MeV.

  10. Residue cross sections of {sup 50}Ti-induced fusion reactions based on the two-step model

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Ling; Tu, Ya [Shenyang Normal University, College of Physics Science and Technology, Liaoning (China); Shen, Caiwan; Li, Qingfeng; Wang, Xiaobao; Wang, Yongjia [Huzhou University, School of Science, Zhejiang (China)

    2016-02-15

    {sup 50}Ti-induced fusion reactions to synthesize superheavy elements are studied systematically with the two-step model developed recently, where fusion process is divided into approaching phase and formation phase. Furthermore, the residue cross sections for different neutron evaporation channels are evaluated with the statistical evaporation model. In general, the calculated cross sections are much smaller than that of {sup 48}Ca-induced fusion reactions, but the results are within the detection capability of experimental facilities nowadays. The maximum calculated residue cross section for producing superheavy element Z=119 is in the reaction {sup 50}Ti + {sup 247}Bk in 3n channels with σ{sub res}(3n) = 0.043 pb at E* = 37.0 MeV. (orig.)

  11. Thermal photon production from gluon fusion induced by magnetic fields in relativistic heavy-ion collisions

    CERN Document Server

    Ayala, Alejandro; Dominguez, C A; Hernandez, L A

    2016-01-01

    We compute the production of thermal photons in relativistic heavy-ion collisions by gluon fusion in the presence of an intense magnetic field, and during the early stages of the reaction. This photon yield is an excess over calculations that do not consider magnetic field effects. We add this excess to recent hydrodynamic calculations that are close to describing the experimental transverse momentum distribution in RHIC and LHC. We then show that with reasonable values for the temperature, magnetic field strength, and strong coupling constant, our results provide a very good description of such excess. These results support the idea that the origin of at least some of the photon excess observed in heavy-ion experiments may arise from magnetic field induced processes.

  12. 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

  13. Polarized exocyst-mediated vesicle fusion directs intracellular lumenogenesis within the C. elegans excretory cell.

    Science.gov (United States)

    Armenti, Stephen T; Chan, Emily; Nance, Jeremy

    2014-10-01

    Lumenogenesis of small seamless tubes occurs through intracellular membrane growth and directed vesicle fusion events. Within the Caenorhabditis elegans excretory cell, which forms seamless intracellular tubes (canals) that mediate osmoregulation, lumens grow in length and diameter when vesicles fuse with the expanding lumenal surface. Here, we show that lumenal vesicle fusion depends on the small GTPase RAL-1, which localizes to vesicles and acts through the exocyst vesicle-tethering complex. Loss of either the exocyst or RAL-1 prevents excretory canal lumen extension. Within the excretory canal and other polarized cells, the exocyst co-localizes with the PAR polarity proteins PAR-3, PAR-6 and PKC-3. Using early embryonic cells to determine the functional relationships between the exocyst and PAR proteins, we show that RAL-1 recruits the exocyst to the membrane, while PAR proteins concentrate membrane-localized exocyst proteins to a polarized domain. These findings reveal that RAL-1 and the exocyst direct the polarized vesicle fusion events required for intracellular lumenogenesis of the excretory cell, suggesting mechanistic similarities in the formation of topologically distinct multicellular and intracellular lumens. PMID:25102190

  14. Increased chemosensitivity of paclitaxel by telomeric fusion-induced genomic instability

    Energy Technology Data Exchange (ETDEWEB)

    Woo, Seon Rang; Juhn, Kyoung Mi; Park, Jeong Eun; Ju, Yeun Jin; Yun, Mi Yong; Lee, Kee Ho [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Park, Gil Hong; Kim, Joon [College of Medicine, Korea University, Seoul (Korea, Republic of)

    2009-05-15

    A telomere is a region of repetitive DNA at the end of chromosomes. They protect a cell's chromosomes from fusing with each other or rearranging and so cells are normally destroyed when their telomeres are consumed. Most normal somatic cells lose telomeric repeats after each cell division. Telomeric shortening in humans can induce replicative senescence which blocks cell division. This mechanism appears to prevent genomic instability by limiting the number of cell divisions. Telomerase is an attractive molecular target, since its activity has been found in more than 85% of human cancers. Combination therapy with chemotherapeutic agent is superior to single in overall response rate and progression free survival. In this study, we showed that telomerase null cells are more hypersensitive by paclitaxel treatment than at wild type cells.

  15. Fusion protein Isl1-Lhx3 specifies motor neuron fate by inducing motor neuron genes and concomitantly suppressing the interneuron programs.

    Science.gov (United States)

    Lee, Seunghee; Cuvillier, James M; Lee, Bora; Shen, Rongkun; Lee, Jae W; Lee, Soo-Kyung

    2012-02-28

    Combinatorial transcription codes generate the myriad of cell types during development and thus likely provide crucial insights into directed differentiation of stem cells to a specific cell type. The LIM complex composed of Isl1 and Lhx3 directs the specification of spinal motor neurons (MNs) in embryos. Here, we report that Isl1-Lhx3, a LIM-complex mimicking fusion, induces a signature of MN transcriptome and concomitantly suppresses interneuron differentiation programs, thereby serving as a potent and specific inducer of MNs in stem cells. We show that an equimolar ratio of Isl1 and Lhx3 and the LIM domain of Lhx3 are crucial for generating MNs without up-regulating interneuron genes. These led us to design Isl1-Lhx3, which maintains the desirable 1:1 ratio of Isl1 and Lhx3 and the LIM domain of Lhx3. Isl1-Lhx3 drives MN differentiation with high specificity and efficiency in the spinal cord and embryonic stem cells, bypassing the need for sonic hedgehog (Shh). RNA-seq analysis revealed that Isl1-Lhx3 induces the expression of a battery of MN genes that control various functional aspects of MNs, while suppressing key interneuron genes. Our studies uncover a highly efficient method for directed MN generation and MN gene networks. Our results also demonstrate a general strategy of using embryonic transcription complexes for producing specific cell types from stem cells. PMID:22343290

  16. CA2+-INDUCED FUSION OF PHOSPHOLIPID-VESICLES CONTAINING FREE FATTY-ACIDS - MODULATION BY TRANSMEMBRANE PH GRADIENTS

    NARCIS (Netherlands)

    WILSCHUT, J; SCHOLMA, J; EASTMAN, SJ; HOPE, MJ; CULLIS, PR

    1992-01-01

    The influence of a transmembrane pH gradient on the Ca2+-induced fusion of phospholipid vesicles, containing free fatty acids, has been investigated. Large unilamellar vesicles composed of an equimolar mixture of cardiolipin, dioleoylphosphatidylcholine, and cholesterol, containing 20 mol % oleic ac

  17. Vicenistatin induces early endosome-derived vacuole formation in mammalian cells.

    Science.gov (United States)

    Nishiyama, Yuko; Ohmichi, Tomohiro; Kazami, Sayaka; Iwasaki, Hiroki; Mano, Kousuke; Nagumo, Yoko; Kudo, Fumitaka; Ichikawa, Sosaku; Iwabuchi, Yoshiharu; Kanoh, Naoki; Eguchi, Tadashi; Osada, Hiroyuki; Usui, Takeo

    2016-05-01

    Homotypic fusion of early endosomes is important for efficient protein trafficking and sorting. The key controller of this process is Rab5 which regulates several effectors and PtdInsPs levels, but whose mechanisms are largely unknown. Here, we report that vicenistatin, a natural product, enhanced homotypic fusion of early endosomes and induced the formation of large vacuole-like structures in mammalian cells. Unlike YM201636, another early endosome vacuolating compound, vicenistatin did not inhibit PIKfyve activity in vitro but activated Rab5-PAS pathway in cells. Furthermore, vicenistatin increased the membrane surface fluidity of cholesterol-containing liposomes in vitro, and cholesterol deprivation from the plasma membrane stimulated vicenistatin-induced vacuolation in cells. These results suggest that vicenistatin is a novel compound that induces the formation of vacuole-like structures by activating Rab5-PAS pathway and increasing membrane fluidity. PMID:27104762

  18. Integrated cell and process engineering for improved transient production of a "difficult-to-express" fusion protein by CHO cells.

    Science.gov (United States)

    Johari, Yusuf B; Estes, Scott D; Alves, Christina S; Sinacore, Marty S; James, David C

    2015-12-01

    Based on an optimized electroporation protocol, we designed a rapid, milliliter-scale diagnostic transient production assay to identify limitations in the ability of Chinese hamster ovary (CHO) cells to produce a model "difficult-to-express" homodimeric Fc-fusion protein, Sp35Fc, that exhibited very low volumetric titer and intracellular formation of disulfide-bonded oligomeric aggregates post-transfection. As expression of Sp35Fc induced an unfolded protein response in transfected host cells, we utilized the transient assay to compare, in parallel, multiple functionally diverse strategies to engineer intracellular processing of Sp35Fc in order to increase production and reduce aggregation as two discrete design objectives. Specifically, we compared the effect of (i) co-expression of ER-resident molecular chaperones (BiP, PDI, CypB) or active forms of UPR transactivators (ATF6c, XBP1s) at varying recombinant gene load, (ii) addition of small molecules known to act as chemical chaperones (PBA, DMSO, glycerol, betaine, TMAO) or modulate UPR signaling (PERK inhibitor GSK2606414) at varying concentration, (iii) a reduction in culture temperature to 32°C. Using this information, we designed a biphasic, Sp35Fc-specific transient manufacturing process mediated by lipofection that utilized CypB co-expression at an optimal Sp35Fc:CypB gene ratio of 5:1 to initially maximize transfected cell proliferation, followed by addition of a combination of PBA (0.5 mM) and glycerol (1% v/v) at the onset of stationary phase to maximize cell specific production and eliminate Sp35Fc aggregation. Using this optimal, engineered process transient Sp35Fc production was significantly increased sixfold over a 12 day production process with no evidence of disulfide-bonded aggregates. Finally, transient production in clonally derived sub-populations (derived from parental CHO host) screened for a heritably improved capability to produce Sp35Fc was also significantly improved by the optimized

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

    Directory of Open Access Journals (Sweden)

    Janine Kimpel

    Full Text Available 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.

  20. 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...... 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...... light as heat to their surroundings. The strong and localized generated heating is sufficient for local melting and/or expansion of lipids within the two adjoining membranes. Since exposure of the hydrophobic bilayer core to the aqueous medium is energetically unfavorable, immediate rearrangements...

  1. Regulated vesicle fusion generates signaling nanoterritories that control T cell activation at the immunological synapse.

    Science.gov (United States)

    Soares, Helena; Henriques, Ricardo; Sachse, Martin; Ventimiglia, Leandro; Alonso, Miguel A; Zimmer, Christophe; Thoulouze, Maria-Isabel; Alcover, Andrés

    2013-10-21

    How the vesicular traffic of signaling molecules contributes to T cell receptor (TCR) signal transduction at the immunological synapse remains poorly understood. In this study, we show that the protein tyrosine kinase Lck, the TCRζ subunit, and the adapter LAT traffic through distinct exocytic compartments, which are released at the immunological synapse in a differentially regulated manner. Lck vesicular release depends on MAL protein. Synaptic Lck, in turn, conditions the calcium- and synaptotagmin-7-dependent fusion of LAT and TCRζ containing vesicles. Fusion of vesicles containing TCRζ and LAT at the synaptic membrane determines not only the nanoscale organization of phosphorylated TCRζ, ZAP70, LAT, and SLP76 clusters but also the presence of phosphorylated LAT and SLP76 in interacting signaling nanoterritories. This mechanism is required for priming IL-2 and IFN-γ production and may contribute to fine-tuning T cell activation breadth in response to different stimulatory conditions.

  2. KAR5 Encodes a Novel Pheromone-inducible Protein Required for Homotypic Nuclear Fusion

    OpenAIRE

    Beh, Christopher T.; Brizzio, Valeria; Rose, Mark D.

    1997-01-01

    KAR5 is required for membrane fusion during karyogamy, the process of nuclear fusion during yeast mating. To investigate the molecular mechanism of nuclear fusion, we cloned and characterized the KAR5 gene and its product. KAR5 is a nonessential gene, and deletion mutations produce a bilateral defect in the homotypic fusion of yeast nuclei. KAR5 encodes a novel protein that shares similarity with a protein in Schizosaccharomyces pombe that may play a similar role in nuclear fusion. Kar5p is i...

  3. 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.

  4. 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. PMID:27515077

  5. Detection of EMI4-ALK fusion gene in non-small cell lung cancer and its clinicopathologic correlation

    Institute of Scientific and Technical Information of China (English)

    钟山

    2013-01-01

    Objective To investigate the frequency of EML4-ALK fusion gene in non small-cell lung cancer NSCLC patients,and its correlation with clinicopathologic features.Methods Real-time PCR was used to detect

  6. 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%.

  7. 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.

  8. 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.

  9. Diamond photovoltaic cells as a first-wall material and energy conversion system for inertial confinement fusion

    International Nuclear Information System (INIS)

    Diamond film technology has advanced to the point where electronic devices are now becoming feasible. In addition, diamond has outstanding mechanical properties. The energy given off in fusion reactions may be converted to a narrow-band light spectrum that can be absorbed by wide-bandgap photovoltaic cells to directly produce electricity. The properties of possible wide-bandgap photovoltaic cells are examined for the purpose of fusion energy conversion. (author)

  10. Tributyltin induces mitochondrial fission through Mfn1 degradation in human induced pluripotent stem cells.

    Science.gov (United States)

    Yamada, Shigeru; Asanagi, Miki; Hirata, Naoya; Itagaki, Hiroshi; Sekino, Yuko; Kanda, Yasunari

    2016-08-01

    Organotin compounds, such as tributyltin (TBT), are well-known endocrine disruptors. TBT is also known to cause various forms of cytotoxicity, including neurotoxicity and immunotoxicity. However, TBT toxicity has not been identified in normal stem cells. In the present study, we examined the effects of TBT on cell growth in human induced pluripotent stem cells (iPSCs). We found that exposure to nanomolar concentrations of TBT decreased intracellular ATP levels and inhibited cell viability in iPSCs. Because TBT suppressed energy production, which is a critical function of the mitochondria, we further assessed the effects of TBT on mitochondrial dynamics. Staining with MitoTracker revealed that nanomolar concentrations of TBT induced mitochondrial fragmentation. TBT also reduced the expression of mitochondrial fusion protein mitofusin 1 (Mfn1), and this effect was abolished by knockdown of the E3 ubiquitin ligase membrane-associated RING-CH 5 (MARCH5), suggesting that nanomolar concentrations of TBT could induce mitochondrial dysfunction via MARCH5-mediated Mfn1 degradation in iPSCs. Thus, mitochondrial function in normal stem cells could be used to assess cytotoxicity associated with metal exposure. PMID:27133438

  11. 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.

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

    Directory of Open Access Journals (Sweden)

    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

  13. Compactness of the 48Ca induced hot fusion reactions and the magnitudes of quadrupole and hexadecapole deformations

    International Nuclear Information System (INIS)

    Based on fragmentation theory extended to include the orientation degrees of freedom and higher multipole deformations up to hexadecapole deformations, the compactness of 48Ca induced reactions on various actinides is studied for Ds (Z=110) to 118 nuclei. It is shown that the reactions leading to Z≥114 nuclei are 'compact' hot fusion reactions at θ=90 deg. orientation angles (equatorial compact or ec; collisions that are in the direction of the minor axis of the deformed reaction partner), but the ones for Z48Ca beam and to Pb based reactions. Furthermore, for compact hot fusion reactions, in addition to the 48Ca reaction valley, a number of other new reaction valleys (target-projectile combinations) are obtained, the most important one (next to 48Ca) being the 54Ti nucleus used previously in Pb based cold fusion reaction studies but now proposed with deformed actinide nuclei such as 226Ra, 232Th, 238U, and 242Pu

  14. 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.

  15. Small Mismatches in Fatty Acyl Tail Lengths Can Effect Non Steroidal Anti-Inflammatory Drug Induced Membrane Fusion.

    Science.gov (United States)

    Majumdar, Anupa; Sarkar, Munna

    2016-06-01

    Biological membranes are made up of a variety of lipids with diverse physicochemical properties. The lipid composition modulates different lipidic parameters, such as hydration, dynamics, lipid packing, curvature strain, etc. Changes in these parameters affect various membrane-mediated processes, such as membrane fusion which is an integral step in many biological processes. Packing defects, which originate either from mismatch in the headgroup region or in the hydrophobic acyl tail region, play a major role in modulating membrane dynamics. In this study, we demonstrate how even a small mismatch in the fatty acyl chain length, achieved by incorporation of low concentrations (up to 30 mol %) of dipalmitoylphosphatidylcholine (DPPC) into dimyristoylphosphatidylcholine (DMPC) small unilamellar vesicles (SUVs), alters several lipidic parameters like packing, dynamics, and headgroup hydration. This in turn affects non steroidal anti-inflammatory drug (NSAID) induced membrane fusion. Dynamic light scattering, differential scanning calorimetry, second-derivative absorption spectrophotometry, and steady-state and time-resolved fluorescence have been used to elucidate the effect of small mismatch in the tails in DMPC/DPPC mixed vesicles and how it modulates membrane fusion induced by the oxicam NSAIDs, meloxicam (Mx), piroxicam (Px), and tenoxicam (Tx). Fusion kinetics was monitored using fluorescence based fusion assays. At low DPPC concentration of 10 mol %, additional fluidization promotes lipid mixing to some extent for Mx, but at higher mol % of DPPC, subsequent increase in rigidity of membrane interior along with increase in headgroup hydration, synergistically inhibits fusion to various extents for the three different drugs, Mx, Px, and Tx. PMID:27153337

  16. 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.

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

    International Nuclear Information System (INIS)

    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. A lab-on-a-chip device for investigating the fusion process of olfactory ensheathing cell spheroids.

    Science.gov (United States)

    Munaz, Ahmed; Vadivelu, Raja K; John, James A St; Nguyen, Nam-Trung

    2016-08-01

    Understanding the process of fusion of olfactory ensheathing cell spheroids will lead to improvement of cell transplantation therapies to repair spinal cord injuries. The successful fusion of transplanted spheroids will enable alternative transplantation strategies to be developed for in vivo applications. This paper describes the use of a microfluidic device to trap and fuse olfactory ensheathing cell spheroids. The velocity, the pressure distribution in the device were simulated numerically to predict the trapping location. The simulation predicted the optimum flow rates for trapping the spheroids in the later experiments. Simulated particle trajectories were verified experimentally with tracing of fluorescent micro particles. The fusion process of the spheroids was investigated over a period of 48 hours. The microfluidic platform presented here can be used for testing potential drugs that can promote the fusion process and improve the transplantation therapy. PMID:27387270

  19. Fusion pore expansion is a slow, discontinuous, and Ca2+-dependent process regulating secretion from alveolar type II cells.

    Science.gov (United States)

    Haller, T; Dietl, P; Pfaller, K; Frick, M; Mair, N; Paulmichl, M; Hess, M W; Furst, J; Maly, K

    2001-10-15

    In alveolar type II cells, the release of surfactant is considerably delayed after the formation of exocytotic fusion pores, suggesting that content dispersal may be limited by fusion pore diameter and subject to regulation at a postfusion level. To address this issue, we used confocal FRAP and N-(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl) pyridinium dibromide (FM 1-43), a dye yielding intense localized fluorescence of surfactant when entering the vesicle lumen through the fusion pore (Haller, T., J. Ortmayr, F. Friedrich, H. Volkl, and P. Dietl. 1998. Proc. Natl. Acad. Sci. USA. 95:1579-1584). Thus, we have been able to monitor the dynamics of individual fusion pores up to hours in intact cells, and to calculate pore diameters using a diffusion model derived from Fick's law. After formation, fusion pores were arrested in a state impeding the release of vesicle contents, and expanded at irregular times thereafter. The expansion rate of initial pores and the probability of late expansions were increased by elevation of the cytoplasmic Ca2+ concentration. Consistently, content release correlated with the occurrence of Ca2+ oscillations in ATP-treated cells, and expanded fusion pores were detectable by EM. This study supports a new concept in exocytosis, implicating fusion pores in the regulation of content release for extended periods after initial formation. PMID:11604423

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

    International Nuclear Information System (INIS)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Boschi, Federico, E-mail: federico.boschi@univr.it [Department of Neurological and Movement Sciences, University of Verona, Strada Le Grazie 8, 37134 Verona (Italy); Department of Computer Science, University of Verona, Strada Le Grazie 15, 37134 Verona (Italy); Rizzatti, Vanni; Zamboni, Mauro [Department of Medicine, Geriatric Section, University of Verona, Piazzale Stefani 1, 37126 Verona (Italy); Sbarbati, Andrea [Department of Neurological and Movement Sciences, University of Verona, Strada Le Grazie 8, 37134 Verona (Italy)

    2014-02-15

    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

  2. Mutations in the Amino Terminus of Herpes Simplex Virus Type 1 gL Can Reduce Cell-Cell Fusion without Affecting gH/gL Trafficking

    OpenAIRE

    Zhou, Wenbo; Chen, Feng; Klyachkin, Yuri; Sham, Yuk Y.; Geraghty, Robert J.

    2014-01-01

    The gH/gL heterodimer represents two of the four herpes simplex virus glycoproteins necessary and sufficient for membrane fusion. We generated deletions and point mutations covering gL residues 24 to 43 to investigate that region's role in gH/gL intracellular trafficking and in membrane fusion. Multiple mutants displayed a 40 to 60% reduction in cell fusion with no effect on gH/gL trafficking. The amino terminus of gL plays an important role in the gH/gL contribution to membrane fusion.

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

    Directory of Open Access Journals (Sweden)

    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.

  4. How the stimulus defines the dynamics of vesicle pool recruitment, fusion mode, and vesicle recycling in neuroendocrine cells.

    Science.gov (United States)

    Cárdenas, Ana María; Marengo, Fernando D

    2016-06-01

    The pattern of stimulation defines important characteristics of the secretory process in neurons and neuroendocrine cells, including the pool of secretory vesicles being recruited, the type and amount of transmitters released, the mode of membrane retrieval, and the mechanisms associated with vesicle replenishment. This review analyzes the mechanisms that regulate these processes in chromaffin cells, as well as in other neuroendocrine and neuronal models. A common factor in these mechanisms is the spatial and temporal distribution of the Ca(2+) signal generated during cell stimulation. For instance, neurosecretory cells and neurons have pools of vesicles with different locations with respect to Ca(2+) channels, and those pools are therefore differentially recruited following different patterns of stimulation. In this regard, a brief stimulus will induce the exocytosis of a small pool of vesicles that is highly coupled to voltage-dependent Ca(2+) channels, whereas longer or more intense stimulation will provoke a global Ca(2+) increase, promoting exocytosis irrespective of vesicle location. The pattern of stimulation, and therefore the characteristics of the Ca(2+) signal generated by the stimulus also influence the mode of exocytosis and the type of endocytosis. Indeed, low-frequency stimulation favors kiss-and-run exocytosis and clathrin-independent fast endocytosis, whereas higher frequencies promote full fusion and clathrin-dependent endocytosis. This latter type of endocytosis is accelerated at high-frequency stimulation. Synaptotagmins, calcineurin, dynamin, complexin, and actin remodeling, appear to be involved in the mechanisms that determine the response of these processes to Ca(2+) . In chromaffin cells, a brief stimulus induces the exocytosis of a small pool of vesicles that is highly coupled to voltage-dependent Ca(2+) channels (A), whereas longer or high-frequency stimulation provokes a global Ca(2+) increase, promoting exocytosis irrespective of

  5. DELIVERY OF siRNA INTO BREAST CANCER CELLS VIA PHAGE FUSION PROTEIN-TARGETED LIPOSOMES

    Science.gov (United States)

    Bedi, Deepa; Musacchio, Tiziana; Fagbohun, Olusegun A.; Gillespie, James W.; Deinnocentes, Patricia; Bird, R. Curtis; Bookbinder, Lonnie; Torchilin, Vladimir P.; Petrenko, Valery A.

    2011-01-01

    Efficacy of siRNAs as potential anticancer therapeutics can be increased by their targeted delivery into cancer cells via tumor-specific ligands. Phage display offers an unique approach to identify highly specific and selective ligands that can deliver nanocarriers to the site of disease. In this study, we proved a novel approach for intracellular delivery of siRNAs into breast cancer cells through their encapsulation into liposomes targeted to the tumor cells with preselected intact phage proteins. The targeted siRNA liposomes were obtained by a fusion of two parental liposomes containing spontaneously inserted siRNA and fusion phage proteins. The presence of pVIII coat protein fused to a MCF-7 cell-targeting peptide DMPGTVLP in the liposomes was confirmed by Western blotting. The novel phage-targeted siRNA-nanopharmaceuticals demonstrate significant down-regulation of PRDM14 gene expression and PRDM14 protein synthesis in the target MCF- 7 cells. This approach offers the potential for development of new anticancer siRNA-based targeted nanomedicines. PMID:21050894

  6. Dendritic-tumor fusion cells derived heat shock protein70-peptide complex has enhanced immunogenicity.

    Science.gov (United States)

    Zhang, Yunfei; Zhang, Yong; Chen, Jun; Liu, Yunyan; Luo, Wen

    2015-01-01

    Tumor-derived heat shock protein70-peptide complexes (HSP70.PC-Tu) have shown great promise in tumor immunotherapy due to numerous advantages. However, large-scale phase III clinical trials showed that the limited immunogenicity remained to be enhanced. In previous research, we demonstrated that heat shock protein 70-peptide complexes (HSP70.PC-Fc) derived from dendritic cell (DC)-tumor fusions exhibit enhanced immunogenicity compared with HSP70.PCs from tumor cells. However, the DCs used in our previous research were obtained from healthy donors and not from the patient population. In order to promote the clinical application of these complexes, HSP70.PC-Fc was prepared from patient-derived DC fused directly with patient-derived tumor cells in the current study. Our results showed that compared with HSP70.PC-Tu, HSP70.PC-Fc elicited much more powerful immune responses against the tumor from which the HSP70 was derived, including enhanced T cell activation, and CTL responses that were shown to be antigen specific and HLA restricted. Our results further indicated that the enhanced immunogenicity is related to the activation of CD4+ T cells and increased association with other heat shock proteins, such as HSP90. Therefore, the current study confirms the enhanced immunogenicity of HSP70.PC derived from DC-tumor fusions and may provide direct evidence promoting their future clinical use.

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

    Science.gov (United States)

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

    2008-06-20

    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 YxxPhi 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. Herpesvirus 6 Glycoproteins B (gB), gH, gL, and gQ Are Necessary and Sufficient for Cell-to-Cell Fusion

    OpenAIRE

    Tanaka, Yuki; Suenaga, Tadahiro; Matsumoto, Misako; Seya, Tsukasa; Arase, Hisashi

    2013-01-01

    The human herpesvirus 6 (HHV-6) envelope glycoprotein gH/gL/gQ1/gQ2 complex associates with host cell CD46 as its cellular receptor. Although gB has been suggested to be involved in HHV-6 infection, its function in membrane fusion has remained unclear. Here, we have developed an HHV-6A (strain GS)and HHV-6B (strain Z29) virus-free cell-to-cell fusion assay and demonstrate that gB and the gH/gL/gQ1/gQ2 complex are the minimum components required for membrane fusion by HHV-6.

  9. Metformin induces apoptosis of pancreatic cancer cells

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    AIM: To assess the role and mechanism of mefformin in inducing apoptosis of pancreatic cancer cells. METHODS: The human pancreatic cancer cell lines ASPC-1, BxPc-3, PANC-1 and SW1990 were exposed to mefformin. The inhibition of cell proliferation and colony formation via apoptosis induction and S phase arrest in pancreatic cancer cell lines of mefformin was tested.RESULTS: In each pancreatic cancer cell line tested, metformin inhibited cell proliferation in a dose dependent manner in MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assays). Flow cytometric analysis showed that metformin reduced the number of cells in G1 and increased the percentage of cells in S phase as well as the apoptotic fraction. Enzymelinked immunosorbent assay (EUSA) showed that metformin induced apaptosis in all pancreatic cancer cell lines. In Western blot studies, metformin induced oly-ADP-ribose polymerase(PARP) cleavage (an indicator of aspase activation) in all pancreatic cancer cell lines. The general caspase inhibitor (VAD-fmk) completely abolished metformin-induced PARP cleavage and apoptosis in ASPC-1 BxPc-3 and PANC-1, the caspase-8 specific inhibitor (IETD-fmk) and the caspase-9 specific inhibitor (LEHD-fmk) only partially abrogated metformin-induced apoptosis and PARP cleavage in BxPc-3 and PANC-1 cells. We also observed that metformin treatment ramatically reduced epidermal growth factor receptor (EGFR) and phosphorylated mitogen activated protein kinase (P-MAPK) in both a time- and dose-dependent manner in all cell lines tested.CONCLUSION: Metformin significantly inhibits cell proliferation and apoptosis in all pancreatic cell lines. And the metformin-induced apoptosis is associated with PARP leavage, activation of caspase-3, -8, and -9 in a time- and dose-dependent manner. Hence, both caspase-8 and -9-initiated apoptotic signaling pathways contribute to metforrnin-induced apoptosis in pancreatic cell lines.

  10. Specific Targeting of Tumor Endothelial Cells by a Shiga-like Toxin-Vascular Endothelial Growth Factor Fusion Protein as a Novel Treatment Strategy for Pancreatic Cancer

    Directory of Open Access Journals (Sweden)

    Birgit Hotz

    2010-10-01

    Full Text Available PURPOSE: Tumor endothelial cells express vascular endothelial growth factor receptor 2 (VEGFR-2. VEGF can direct toxins to tumor vessels through VEGFR-2 for antiangiogenic therapy. This study aimed to selectively damage the VEGFR-2-overexpressing vasculature of pancreatic cancer by SLT-VEGF fusion protein comprising VEGF and the A subunit of Shiga-like toxin which inhibits protein synthesis of cells with high VEGFR-2 expression. EXPERIMENTAL DESIGN: Expression of VEGF and VEGF receptors was evaluated in human pancreatic cancer cells (AsPC-1, HPAF-2 and in normal human endothelial cells (HUVEC by reverse transcription-polymerase chain reaction. Cells were treated with SLT-VEGF (0.1–10 nM, and cell viability, proliferation, and endothelial tube formation were assessed. Orthotopic pancreatic cancer (AsPC-1, HPAF-2 was induced in nude mice. Animals were treated with SLT-VEGF fusion protein alone or in combination with gemcitabine. Treatment began 3 days or 6 weeks after tumor induction. Primary tumor volume and dissemination were determined after 14 weeks. Microvessel density and expression of VEGF and VEGF receptors were analyzed by immunohistochemistry. RESULTS: SLT-VEGF did not influence proliferation of pancreatic cancer cells; HUVECs (low-level VEGFR-2 reduced their proliferation rate and tube formation but not their viability. SLT-VEGF fusion protein reduced tumor growth and dissemination, increasing 14-week survival (AsPC-1, up to 75%; HPAF-2, up to 83%. Results of gemcitabine were comparable with SLT-VEGF monotherapy. Combination partly increased the therapeutic effects in comparison to the respective monotherapies. Microvessel density was reduced in all groups. Intratumoral VEGFR-2 expression was found in endothelial but not in tumor cells. CONCLUSIONS: SLT-VEGF is toxic for tumor vasculature rather than for normal endothelial or pancreatic cancer cells. SLT-VEGF treatment in combination with gemcitabine may provide a novel approach for

  11. 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.

  12. Early Events in Chikungunya Virus Infection-From Virus Cell Binding to Membrane Fusion.

    Science.gov (United States)

    van Duijl-Richter, Mareike K S; Hoornweg, Tabitha E; Rodenhuis-Zybert, Izabela A; Smit, Jolanda M

    2015-07-07

    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.

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

    Science.gov (United States)

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

    2015-11-15

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

  14. Near-field nonuniformities in angularly multiplexed KrF fusion lasers with induced spatial incoherence

    Science.gov (United States)

    Lehmberg, Robert H.; Chan, Yung

    2005-05-01

    Induced spatial incoherence (ISI) has been proposed for KrF laser drivers to achieve the high degree of spatial beam uniformity required for direct-drive inertial confinement fusion. Although ISI provides ultrasmooth illumination at the far field of the laser, where the target is located, it can still allow the beams in the quasi-near field to develop a time-averaged spatial structure. This speckle, which arises primarily from random-phase aberration, builds up as the laser beams propagate away from the pupil plane located at the final amplifier stage; it is distinct from any structure imposed by gain nonuniformities in the amplifiers. Because of the spatial incoherence, the speckle is significantly smaller than that experienced by coherent beams. Nevertheless, it remains a damage issue, especially for the long beam delay paths required in angularly multiplexed KrF lasers. We develop a novel algorithm for calculating the time-integrated intensities; compare simulations and measurements of the near-field speckle in the Nike KrF laser; and explore options, such as aberration reduction and optical relaying, for controlling the problem in future angularly multiplexed KrF drivers. © Optical Society of America

  15. UVC-induced stress granules in mammalian cells.

    Directory of Open Access Journals (Sweden)

    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.

  16. Efficacy of an adapted granzyme B-based anti-CD30 cytolytic fusion protein against PI-9-positive classical Hodgkin lymphoma cells in a murine model

    International Nuclear Information System (INIS)

    Tumors develop when infiltrating immune cells contribute growth stimuli, and cancer cells are selected to survive within such a cytotoxic microenvironment. One possible immune-escape mechanism is the upregulation of PI-9 (Serpin B9) within cancer cells. This serine proteinase inhibitor selectively inactivates apoptosis-inducing granzyme B (GrB) from cytotoxic granules of innate immune cells. We demonstrate that most classical Hodgkin lymphoma (cHL)-derived cell lines express PI-9, which protects them against the GrB attack and thereby renders them resistant against GrB-based immunotherapeutics. To circumvent this disadvantage, we developed PI-9-insensitive human GrB mutants as fusion proteins to target the Hodgkin-selective receptor CD30. In contrast to the wild-type GrB, a R201K point-mutated GrB construct most efficiently killed PI-9-positive and -negative cHL cells. This was tested in vitro and also in vivo whereby a novel optical imaging-based tumor model with HL cell line L428 was applied. Therefore, this variant, as part of the next generation immunotherapeutics, also named cytolytic fusion proteins showing reduced immunogenicity, is a promising molecule for (targeted) therapy of patients with relapsing malignancies, such as cHL, and possibly other PI-9-positive malignancies, such as breast or lung carcinoma

  17. Radiation induced surface modification and contamination for EUV lithography and fusion applications

    Science.gov (United States)

    Al-Ajlony, Al-Montaser Bellah

    Al-Montaser Bellah Al-Ajlony. Ph.D., Purdue University, May 2014. Radiation Induced Surface Modification and Contamination for EUV Lithography and Fusion Applications. Major Professor: Ahmed Hassanein. The effect of ionizing radiation on materials surfaces is of major interest for many engineering applications. The importance of this topic rises from the severity of the implications that a surface at a certain application might suffer due its interaction with some sort of ionizing radiation. The severity of implication is not always related to the severity of the radiation, in many applications the concern comes from the over-sensitivity of the surface to a low doses of radiations. One example of these sensitive applications is the extreme ultraviolet (EUV) induced surface contaminations of the optics in EUV lithography devices. In this application, a small dose of ionizing radiation (EUV at 13.5 nm wavelength) can cause slight change in the chemical composition of the irradiated surface. This change in chemical composition can cause large change in the surface optical properties of the irradiated surface (EUV optics). This degradation in reflectivity is an issue that needs to be avoided. On the other extreme where intense radiation is implemented, the main concern of the radiation-surface interaction comes from the severity of the irradiation process. The plasma-facing component (PFC) in future thermonuclear devices represent the ultimate example where the materials might be exposed to severe irradiation processes. Under such extreme irradiation processes, some candidate PFC materials exhibit the formation of very fine and fragile nanostructure (Fuzz) that can be washed out into the fusion device plasma and stop the fusion reaction. These two extreme examples of the radiation-surface interaction were selected to be my PhD research topic. The change in chemical properties of Ru surface during exposure to a 13.5 nm wavelength of EUV light radiation was investigated

  18. 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.

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

    International Nuclear Information System (INIS)

    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.

  20. 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.

  1. Transcriptomic and epigenomic landscapes during cell fusion in BeWo trophoblast cells

    Science.gov (United States)

    Syncytialization is a process essential to the genesis and vitality of the decisive maternal-fetal interface, the syncytiotrophoblast. While the role of specific genes important in syncytial fusion is appreciated, an integrated global analysis of syncytialization is absent. We leveraged a variety of...

  2. Dolichol phosphate induces non-bilayer structures, vesicle fusion and transbilayer movements of lipids in model membranes

    Energy Technology Data Exchange (ETDEWEB)

    de Kruijff, B.; Van Duijn, G.; Valtersson, C.; Chojnacki, T.; Verkleij, A.J.; Dallner, G.

    1987-05-01

    The effect of dolichols, polyprenols, dolichol esterified with fatty acids, and dolichol phosphate on the structure and fluidity of model membranes was studied using different biophysical techniques. These studies suggest that (1) dolichol and dolichol derivatives destabilize unsaturated PE-containing bilayers and promote hexagonal II phase formation; (2) high concentrations of dolichol induce lipid structures characterized by isotropic T P NMR and particulate fracture faces. The effect of dolichol and dolichyl phosphate on fusion between large unilamellar vesicles of DOPC and DOPE was studied using a fluroescence resonance energy transfer assay. The influence of dolichyl phosphate on the transbilary movement of DOPC in multilamellar vesicles (MLV) and large unilamellar vesicles (LUV) composed of DOPC and DOPE (1:2) was investigated by using the PC-specified transfer protein. The results indicate that: (1) both dolichol and dolichyl phosphate enhance vesicle fusion in a comparable and concentration-dependent way; (2) the amount of exchangeable PC from MLVs is increased by dolichyl phosphate probably as a result of fusion processes. It thus appears that these polyprenols are potent destabilizers of bilayer structure and that this process is accompanied by membrane fusion and transbilayer transport of phospholipids.

  3. Dolichol phosphate induces non-bilayer structures, vesicle fusion and transbilayer movements of lipids in model membranes

    International Nuclear Information System (INIS)

    The effect of dolichols, polyprenols, dolichol esterified with fatty acids, and dolichol phosphate on the structure and fluidity of model membranes was studied using different biophysical techniques. These studies suggest that (1) dolichol and dolichol derivatives destabilize unsaturated PE-containing bilayers and promote hexagonal II phase formation; (2) high concentrations of dolichol induce lipid structures characterized by isotropic 31P NMR and particulate fracture faces. The effect of dolichol and dolichyl phosphate on fusion between large unilamellar vesicles of DOPC and DOPE was studied using a fluroescence resonance energy transfer assay. The influence of dolichyl phosphate on the transbilary movement of DOPC in multilamellar vesicles (MLV) and large unilamellar vesicles (LUV) composed of DOPC and DOPE (1:2) was investigated by using the PC-specified transfer protein. The results indicate that: (1) both dolichol and dolichyl phosphate enhance vesicle fusion in a comparable and concentration-dependent way; (2) the amount of exchangeable PC from MLVs is increased by dolichyl phosphate probably as a result of fusion processes. It thus appears that these polyprenols are potent destabilizers of bilayer structure and that this process is accompanied by membrane fusion and transbilayer transport of phospholipids

  4. Bidirectional reprogramming of fusion cells of pluripotent stem cells/primary cardiac myocytes%诱导多能干细胞/原代心肌细胞的融合细胞表现出双向重建

    Institute of Scientific and Technical Information of China (English)

    熊挺淋; 张晓刚; 赵霞; 马红芬

    2011-01-01

    Objective To construct fusion cells with induced pluripotent stem cells (iPSc) and primary cardiac myocytes in vitro, and to investigate biological features of the fusion cells. Methods Polyethylene glycol (PEG-4000) was used to mediate the cell fusion of iPSc derived from green fluorescent protein (GFP) transgenes (octamer-binding transcription factor-4, Oct-4) mouse and cardiac myocytes from neonatal mouse. Morphological changes of the fusion cells were observed dynamically after alkaline phosphatase (AKP) staining. Specific proteins of stem cells and cardiac myocytes in fusion cells were detected by immunofluores-cence. Chromosome karyotype analysis were performed to determine whether the occurrence of nuclear fusion and degree of integration. Results Fusion cells were constructed successfully by polyethylene glycol mediation. Colony-like cell clusters appeared in 4 d after fusion. The AKP positive rate of iPSc were 0.935 ±0.039, 0.939 ± 0.022, 0.954 ± 0.017, and 0.944 ± 0.027 at the 2nd, 3rd, 4th and 5th days respectively, and that of fusion cells were 0.761 ±0.044, 0.740 ±0.023, 0.681 ±0.034, and 0.748 ±0.045 at the corresponding days respectively. At the same time points, there were significant differences between iPSc AKP-positive rates and those of fusion cells ( P < 0. 05). In the initial stage, fusion cells mainly displayed iPSc characteristics, with Oct-4 positive while cTnT negative. Then the fusion cells began to display both characteristics of iPSc and cardiac myocytes in 7 d after fusion, with positive expression of Oct-4 and cTnT. More than 80% of fusion cells had 76 to 80 chromosomes. Conclusion Fusion cells from diploid iPSc and diploid myocardial cells display the characteristics of the two parental cells and show bidirectional reprogramming.%目的 体外构建诱导多能干细胞(induced pluripotent stem cells,iPSc)与原代心肌细胞的融合细胞,初步探讨融合细胞体外生物学特性.方法

  5. 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.

  6. Transduction of the MPG-tagged fusion protein into mammalian cells and oocytes depends on amiloride-sensitive endocytic pathway

    Directory of Open Access Journals (Sweden)

    Cheon Yong-Pil

    2009-08-01

    Full Text Available Abstract Background MPG is a cell-permeable peptide with proven efficiency to deliver macromolecular cargoes into cells. In this work, we examined the efficacy of MPG as an N-terminal tag in a fusion protein to deliver a protein cargo and its mechanism of transduction. Results We examined transduction of MPG-EGFP fusion protein by live imaging, flow cytometry, along with combination of cell biological and pharmacological methods. We show that MPG-EGFP fusion proteins efficiently enter various mammalian cells within a few minutes and are co-localized with FM4-64, a general marker of endosomes. The transduction of MPG-EGFP occurs rapidly and is inhibited at a low temperature. The entry of MPG-EGFP is inhibited by amiloride, but cytochalasin D and methyl-β-cyclodextrin did not inhibit the entry, suggesting that macropinocytosis is not involved in the transduction. Overexpression of a mutant form of dynamin partially reduced the transduction of MPG-EGFP. The partial blockade of MPG-EGFP transduction by a dynamin mutant is abolished by the treatment of amiloride. MPG-EGFP transduction is also observed in the mammalian oocytes. Conclusion The results show that the transduction of MPG fusion protein utilizes endocytic pathway(s which is amiloride-sensitive and partially dynamin-dependent. Collectively, the MPG fusion protein could be further developed as a novel tool of "protein therapeutics", with potentials to be used in various cell systems including mammalian oocytes.

  7. Electro Protoplast Fusion between Solanum sisymbriifolium and Other Solanum Species

    OpenAIRE

    ODA, Naoki; Isshiki, Shiro; Sadohara, Takeshi; Ozaki, Yukio; Okubo, Hiroshi

    2006-01-01

    Symmetric or asymmetric protoplast fusions between Solanum sisymbriidorium and othe Solanum species (S. integrifolium and S. toxicarium) were performed to obtain the perfectly soil-borne disease resistant rootstocks for eggplant cultivation. Somatic hybrid was not obtained from symmetric fusion of iodoacetamide-induced inactivation of S. integrifolium and no cell division ability of S. sisymbriifolium on a selective medium in asymmetric fusion, the hybridity of two calli was eluciated by ana...

  8. The natural product peiminine represses colorectal carcinoma tumor growth by inducing autophagic cell death

    International Nuclear Information System (INIS)

    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. 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.

  10. 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.

  11. Effective myotube formation in human adipose tissue-derived stem cells expressing dystrophin and myosin heavy chain by cellular fusion with mouse C2C12 myoblasts

    International Nuclear Information System (INIS)

    Highlights: → hASCs were differentiated into skeletal muscle cells by treatment with 5-azacytidine, FGF-2, and the supernatant of cultured hASCs. → Dystrophin and MyHC were expressed in late differentiation step by treatment with the supernatant of cultured hASCs. → 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.

  12. Effect of hyperbaric oxygen on mesenchymal stem cells for lumbar fusion in vivo

    Directory of Open Access Journals (Sweden)

    Lin Song-Shu

    2010-03-01

    Full Text Available Abstract Background Hyperbaric oxygen (HBO therapy has been proved in improving bone healing, but its effects on mesenchymal stem cells (MSCs in vivo is not clear. The aims of this study are to clarify whether the HBO therapy has the same enhancing effect on MSCs with regard to bone formation and maturation and to ascertain whether the transplanted MSCs survive in the grafted area and contribute to new bone formation. Methods Twenty-three adult rabbits underwent posterolateral fusion at L4-L5 level. The animals were divided into three groups according to the material implanted and subsequent treatment: (1 Alginate carrier (n = 6; (2 Alginate-MSCs composite (n = 11; and (3 Alginate-MSCs composite with HBO therapy (n = 6. After 12 weeks, spine fusion was examined using radiographic examination, manual testing, and histological examination. Using a PKH fluorescence labeling system, whether the transplanted MSCs survived and contributed to new bone formation in the grafted area after HBO therapy was also examined. Results The bilateral fusion areas in each animal were evaluated independently. By radiographic examination and manual palpation, union for the Alginate, Alginate-MSCs, and Alginate-MSCs-HBO groups was 0 of 12, 10 of 22, and 6 of 12 respectively. The difference between the Alginate-MSCs and Alginate-MSCs-HBO groups was not significant (P = 0.7997. The fluorescence microscopy histological analysis indicated that the transplanted PKH67-labeled MSCs survived and partly contributed to new bone formation in the grafted area. Conclusions This study demonstrated that the preconditioned MSCs could survive and yield bone formation in the grafted area. HBO therapy did not enhance the osteogenic ability of MSCs and improve the success of spine fusion in the rabbit model. Although there was no significant effect of HBO therapy on MSCs for spine fusion, the study encourages us to research a more basic approach for determining the optimal oxygen tension

  13. An adjuvanted respiratory syncytial virus fusion protein induces protection in aged BALB/c mice

    Directory of Open Access Journals (Sweden)

    Cherukuri Anu

    2012-10-01

    Full Text Available Abstract Background Respiratory Syncytial Virus (RSV causes significant disease in the elderly, in part, because immunosenescence impairs protective immune responses to infection in this population. Despite previous and current efforts, there is no RSV vaccine currently licensed in infants or elderly adults. Adjuvanted RSV subunit vaccines have the potential to boost waning immune responses and reduce the burden of RSV disease in the elderly population. Results We used an aged BALB/c mouse model to evaluate immune responses to RSV Fusion (F protein in the absence and presence of an alum adjuvant. We demonstrate that aged BALB/c mice immunized with alum-adjuvanted RSV F protein had significantly reduced lung viral titers at day 4 following challenge with wild-type (wt RSV. Serum neutralizing antibody titers measured on day 27 correlated with protection in both young and aged vaccinated mice, although the magnitude of antibody titers was lower in aged mice. Unlike young mice, in aged mice, alum-adjuvanted RSV F did not induce lung TH2-type cytokines or eosinophil infiltration compared to non-adjuvanted F protein following wt RSV challenge. Conclusion Our studies demonstrate that neutralizing anti-RSV antibody titers correlate with protection in both young and aged BALB/c mice vaccinated with RSV F protein vaccines. The F + alum formulation mediated greater protection compared to the non-adjuvanted F protein in both young and aged mice. However, while alum can boost F-specific antibody responses in aged mice, it does not completely overcome the reduced ability of a senescent immune system to respond to the RSV F antigen. Thus, our data suggest that a stronger adjuvant may be required for the prevention of RSV disease in immunosenescent populations, to achieve the appropriate balance of protective neutralizing antibodies and effective TH1-type cytokine response along with minimal lung immunopathology.

  14. Systematic study of 16O-induced fusions with the improved quantum molecular dynamics model

    CERN Document Server

    Wang, Ning; Li, Zhuxia

    2014-01-01

    The heavy-ion fusion reactions with 16O bombarding on 62Ni, 65Cu, 74Ge, 148Nd, 180Hf, 186W, 208Pb, 238U are systematically investigated with the improved quantum molecular dynamics (ImQMD) model. The fusion cross sections at energies near and above the Coulomb barriers can be reasonably well reproduced by using this semi-classical microscopic transport model with the parameter sets SkP* and IQ3a. The dynamical nucleus-nucleus potentials and the influence of Fermi constraint on the fusion process are also studied simultaneously. In addition to the mean field, the Fermi constraint also plays a key role for the reliable description of fusion process and for improving the stability of fragments in heavy-ion collisions.

  15. A model for cell wall dissolution in mating yeast cells: polarized secretion and restricted diffusion of cell wall remodeling enzymes induces local dissolution.

    Directory of Open Access Journals (Sweden)

    Lori B Huberman

    Full Text Available Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells.

  16. A model for cell wall dissolution in mating yeast cells: polarized secretion and restricted diffusion of cell wall remodeling enzymes induces local dissolution.

    Science.gov (United States)

    Huberman, Lori B; Murray, Andrew W

    2014-01-01

    Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells.

  17. 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)

    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 [3H]thymidine into cell nuclei as measured by autoradiography

  18. An induced pocket for the binding of potent fusion inhibitor CL-385319 with H5N1 influenza virus hemagglutinin.

    Directory of Open Access Journals (Sweden)

    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.

  19. Fusability and fissionability in 86Kr induced reactions near and below the fusion barrier

    International Nuclear Information System (INIS)

    Evaporation-residue excitation functions for the reactions 86Kr + sup(70,76)Ge, sup(92,100)Mo, sup(99,102,104)Ru have been measured using activation methods and the velocity filter SHIP. The data span the region from well below the fusion barrier up to and beyond the energy where limitation by fission competition takes place. The data are shown to be compatible with the concept of complete fusion followed by the statistical decay of the equilibrated compound nucleus. Information on both the fusion probability at and below the fusion threshold and the fissionability of the compound nuclei formed is extracted. The model dependence of the extracted fission barriers is discussed in detail. In analogy to studies involving lighter projectiles, strong correlations between the low-energy nuclear-structure properties of the nuclei and the subbarrier fusion probability are found. A relative shift of the fusion barrier to higher energies, that increases with the number of valence neutrons in the target nuclei, is observed. (orig.)

  20. The Effects of Weak Combined Magnetic Field on Cell Wall Regeneration and Frequency of Plant Protoplasts Fusion

    Science.gov (United States)

    Nedukha, Olena

    The major purpose of these experiments was to investigate plant protoplast fusion frequency and regeneration of a cell wall by protoplasts at weak combined magnetic field (CMF) with the frequency resonance to the cyclotron frequency of Mg2+, Ca2+ and K+ ions. The protoplasts were isolated from Nicotiana lumbaginifolia and N. silvestris leaf mesophyll and from callus tissues (Nicotiana tabacum and Glycine max). The special extra apparatus with ferromagnetic shield was used for estimate of CMF with the frequency resonance to the cyclotron frequency of Mg2+, Ca2+ and K+ ions. The fusion of protoplasts is realized by using of parent protoplasts isolated from one plant species, as well as from various plant species. Control samples were situated near the apparatus with CMF. The laser confocal microscopy was used for study of cell wall regeneration by single and fused protoplasts. The cytochemical methods with DAPI and calcofluor dye were also applied as the detectors for protoplast fusion and regeneration of cell wall. We have been established that CMF with frequency adjusted to the cyclotron frequency Mg2+ ions have shown the most positive influence on regeneration of cell wall by protoplasts. CMF adjusted to the cyclotron frequency of K+ ions very weakly affected on the frequency of protoplast fusion. Largest frequency of protoplasts fusion is noted in the CMF adjusted to the cyclotron frequency of Ca2+ in comparison with the control samples.

  1. 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

    1995-06-01

    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. 14 refs., 4 figs., 1 tab.

  2. 5-Fluorocytosine combined with Fcy-hEGF fusion protein targets EGFR-expressing cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Lan, Keng-Hsueh [Division of Radiation Oncology, Department of Oncology, National Taiwan University Hospital, Taipei 100, Taiwan (China); Shih, Yi-Sheng [Cancer Center, Taipei Veterans General Hospital, Taipei 112, Taiwan (China); Chang, Cheng Allen [Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei 112, Taiwan (China); School of Biomedical Science and Engineering, National Yang-Ming University, Taipei 112, Taiwan (China); Yen, Sang-Hue [Cancer Center, Taipei Veterans General Hospital, Taipei 112, Taiwan (China); Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei 112, Taiwan (China); Lan, Keng-Li, E-mail: kllan@vghtpe.gov.tw [Cancer Center, Taipei Veterans General Hospital, Taipei 112, Taiwan (China); Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei 112, Taiwan (China)

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer EGFR-expressing epithelial cancers account for significant portion of cancer deaths. Black-Right-Pointing-Pointer EGF-EGFR signaling pathway is validated as an important anticancer drug target. Black-Right-Pointing-Pointer EGF and Fcy fusion protein (Fcy-hEGF) can bind to EGFR and convert 5-FC to 5-FU. Black-Right-Pointing-Pointer 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{sub 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

  3. 5-Fluorocytosine combined with Fcy–hEGF fusion protein targets EGFR-expressing cancer cells

    International Nuclear Information System (INIS)

    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 IC50 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.

  4. Optimization of Landscape Phage Fusion Protein-Modified Polymeric PEG-PE Micelles for Improved Breast Cancer Cell Targeting

    Science.gov (United States)

    Wang, Tao; Petrenko, Valery A.; Torchilin, Vladimir P.

    2015-01-01

    Amphiphilic landscape phage fusion proteins with high affinity and selectivity towards breast cancer MCF-7 (Michigan Cancer Foundation-7) cells self-assemble with polymeric PEG-PE conjugates to form mixed micelles (phage-micelles) capable of cancer cell-targeted delivery of poorly-soluble drugs. While the PEG corona provides the stability and longevity to the micelles, its presence is a potential steric difficulties for the interaction of phage fusion protein with cell surface targets. We attempted to address this problem by controlling the length of the PEG block and the phage fusion protein quantity, selecting the optimal ones to produce a reasonable retention of the targeting affinity and selectivity of the MCF-7-specific phage fusion protein. Three PEG-PE conjugates with different PEG lengths were used to construct phage- and plain-micelles, followed by FACS analysis of the effect of the PEG length on their binding affinity and selectivity towards target MCF-7 cells using either a MCF-7 cell monoculture or a cell co-culture model composed of target cancer MCF-7 cells and non-target, non-cancer C166 cells expressing GFP (Green Fluorescent Protein). Both, the length of PEG and quantity of phage fusion protein had a profound impact on the targetability of the phage-micelles. Phage-micelles prepared with PEG2k-PE achieved a desirable binding affinity and selectivity. Incorporation of a minimal concentration of phage protein, up to 0.5%, produced maximal targeting efficiency towards MCF-7 cells. Overall, phage-micelles with PEG2k-PE and 0.5% of phage protein represent the optimal formulation for targeting towards breast cancer cells. PMID:26451274

  5. 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.

  6. Cytoplasmic Delivery of Liposomes into MCF-7 Breast Cancer Cells Mediated by Cell-Specific Phage Fusion Coat Protein

    Science.gov (United States)

    Wang, Tao; Yang, Shenghong; Petrenko, Valery A; Torchilin, Vladimir P

    2010-01-01

    Earlier, we have shown that doxorubicin-loaded liposomes (Doxil) modified with a chimeric phage fusion coat protein specific towards MCF-7 breast cancer cells identified from a phage landscape library demonstrated a significantly enhanced association with target cells and an increased cytotoxicity. Based on some structural similarities between the N-terminus of the phage potein and known fusogenic peptides, we hypothesized that, in addition to the specific targeting, the phage protein may possess endosome-escaping potential and an increased cytotoxicity of drug-loaded phage protein-targeted liposomes may be explained by an advantageous combination of both, cell targeting and endosomal escape of drug-loaded nanocarrier. The use of the fluorescence resonance energy transfer (FRET) technique allowed us to clearly demonstrate the pH-dependent membrane fusion activity of the phage protein. Endosomal escape and cytosolic delivery of phage-liposomes was visualized with fluorescence microscopy. Endosome acidification inhibition by bafilomycin A 1 resulted in decreased cytotoxicity of the phage-Doxil, while the endosome disruption by chloroquine had a negligible effect on efficacy of phage-Doxil, confirming its endosomal escape. Our results demonstrated an endosome-escaping property of the phage protein and provided an insight on mechanism of the enhanced cytotoxicity of phage-Doxil. PMID:20438086

  7. Fuel cells: Hydrogen induced insulation

    Science.gov (United States)

    Zhou, Wei; Shao, Zongping

    2016-06-01

    Coupling high ionic and low electronic conductivity in the electrolyte of low-temperature solid-oxide fuel cells remains a challenge. Now, the electronic conductivity of a perovskite electrolyte, which has high proton conductivity, is shown to be heavily suppressed when exposed to hydrogen, leading to high fuel cell performance.

  8. 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.

  9. Mechanisms of cell transformation induced by polyomavirus

    Directory of Open Access Journals (Sweden)

    M.L.S. Oliveira

    1999-07-01

    Full Text Available Polyomavirus is a DNA tumor virus that induces a variety of tumors in mice. Its genome encodes three proteins, namely large T (LT, middle T (MT, and small T (ST antigens, that have been implicated in cell transformation and tumorigenesis. LT is associated with cell immortalization, whereas MT plays an essential role in cell transformation by binding to and activating several cytoplasmic proteins that participate in growth factor-induced mitogenic signal transduction to the nucleus. The use of different MT mutants has led to the identification of MT-binding proteins as well as analysis of their importance during cell transformation. Studying the molecular mechanisms of cell transformation by MT has contributed to a better understanding of cell cycle regulation and growth control.

  10. Acidification triggers Andes hantavirus membrane fusion and rearrangement of Gc into a stable post-fusion homotrimer.

    Science.gov (United States)

    Acuña, Rodrigo; Bignon, Eduardo A; Mancini, Roberta; Lozach, Pierre-Yves; Tischler, Nicole D

    2015-11-01

    The hantavirus membrane fusion process is mediated by the Gc envelope glycoprotein from within endosomes. However, little is known about the specific mechanism that triggers Gc fusion activation, and its pre- and post-fusion conformations. We established cell-free in vitro systems to characterize hantavirus fusion activation. Low pH was sufficient to trigger the interaction of virus-like particles with liposomes. This interaction was dependent on a pre-fusion glycoprotein arrangement. Further, low pH induced Gc multimerization changes leading to non-reversible Gc homotrimers. These trimers were resistant to detergent, heat and protease digestion, suggesting characteristics of a stable post-fusion structure. No acid-dependent oligomerization rearrangement was detected for the trypsin-sensitive Gn envelope glycoprotein. Finally, acidification induced fusion of glycoprotein-expressing effector cells with non-susceptible CHO cells. Together, the data provide novel information on the Gc fusion trigger and its non-reversible activation involving lipid interaction, multimerization changes and membrane fusion which ultimately allow hantavirus entry into cells.

  11. 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

  12. Reprogramming Müller glia via in vivo cell fusion regenerates murine photoreceptors.

    Science.gov (United States)

    Sanges, Daniela; Simonte, Giacoma; Di Vicino, Umberto; Romo, Neus; Pinilla, Isabel; Nicolás, Marta; Cosma, Maria Pia

    2016-08-01

    Vision impairments and blindness caused by retinitis pigmentosa result from severe neurodegeneration that leads to a loss of photoreceptors, the specialized light-sensitive neurons that enable vision. Although the mammalian nervous system is unable to replace neurons lost due to degeneration, therapeutic approaches to reprogram resident glial cells to replace retinal neurons have been proposed. Here, we demonstrate that retinal Müller glia can be reprogrammed in vivo into retinal precursors that then differentiate into photoreceptors. We transplanted hematopoietic stem and progenitor cells (HSPCs) into retinas affected by photoreceptor degeneration and observed spontaneous cell fusion events between Müller glia and the transplanted cells. Activation of Wnt signaling in the transplanted HSPCs enhanced survival and proliferation of Müller-HSPC hybrids as well as their reprogramming into intermediate photoreceptor precursors. This suggests that Wnt signaling drives the reprogrammed cells toward a photoreceptor progenitor fate. Finally, Müller-HSPC hybrids differentiated into photoreceptors. Transplantation of HSPCs with activated Wnt functionally rescued the retinal degeneration phenotype in rd10 mice, a model for inherited retinitis pigmentosa. Together, these results suggest that photoreceptors can be generated by reprogramming Müller glia and that this approach may have potential as a strategy for reversing retinal degeneration. PMID:27427986

  13. 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.

  14. 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

    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....... 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...

  15. The measurement of neutron and neutron induced photon spectra in fusion reactor related assemblies

    CERN Document Server

    Unholzer, S; Klein, H; Seidel, K

    2002-01-01

    The spectral neutron and photon fluence (or flux) measured outside and inside of assemblies related to fusion reactor constructions are basic quantities of fusion neutronics. The comparison of measured spectra with the results of MCNP neutron and photon transport calculations allows a crucial test of evaluated nuclear data as generally used in fusion applications to be carried out. The experiments concern mixed neutron/photon fields with about the same intensity of the two components. An NE-213 scintillation spectrometer, well described by response matrices for both neutrons and photons, is used as proton-recoil and Compton spectrometer. The experiments described here in more detail address the background problematic of two applications, an iron benchmark experiment with an ns-pulsed neutron source and a deep penetration mock-up experiment for the investigation of the ITER in-board shield system. The measured spectral neutron and photon fluences are compared with spectra calculated with the MCNP code on the b...

  16. 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...... 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....... 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...

  17. No-capture breakup and incomplete fusion reactions induced by stable weakly bound nucleus 9Be

    Science.gov (United States)

    Seyyedi, S. A.

    2016-06-01

    The reactions including the stable weakly bound nucleus 9Be have been studied using the classical trajectory model accompanied with the experimental breakup function and the Aage-Winther interaction potential (AW95). In these calculations, the no-capture breakup and the incomplete fusion cross-sections as well as their competition at around the Coulomb barrier have been investigated. Our calculations showed that at a given far-Coulomb-barrier energy the incomplete fusion reaction in different distributions of angular momentum and energies can dominate the no-capture breakup reaction. This dominating process is reversed at the near-barrier energies.

  18. Characterization of fusion genes and the significantly expressed fusion isoforms in breast cancer by hybrid sequencing

    OpenAIRE

    Weirather, Jason L.; Afshar, Pegah Tootoonchi; Clark, Tyson A.; Tseng, Elizabeth; Powers, Linda S.; Underwood, Jason G; Zabner, Joseph; Korlach, Jonas; Wong, Wing Hung; Au, Kin Fai

    2015-01-01

    We developed an innovative hybrid sequencing approach, IDP-fusion, to detect fusion genes, determine fusion sites and identify and quantify fusion isoforms. IDP-fusion is the first method to study gene fusion events by integrating Third Generation Sequencing long reads and Second Generation Sequencing short reads. We applied IDP-fusion to PacBio data and Illumina data from the MCF-7 breast cancer cells. Compared with the existing tools, IDP-fusion detects fusion genes at higher precision and ...

  19. Radius Intermedius Stenosis Induced Myocardial Perfusion Defect : Provened by the Fusion Images of Myocardial Perfusion SPECT and 64 Channel CTA

    Energy Technology Data Exchange (ETDEWEB)

    Kong, Eun Jung; Cho, Ihn Ho; Chun, Kyung Ah; Won, Kyu Chang; Lee, Hyung Woo; Park, Jong Seon [College of Medicine, Yeungnam University, Daegu (Korea, Republic of)

    2008-02-15

    A 71-year-old woman was assigned to our department for Tc-99m myocardial perfusion SPECT(MPS) and coronary CT angiography. She admitted for substernal pain, via the ER, 2 days ago. The heart was scanned after intravenous injection of 925 MBq of {sup 99m}Tc-sestamibi adenosine-induced stress SPECT using dual head gamma camera (Hawkeye, GE healthcare. USA). The MPS shows decreased tracer uptake in the apical and mid area of anterior and lateral wall and mid and basal inferior wall. Coronary CT angiography was obtained using Discovery VCT (GE healthcare). 3D angiography portrayed significant stenosis of ramus intermedius(RI) and posterolateral branch of right coronary artery(PLB) with fibrocalcified plaque. Two images were fused using Cardiac IQ fusion software package (Advantage workstation 4.4, GE healthcare) The fusion images explain the perfusion defect of anterior, lateral and inferior wall is due to stenosis of the RI and PLB. And 3 days later, coronary angiography was done and revealed the marked stenosis of RI and PLB. Then balloon angioplasty and stent was instituted in RI. Cardiac SPECT/CT fusion imaging provides additional information about hemodynamic relevance and facilitates lesion interpretation by allowing exact allocation of perfusion defects to its subtending coronary artery.

  20. Glucocorticoid induced osteopenia in cancellous bone of sheep: validation of large animal model for spine fusion and biomaterial research

    DEFF Research Database (Denmark)

    Ding, Ming; Cheng, Liming; Bollen, Peter;

    2010-01-01

    STUDY DESIGN: Glucocorticoid with low calcium and phosphorus intake induces osteopenia in cancellous bone of sheep. OBJECTIVE: To validate a large animal model for spine fusion and biomaterial research. SUMMARY OF BACKGROUND DATA: A variety of ovariectomized animals has been used to study......-1) received prednisolone (GC) treatment (0.60 mg/kg/day, 5 times weekly) for 7 months. Group 2 (GC-2) received the same treatment as GC-1 for 7 months followed by 3 months without treatment. Group 3 was left untreated and served as the controls. All sheep received restricted diet with low calcium...

  1. Acetylsalicylic acid induces programmed cell death in Arabidopsis cell cultures.

    Science.gov (United States)

    García-Heredia, José M; Hervás, Manuel; De la Rosa, Miguel A; Navarro, José A

    2008-06-01

    Acetylsalicylic acid (ASA), a derivative from the plant hormone salicylic acid (SA), is a commonly used drug that has a dual role in animal organisms as an anti-inflammatory and anticancer agent. It acts as an inhibitor of cyclooxygenases (COXs), which catalyze prostaglandins production. It is known that ASA serves as an apoptotic agent on cancer cells through the inhibition of the COX-2 enzyme. Here, we provide evidences that ASA also behaves as an agent inducing programmed cell death (PCD) in cell cultures of the model plant Arabidopsis thaliana, in a similar way than the well-established PCD-inducing agent H(2)O(2), although the induction of PCD by ASA requires much lower inducer concentrations. Moreover, ASA is herein shown to be a more efficient PCD-inducing agent than salicylic acid. ASA treatment of Arabidopsis cells induces typical PCD-linked morphological and biochemical changes, namely cell shrinkage, nuclear DNA degradation, loss of mitochondrial membrane potential, cytochrome c release from mitochondria and induction of caspase-like activity. However, the ASA effect can be partially reverted by jasmonic acid. Taking together, these results reveal the existence of common features in ASA-induced animal apoptosis and plant PCD, and also suggest that there are similarities between the pathways of synthesis and function of prostanoid-like lipid mediators in animal and plant organisms.

  2. Production of Hev b5 as a fluorescent biotin-binding tripartite fusion protein in insect cells

    International Nuclear Information System (INIS)

    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

  3. Convergent mutations and kinase fusions lead to oncogenic STAT3 activation in anaplastic large cell lymphoma.

    Science.gov (United States)

    Crescenzo, Ramona; Abate, Francesco; Lasorsa, Elena; Tabbo', Fabrizio; Gaudiano, Marcello; Chiesa, Nicoletta; Di Giacomo, Filomena; Spaccarotella, Elisa; Barbarossa, Luigi; Ercole, Elisabetta; Todaro, Maria; Boi, Michela; Acquaviva, Andrea; Ficarra, Elisa; Novero, Domenico; Rinaldi, Andrea; Tousseyn, Thomas; Rosenwald, Andreas; Kenner, Lukas; Cerroni, Lorenzo; Tzankov, Alexander; Ponzoni, Maurilio; Paulli, Marco; Weisenburger, Dennis; Chan, Wing C; Iqbal, Javeed; Piris, Miguel A; Zamo', Alberto; Ciardullo, Carmela; Rossi, Davide; Gaidano, Gianluca; Pileri, Stefano; Tiacci, Enrico; Falini, Brunangelo; Shultz, Leonard D; Mevellec, Laurence; Vialard, Jorge E; Piva, Roberto; Bertoni, Francesco; Rabadan, Raul; Inghirami, Giorgio

    2015-04-13

    A systematic characterization of the genetic alterations driving ALCLs has not been performed. By integrating massive sequencing strategies, we provide a comprehensive characterization of driver genetic alterations (somatic point mutations, copy number alterations, and gene fusions) in ALK(-) ALCLs. We identified activating mutations of JAK1 and/or STAT3 genes in ∼20% of 88 [corrected] ALK(-) ALCLs and demonstrated that 38% of systemic ALK(-) ALCLs displayed double lesions. Recurrent chimeras combining a transcription factor (NFkB2 or NCOR2) with a tyrosine kinase (ROS1 or TYK2) were also discovered in WT JAK1/STAT3 ALK(-) ALCL. All these aberrations lead to the constitutive activation of the JAK/STAT3 pathway, which was proved oncogenic. Consistently, JAK/STAT3 pathway inhibition impaired cell growth in vitro and in vivo. PMID:25873174

  4. Fusion Machinery

    DEFF Research Database (Denmark)

    Sørensen, Jakob Balslev; Milosevic, Ira

    2015-01-01

    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...

  5. Induced pluripotent stem cells and neurodegenerative diseases.

    Science.gov (United States)

    Chen, Chao; Xiao, Shi-Fu

    2011-04-01

    Neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease and Amyotrophic Lateral Sclerosis, are characterized by idiopathic neuron loss in different regions of the central nervous system, which contributes to the relevant dysfunctions in the patients. The application of cell replacement therapy using human embryonic stem (hES) cells, though having attracted much attention, has been hampered by the intrinsic ethical problems. It has been demonstrated that adult somatic cells can be reprogrammed into the embryonic state, called induced pluripotent stem (iPS) cells. It is soon realized that iPS cells may be an alternative source for cell replacement therapy, because it raises no ethical problems and using patient-specific iPS cells for autologous transplantation will not lead to immunological rejection. What's more, certain types of neurons derived from patient-specific iPS cells may display disease-relevant phenotypes. Thus, patient-specific iPS cells can provide a unique opportunity to directly investigate the pathological properties of relevant neural cells in individual patient, and to study the vulnerability of neural cells to pathogenic factors in vitro, which may help reveal the pathogenesis of many neurodegenerative diseases. In this review, the recent development in cellular treatment of neurodegenerative diseases using iPS cells was summarized, and the potential value of iPS cells in the modeling of neurodegenerative disease was discussed.

  6. Mast cell degranulation induced by chlorogenic acid

    OpenAIRE

    Huang, Fang-hua; Zhang, Xin-yue; Zhang, Lu-Yong; Li, Qin; Ni, Bin; Zheng, Xiao-liang; CHEN, AI-JUN

    2010-01-01

    Aim: To investigate the mechanism of chlorogenic acid (CA)-induced anaphylactoid reactions. Methods: Degranulation of peritoneal mast cells was assayed by using alcian blue staining in guinea pigs, and the degranulation index (DI) was calculated. CA-induced degranulation of RBL-2H3 cells was also observed and assayed using light microscopy, transmission electron microscopy, flow cytometry, and β-hexosaminidase release. Results: CA 0.2, 1.0, and 5.0 mmol/L was able to promote degranulation of ...

  7. Evaluation of immune effect of recombinant fusion protein targeting the prostate stem cell antigen based on PSCA and HSP70

    Directory of Open Access Journals (Sweden)

    Lei DONG

    2014-10-01

    Full Text Available Objective To explore the immune effect and antitumor activity of recombinant prostate stem cell protein (PSCA and heat shock protein 70 (HSP70 in a murine model of prostate cancer. Methods Twenty-five healthy male C57BL/6 mice were randomly divided into 5 groups (5 each: PSCA, HSP, PSCA+HSP, PSCA-HSP and control group. Mice in the first 4 groups were vaccinated with the corresponding proteins, and those in control group were faked with injection of phosphate buffer saline (PBS. After immunization with recombinant proteins, the PSCA-specific cellular immune responses were monitored with ELISPOT, intracellular cytokine staining assay, and flow cytometry, and ELISA assay was used to detect humoral immune responses. The tumor growth and survival of vaccined mice were observed. Results ELISPOT revealed that the mice in PSCA-HSP group generated much more IFN-γ spot-forming cells than those in other groups (P<0.05, and they could generate strong anti-PSCA antibody response. Results of flow cytometry showed that the number of CD8+/IFN-γ+ T cells was significantly higher in PSCAHSP group than that in other groups (P<0.05. ELISA results revealed that all the mice in PSCA, PSCA+HSP and PSCA-HSP group were induced to generate the PSCA-specific humoral immune response, and no statistical difference was found on the antibody levels among the three groups. Animal experiment showed that PSCA-HSP could inhibit the growth of PSCA-expressing tumors and prolong the survival time of vaccinated mice. Conclusion HSP70 is a chaperone with significant effect for protein vaccines, and the recombinant fusion protein PSCA-HSP70 could be of potential value for prostate cancer treatment. DOI: 10.11855/j.issn.0577-7402.2014.09.08

  8. Using a GFP-gene fusion technique to study the cell cycle-dependent distribution of calmodulin in living cells

    Institute of Scientific and Technical Information of China (English)

    李朝军; 吕品; 张东才

    1999-01-01

    In this study, a green fluorescent protein (GFP)-calmodulin (CaM) fusion gene method was used to examine the distribution of calmodulin during various stages of cell cycle. First, it was found that the distribution of CaM in living cells changes with the cell cycle. CaM was found mainly in the cytoplasm during G1 phase. It began to move into the nucleus when the cell entered S phase. At G2 phase, CaM became more concentrated in the nucleus than in cytoplasm. Second, the accumulation of CaM in the nucleus during G2 phase appeared to be related to the onset of mitosis, since inhibiting the activation of CaM at this stage resulted in blocking the nuclear membrane breakdown and chromatin condensation. Finally, after the cell entered mitosis, a high concentration of CaM was found at the polar regions of the mitotic spindle. At this time, inhibiting the activity of CaM would cause a disruption of the spindle structure. The relationship between the stage-specific distribution of CaM and its function in regulat

  9. Establishment of cells to monitor Microprocessor through fusion genes of microRNA and GFP.

    Science.gov (United States)

    Tsutsui, Motomu; Hasegawa, Hitoki; Adachi, Koichi; Miyata, Maiko; Huang, Peng; Ishiguro, Naoki; Hamaguchi, Michinari; Iwamoto, Takashi

    2008-08-01

    Microprocessor, the complex of Drosha and DGCR8, promotes the processing of primary microRNA to precursor microRNA, which is a crucial step for microRNA maturation. So far, no convenient assay systems have been developed for observing this step in vivo. Here we report the establishment of highly sensitive cellular systems where we can visually monitor the function of Microprocessor. During a series of screening of transfectants with fusion genes of the EGFP cDNA and primary microRNA genes, we have obtained certain cell lines where introduction of siRNA against DGCR8 or Drosha strikingly augments GFP signals. In contrast, these cells have not responded to Dicer siRNA; thus they have a unique character that GFP signals should be negatively and specifically correlated to the action of Microprocessor among biogenesis of microRNA. These cell lines can be useful tools for real-time analysis of Microprocessor action in vivo and identifying its novel modulators.

  10. EML4-ALK induces epithelial–mesenchymal transition consistent with cancer stem cell properties in H1299 non-small cell lung cancer cells

    International Nuclear Information System (INIS)

    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

  11. 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.

  12. Cell-cycle dependent expression of a translocation-mediated fusion oncogene mediates checkpoint adaptation in rhabdomyosarcoma.

    Science.gov (United States)

    Kikuchi, Ken; Hettmer, Simone; Aslam, M Imran; Michalek, Joel E; Laub, Wolfram; Wilky, Breelyn A; Loeb, David M; Rubin, Brian P; Wagers, Amy J; Keller, Charles

    2014-01-01

    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. PMID:24453992

  13. 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.

  14. Cloned s-Lap Gene Coding Area, Expression and Localizationof s-Lap/GFP Fusion Protein in Mammal Cells

    Institute of Scientific and Technical Information of China (English)

    SONG Yi-shu; SONG Zhi-yu; LI Hong-jun; Wu Yin; BAO Yong-li; TAN Da-peng; LI Yu-xin

    2005-01-01

    s-Lap is a new gene sequence from pig retinal pigment epithelial(RPE) cells, which was found and cloned in the early period of apoptosis of RPE cells damaged with visible light. We cloned the coding area sequence of the novel gene of s-Lap and constructed its recombinant eukaryotic plasmid pcDNA3.1-GFP/s-lap with the recombinant DNA technique. The expression and localization of s-lap/GFP fusion protein in CHO and B16 cell lines were studied with the instantaneously transfected pcDNA3.1-GFP/s-lap recombinant plasmid. s-Lap/GFP fusion protein can be expressed in CHO and B16 cells with a high rate expression in the nuclei.

  15. Dock mediates Scar- and WASp-dependent actin polymerization through interaction with cell adhesion molecules in founder cells and fusion-competent myoblasts.

    Science.gov (United States)

    Kaipa, Balasankara Reddy; Shao, Huanjie; Schäfer, Gritt; Trinkewitz, Tatjana; Groth, Verena; Liu, Jianqi; Beck, Lothar; Bogdan, Sven; Abmayr, Susan M; Önel, Susanne-Filiz

    2013-01-01

    The formation of the larval body wall musculature of Drosophila depends on the asymmetric fusion of two myoblast types, founder cells (FCs) and fusion-competent myoblasts (FCMs). Recent studies have established an essential function of Arp2/3-based actin polymerization during myoblast fusion, formation of a dense actin focus at the site of fusion in FCMs, and a thin sheath of actin in FCs and/or growing muscles. The formation of these actin structures depends on recognition and adhesion of myoblasts that is mediated by cell surface receptors of the immunoglobulin superfamily. However, the connection of the cell surface receptors with Arp2/3-based actin polymerization is poorly understood. To date only the SH2-SH3 adaptor protein Crk has been suggested to link cell adhesion with Arp2/3-based actin polymerization in FCMs. Here, we propose that the SH2-SH3 adaptor protein Dock, like Crk, links cell adhesion with actin polymerization. We show that Dock is expressed in FCs and FCMs and colocalizes with the cell adhesion proteins Sns and Duf at cell-cell contact points. Biochemical data in this study indicate that different domains of Dock are involved in binding the cell adhesion molecules Duf, Rst, Sns and Hbs. We emphasize the importance of these interactions by quantifying the enhanced myoblast fusion defects in duf dock, sns dock and hbs dock double mutants. Additionally, we show that Dock interacts biochemically and genetically with Drosophila Scar, Vrp1 and WASp. Based on these data, we propose that Dock links cell adhesion in FCs and FCMs with either Scar- or Vrp1-WASp-dependent Arp2/3 activation.

  16. Viral membrane fusion

    Energy Technology Data Exchange (ETDEWEB)

    Harrison, Stephen C., E-mail: harrison@crystal.harvard.edu

    2015-05-15

    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.

  17. Viral membrane fusion

    International Nuclear Information System (INIS)

    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

  18. Cell Death Mechanisms Induced by Cytotoxic Lymphocytes

    Institute of Scientific and Technical Information of China (English)

    Ch(a)vez-Gal(a)n L; Arenas-Del Angel MC; Zenteno E; Ch(a)vez R; Lascurain R

    2009-01-01

    One of the functions of the immune system is to recognize and destroy abnormal or infected cells to maintain homeostasis. This is accomplished by cytotoxic lymphocytes. Cytotoxicity is a highly organized multifactor process. Here, we reviewed the apoptosis pathways induced by the two main cytotoxic lymphocyte subsets, natural killer (NK) cells and CD8+T cells. In base to recent experimental evidence, we reviewed NK receptors involved in recognition of target-cell, as well as lytic molecules such as perforin, granzymes-A and -B, and granulysin. In addition, we reviewed the Fas-FasL intercellular linkage mediated pathway, and briefly the cross-linking of tumor necrosis factor (TNF) and TNF receptor pathway. We discussed three models of possible molecular interaction between lyric molecules from effector cytotoxic cells and target-cell membrane to induction of apoptosis.

  19. Dielectrophoretic forces and potentials induced on pairs of cells in an electric field.

    Science.gov (United States)

    Foster, K R; Sowers, A E

    1995-09-01

    A combined numerical/experimental study is reported of the membrane potentials and dielectrophoretically induced forces between cells, membrane pressures, and velocity of attraction of cells under the influence of an electric field. This study was designed to explore electrical and mechanical effects produced by a field on cells in close proximity or undergoing electrically induced fusion. Laplace's equation for pairs of membrane-covered spheres in close proximity was solved numerically by the boundary element method, and the electrically induced forces on the cells and between cells were obtained by evaluating the Maxwell stress tensor. The velocity of approach of erythrocyte ghosts or fused ghosts in a 60-Hz field of 6 V/mm was measured experimentally, and the data were interpreted by using Batchelor's theory for hydrodynamic interaction of hard spheres. The numerical results show clearly the origin of the dielectrophoretic pressures and forces in fused and unfused cells and the effects of a nearby cell on the induced membrane potentials. The experimental results agree well with predictions based on the simple electrical model of the cell. The analysis shows the strong effect of hydrodynamic interactions between the cells in determining their velocity of approach. PMID:8519978

  20. Overexpression of p27KIP1 induced cell cycle arrest in G1 phase and subsequent apoptosis in HCC-9204 cell line

    Institute of Scientific and Technical Information of China (English)

    Jiang Li; Wen Liang Wang; Xin Ke Yang; Xin Xin Yu; Yun De Hou; Meng Liang Ge; Jie Zhang

    2000-01-01

    AIM We have previously reported that inducible over-expresaion of Bak may prolong cell cycle in G1 phase and lead to apoptosis in HCC-9204 cells. This study is to investigate whether p27KIP1 plays an important role in this process. MEHODS In order to elucidate the exact function of p27KIP1 in this process, a zinc inducible p27KIP1 stable transfectant and transient p27KIP1- GFP fusion transfectant were constructed. The effects of inducible p27KIP1 on cell growth, cell cycle arrest and apoptosis were examined in the mock, control pMD vector, and pMD-KIP1 transfected HCC-9204 cells. RESULTS This p27KIP1-GFP transfectant may transiently express the fusion gene. The cell growth was reduced by 35% at 48 h of p27KIP1 induction with zinc treatment as determined by trypan blue exclusion assay. These differences remained the same after 72 h of p27KIP1 expression, p27KIP1 caused cell cycle arrest after 24 h of induction, with 40% increase in G1 population. Prolonged p27KIP1 expression in this cell line induced apoptotic cell death reflected by TUNEL assay. Fourty-eight h and 72 h of p27KIP1 expression showed a characteristic DNA ladder on agarose gel electrophoresis.

  1. HAM-5 functions as a MAP kinase scaffold during cell fusion in Neurospora crassa.

    Directory of Open Access Journals (Sweden)

    Wilfried Jonkers

    2014-11-01

    Full Text Available 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

  2. 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.

  3. Trophoblast lineage cells derived from human induced pluripotent stem cells

    International Nuclear Information System (INIS)

    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

  4. Baicalin induced dendritic cell apoptosis in vitro

    Directory of Open Access Journals (Sweden)

    Huahua eZhang

    2011-03-01

    Full Text Available This study was aimed to investigate the effects of Baicalin (BA, a major flavonoid constituent found in the herb Baikal skullcap, on dendritic cells (DCs. DCs were generated by culturing murine bone marrow cells for 6 days with granulocyte-macrophage colony-stimulating factor and interleukin-4, and lipopolysaccharide (LPS was added on day 5 to stimulate DCs maturation. The expression levels of DC maturity markers (CD80/CD86 were assessed by flow cytometry using direct immunofluorescence method. Interleukin-12 (IL-12 levels in the culture supernatants were assayed by ELISA. Apoptosis of DCs was analyzed by flow cytometry after Annexin V/propidium iodide staining. The mitochondrial membrane potential changes were measured by using the J-aggregate forming lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1. Exposure of DCs to BA (2-50 microM during bone marrow cell differentiation showed no effects on the up-regulation of CD80/CD86 expression on DCs in response to LPS stimulation, but reduced DCs recovery by inducing apoptosis, and significantly inhibited the release of IL-12 to culture supernatants. BA induced DC apoptosis in a time- and dose-dependent way, and immature DCs were more sensitive for BA-induced apoptosis than mature DC. BA also induced mitochondrial membrane potential changes in DCs. These results demonstrate that BA induces selective apoptosis in immature DCs possibly through mitochondria-mediated pathway.

  5. Dynamical approach to heavy-ion induced fusion using actinide target

    Energy Technology Data Exchange (ETDEWEB)

    Aritomo, Y.; Hagino, K.; Chiba, S.; Nishio, K. [Flerov Laboratory of Nuclear Reactions, JINR, Dubna, 141980 (Russian Federation); Department of Physics, Tohoku University, Sendai 980-8578 (Japan); Research Laboratory for Nuclear Reactors, Tokyo Institute of Technology, Tokyo 152-8550 (Japan); Japan Atomic Energy Agency, Tokai, Ibaraki, 319-1195 (Japan)

    2012-10-20

    To treat heavy-ion reactions using actinide target nucleus, we propose a model which takes into account the coupling to the collective states of interacting nuclei in the penetration of the Coulomb barrier and the dynamical evolution of nuclear shape from the contact configuration. A fluctuation-dissipation model (Langevin equation) was applied in the dynamical calculation, where effect of nuclear orientation at the initial impact on the prolately deformed target nucleus was considered. Using this model, we analyzed the experimental data for the mass distribution of fission fragments (MDFF) in the reaction of {sup 36}S+{sup 238}U at several incident energies. Fusion-fission, quasifission and deep-quasi-fission are separated as different trajectories on the potential energy surface. We estimated the fusion cross section of the reaction.

  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. Neutron-induced dpa, transmutations, gas production, and helium embrittlement of fusion materials

    OpenAIRE

    Gilbert, M. R.; Dudarev, S. L.; Nguyen-Manh, D.; S. Zheng; Packer, L.W.; Sublet, J. -Ch.

    2013-01-01

    In a fusion reactor materials will be subjected to significant fluxes of high-energy neutrons. As well as causing radiation damage, the neutrons also initiate nuclear reactions leading to changes in the chemical composition of materials (transmutation). Many of these reactions produce gases, particularly helium, which cause additional swelling and embrittlement of materials. This paper investigates, using a combination of neutron-transport and inventory calculations, the variation in displace...

  8. 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”.

  9. Heat induces gene amplification in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Bin, E-mail: yanbin@mercyhealth.com [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Mercy Cancer Center, Mercy Medical Center-North Iowa, Mason City, IA 50401 (United States); Ouyang, Ruoyun [Department of Respiratory Medicine, The Second Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410011 (China); Huang, Chenghui [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Department of Oncology, The Third Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410013 (China); Liu, Franklin [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States); Neill, Daniel [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Li, Chuanyuan [Dermatology, Duke University Medical Center, Durham, NC 27710 (United States); Dewhirst, Mark [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States)

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer This study discovered that heat exposure (hyperthermia) results in gene amplification in cancer cells. Black-Right-Pointing-Pointer Hyperthermia induces DNA double strand breaks. Black-Right-Pointing-Pointer DNA double strand breaks are considered to be required for the initiation of gene amplification. Black-Right-Pointing-Pointer 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 Degree-Sign 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) {gamma}H2AX immunostaining to detect {gamma}H2AX foci as an indication for DNA double strand breaks. Results: (1) Heat exposure at 42 or 44 Degree-Sign 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 Degree-Sign C for 30 min induces DNA double strand breaks in HCT116 cells as shown by {gamma}H2AX 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

  10. Heat induces gene amplification in cancer cells

    International Nuclear Information System (INIS)

    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.

  11. Spine Fusion Using Cell Matrix Composites Enriched in Bone Marrow-Derived Cells

    OpenAIRE

    Muschler, George F.; Nitto, Hironori; Matsukura, Yoichi; Boehm, Cynthia; Valdevit, Antonio; Kambic, Helen; Davros, William; Powell, Kimerly; Easley, Kirk

    2003-01-01

    Bone marrow-derived cells including osteoblastic progenitors can be concentrated rapidly from bone marrow aspirates using the surface of selected implantable matrices for selective cell attachment. Concentration of cells in this way to produce an enriched cellular composite graft improves graft efficacy. The current study was designed to test the hypothesis that the biologic milieu of a bone marrow clot will significantly improve the efficacy of such a graft. An established posterior spinal f...

  12. Retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2 in mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Zhang Yingang; Guo Xiong; Liu Zheng; Wang Shijie

    2007-01-01

    Objective To develop retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2 in mesenchymal stem cells. Methods Mesenchymal stem cells from New Zealand white rabbits were transduced with retroviral pLEGFP-BMP2 vector by the optimized retroviral transduction protocol. Fluorescent microscopy's examination was to evaluate the results of the transduction, flow cytometer's analysis was to evaluate the transduction efficiency and the Fluorescence-activated cell sorting method was to sort the transduced cells. Bioactivity test from C2C12K4 cells was to show the expression and bio-activity of the fusion gene. Results Fluorescent microscopy showed the success of the transduction. By flow cytometer's analysis, the mean efficiency of the transduction with EGFP was (42.8±6.1)% SD. Transduced cells were sorted efficiently by the fluorescence-activated cell sorting method and after sorting, almost of those showed the expression of BMP2. Fluorescently and strongly bioactivity test for C2C12K4 cells demonstrated that fluorescent materials were located the surface of cells and the activity of luciferase increased compared with the control. Analysis of long-term expression showed there was no difference between 2 week-time point and 3 month-time point of culture post-sorting. Conclusion Mesenchymal stem cells can be transduced efficiently by retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2, the highly pure transduced cells are obtained by the fluorescence-activated cell sorting technique, the expressed chimeric protein embraced the double bioactivity of EGFP and BMP2, and moreover, the expression had not attenuated over time.

  13. The “Grep” Command But Not FusionMap, FusionFinder or ChimeraScan Captures the CIC-DUX4 Fusion Gene from Whole Transcriptome Sequencing Data on a Small Round Cell Tumor with t(4;19)(q35;q13)

    OpenAIRE

    Ioannis Panagopoulos; Ludmila Gorunova; Bodil Bjerkehagen; Sverre Heim

    2014-01-01

    Whole transcriptome sequencing was used to study a small round cell tumor in which a t(4;19)(q35;q13) was part of the complex karyotype but where the initial reverse transcriptase PCR (RT-PCR) examination did not detect a CIC-DUX4 fusion transcript previously described as the crucial gene-level outcome of this specific translocation. The RNA sequencing data were analysed using the FusionMap, FusionFinder, and ChimeraScan programs which are specifically designed to identify fusion genes. Fusio...

  14. Resveratrol induces apoptosis in pancreatic cancer cells

    Institute of Scientific and Technical Information of China (English)

    ZHOU Jia-hua; CHENG Hai-yan; YU Ze-qian; HE Dao-wei; PAN Zheng; YANG De-tong

    2011-01-01

    Background Pancreatic cancer is one of the most lethal human cancers with a very low survival rate of 5 years.Conventional cancer treatments including surgery, radiation, chemotherapy or combinations of these show little effect on this disease. Several proteins have been proved critical to the development and the progression of pancreatic cancer.The aim of this study was to investigate the effect of resveratrol on apoptosis in pancreatic cancer cells.Methods Several pancreatic cancer cell lines were screened by resveratrol, and its toxicity was tested by normal pancreatic cells. Western blotting was then performed to analyze the molecular mechanism of resveratrol induced apoptosis of pancreatic cancer cell lines.Results In the screened pancreatic cancer cell lines, capan-2 and colo357 showed high sensitivity to resveratrol induced apoptosis. Resveratrol exhibited insignificant toxicity to normal pancreatic cells. In resveratrol sensitive cells,capan-2 and colo357, the activation of caspase-3 was detected and showed significant caspase-3 activation upon resveratrol treatment; p53 and p21 were also detected up-regulated upon resveratrol treatment.Conclusion Resveratrol provides a promising anti-tumor stratagy to fight against pancreatic cancer.

  15. 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.

  16. In search of nuclear fusion in electrolytic cells and in metal/gas systems

    Science.gov (United States)

    McCracken, D. R.; Paquette, J.; Boniface, H. A.; Graham, W. R. C.; Johnson, R. E.; Briden, N. A.; Cross, W. G.; Arneja, A.; Tennant, D. C.; Lone, M. A.; Buyers, W. J. L.; Chambers, K. W.; McIlwain, A. K.; Attas, E. M.; Dutton, R.

    1990-06-01

    It has been reported recently in the literature that unexpected thermal and nuclear effects (production of excess heat, neutrons, γ-rays, and tritium) can occur during the electrolysis of heavy water at palladium or titanium electrodes, or during temperature and pressure cycling of the titanium/deuterium gas system. We have attempted to reproduce some of these experiments. A variety of electrochemical cells having palladium cathodes in the form of wires, tubes, sheets, and rods have been used to electrolyze heavy water containing 0.1 mol.dm-3 LiOH, 0.1 mol.dn-3 LiOD or 0.5 mol.dm-3 D3PO4. Current densities of up to 200 mA.cm-2 were applied. The mass of the palladium cathodes covered the range from 1-40 grams and the surface area varied from 8-140 cm2. Neutron detection systems with low constant backgrounds were used to search for neutron emission during electrolysis. These included3He- and10BF3-based detectors. After running some of the cells for more than 30 days, no neutron emission above background could be detected. This puts upper limits of 0.5 s-1 and 2×10-23 fus. D-D.s-1 on the neutron emission and the fusion rate, respectively. A sensitive and accurate heat-flow calorimeter was built and used to monitor the energy balance of some of the cells during electrolysis. No unexpected heat effects were observed. This puts an upper limit of 0.13 W.cm-3 on the specific excess power. No enrichment of the electrolyte in tritium was evident after electrolysis. Experiments were also performed with the titanium/ deuterium gas system. These consisted of exposing titanium metal to a deuterium gas pressure of 40 atmospheres, lowering the temperature to -196°C, releasing the pressure and gradually warming the titanium to room temperature. No neutron emission above background was observed during these experiments, which puts upper limits of 0.5 s-1 and 4×10-25 fus.D-D.s-1 on the neutron emission and fusion rate, respectively.

  17. 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)

    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

  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. Internalization and fate of silica nanoparticles in C2C12 skeletal muscle cells: evidence of a beneficial effect on myoblast fusion

    Directory of Open Access Journals (Sweden)

    Poussard S

    2015-02-01

    Full Text Available Sylvie Poussard,1,2 Marion Decossas,1,2 Olivier Le Bihan,1,2 Stéphane Mornet,3 Grégoire Naudin,1,2 Olivier Lambert1,2 1Institute of Chemistry and Biology of Membranes and Nanoobjects, University of Bordeaux, UMR5248, Pessac, France; 2Institute of Chemistry and Biology of Membranes and Nanoobjects, Centre National de la Recherche Scientifique, Institute of Chemistry and Biology of Membranes and Nanoobjects, UMR5248, Pessac, France; 3ICMCB, Institut de Chimie de la Matière Condensée de Bordeaux, CNRS UPR9048, Université de Bordeaux, Pessac, France Abstract: 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. Keywords: silica, nanoparticle, muscle, cell encapsulation, transmission electron microscopy, apoptosis

  20. 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.

  1. Retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP_2 in mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Bone marrow mesenchymal stemcells(MSCs)are pluripotential stemcells that have the capacitytodifferentiate into chondrocytes and osteoblasts[1].Ithas been well documented that bone morphogeneticproteins(BMPs),a group of proteins belonging tothe TGF-βsuperfamily,can induce bone for mationbothin vivoandin vitroas well as promote osteo-blastic differentiation of MSC[2].HeterologousBMP2is successfully transferred to MSCs and genetherapy is employed based on repairing bony andcartilage defects,spinal fusion[3-5]....

  2. 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

  3. Expression of Chlamydomonas actin-gfp fusion gene in to-bacco suspension cell and polymerization of the actin-gfp protein in vitro

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The fusion gene of actin (cDNA of Chlamydo- monas reinhardtii) and green fluorescence protein (gfp) had been constructed into two expression vectors which could be expressed in E. coli and tobacco suspension cells BY2. The correct expression was observed in E. coli and BY2 with a fluorescence microscopy. The fusion protein, which took part in the membrane skeleton, was mainly located peripherally along the membrane, specially the fusion protein was dis-tributed around nucleus and cell plate, while the fusion pro-tein also forms F-actin in the cell. The fusion protein was purified from Bl21plus by ammonium sulfate fractionation, ion exchange chromatography and hydrophobic interaction chromatography. The purified production could polymerize into F-actin when the actin polymerizing buffer was added. It was demonstrated that the characteristics and function of actin in Chlamydomonas was similar with those of animals and higher plants.

  4. Prognostic significance of NPM-ALK fusion transcript overexpression in ALK-positive anaplastic large-cell lymphoma.

    Science.gov (United States)

    Li, Chunmei; Takino, Hisashi; Eimoto, Tadaaki; Ishida, Takashi; Inagaki, Atsushi; Ueda, Ryuzo; Suzuki, Ritsuro; Yoshino, Tadashi; Nakagawa, Atsuko; Nakamura, Shigeo; Inagaki, Hiroshi

    2007-06-01

    In anaplastic large-cell lymphomas positive for anaplastic lymphoma kinase (ALK) protein, the ALK gene is most commonly fused to the NPM gene, and less commonly to TPM3, TFG, ATIC, and other rare genes. Although this lymphoma is generally associated with a favorable clinical outcome, 25% of the patients die of the disease within 5 years. In this study, we developed three assays, all of which can be used with archival formalin-fixed, paraffin-embedded tissues: (1) a sensitive reverse transcription-polymerase chain reaction (RT-PCR) assay for various X-ALK fusion genes, (2) a 5' rapid amplification of cDNA ends (RACE) assay to identify unknown fusion partners, and (3) a real-time RT-PCR assay to quantify the amount of the NPM-ALK fusion transcript. In 26 cases of ALK(+) anaplastic large-cell lymphoma, the RT-PCR assay showed that the ALK was fused to NPM in 21 cases, to TPM3 in three, and to TFG in one. The 5' RACE assay detected ATIC-ALK fusion in the remaining case. The real-time quantitative RT-PCR assay showed that the NPM-ALK transcript was over expressed in four of 20 quantifiable cases. Patients with NPM-ALK overexpression showed a significantly unfavorable overall survival compared with those with a low expression of this transcript. The RT-PCR and 5' RACE assays developed here may be useful for identification of known and unknown gene partners fused to the ALK gene. Overexpression of the NPM-ALK fusion transcript may be associated with a poor prognosis of the patients with ALK(+) anaplastic large-cell lymphomas.

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

    OpenAIRE

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

    2013-01-01

    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...

  6. Khz (fusion of Ganoderma lucidum and Polyporus umbellatus mycelia induces apoptosis by increasing intracellular calcium levels and activating JNK and NADPH oxidase-dependent generation of reactive oxygen species.

    Directory of Open Access Journals (Sweden)

    Tae Hwan Kim

    Full Text Available Khz is a compound derived from the fusion of Ganoderma lucidum and Polyporus umbellatus mycelia that inhibits the growth of cancer cells. The results of the present study show that Khz induced apoptosis preferentially in transformed cells and had only minimal effects on non-transformed cells. Furthermore, Khz induced apoptosis by increasing the intracellular Ca(2+ concentration ([Ca(2+](i and activating JNK to generate reactive oxygen species (ROS via NADPH oxidase and the mitochondria. Khz-induced apoptosis was caspase-dependent and occurred via a mitochondrial pathway. ROS generation by NADPH oxidase was critical for Khz-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 demonstrated by the translocation of regulatory subunits p47(phox and p67(phox to the cell membrane and was necessary for ROS generation by Khz. Khz triggered a rapid and sustained increase in [Ca(2+](i, which activated JNK. JNK plays a key role in the activation of NADPH oxidase because inhibition of its expression or activity abrogated membrane translocation of the p47(phox and p67(phox subunits and ROS generation. In summary, these data indicate that Khz preferentially induces apoptosis in cancer cells, and the signaling mechanisms involve an increase in [Ca(2+](i, JNK activation, and ROS generation via NADPH oxidase and mitochondria.

  7. 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.

  8. 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.

  9. 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

  10. Paraquat-induced radiosensitization of mammalian cells

    International Nuclear Information System (INIS)

    The herbicide, paraquat (methyl viologen, 1-1' dimethy1-4, 4'-bipyridinium dichloride), stimulates the production of superoxide anion (O2sup(-.)) in aerobic cells and therefore mimics some effects of ionizing radiation. In addition, concentrations of cellular glutathione are reduced by reaction with O2sup(-.). It is reported here that paraquat, toxic in its own right to aerobic cells, acts as a radiosensitizer when cells are exposed to nontoxic concentrations of the drug prior to and during irradiation. The radiomimetic effect of paraquat, alone and in combination with X-rays, was examined. Paraquat affects aerated cells (hamster lung V79 cells) in a dose-dependent manner. Doses in excess of 1 mM for two hours cause significant cell killing. In combination with radiation, sublethal doses of paraquat, given for two hours prior to irradiation, enhance the lethal effects of radiation. However, if cells are exposed to the same concentration of paraquat following irradiation, no additional lethal effect is observed. Paraquat is a useful tool to study the effects of O2sup(-.) and may lead to better understanding of the mechanisms of radiation-induced energy deposition in cells. (author)

  11. Induced pluripotent stem cells: advances to applications

    Directory of Open Access Journals (Sweden)

    Timothy J Nelson

    2009-12-01

    Full Text Available Timothy J Nelson1, Almudena Martinez-Fernandez1, Satsuki Yamada1, Yasuhiro Ikeda2, Carmen Perez-Terzic1, Andre Terzic11Marriott Heart Disease Research Program, Division of Cardiovascular Diseases, Mayo Clinic, Rochester, Minnesota, USA; 2Department of Molecular Medicine; Mayo Clinic, Rochester, Minnesota, USAAbstract: Induced pluripotent stem cell (iPS technology has enriched the armamentarium of regenerative medicine by introducing autologous pluripotent progenitor pools bioengineered from ordinary somatic tissue. Through nuclear reprogramming, patient-specific iPS cells have been derived and validated. Optimizing iPS-based methodology will ensure robust applications across discovery science, offering opportunities for the development of personalized diagnostics and targeted therapeutics. Here, we highlight the process of nuclear reprogramming of somatic tissues that, when forced to ectopically express stemness factors, are converted into bona fide pluripotent stem cells. Bioengineered stem cells acquire the genuine ability to generate replacement tissues for a wide-spectrum of diseased conditions, and have so far demonstrated therapeutic benefit upon transplantation in model systems of sickle cell anemia, Parkinson’s disease, hemophilia A, and ischemic heart disease. The field of regenerative medicine is therefore primed to adopt and incorporate iPS cell-based advancements as a next generation stem cell platforms.Keywords: iPS, regenerative medicine, individualized medicine, stem cell therapy

  12. Construction of a fusion protein expression vector MK-EGFP and its subcellular localization in different carcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    Li-Cheng Dai; Di-Yong Xu; Xing Yao; Li-Shan Min; Ning Zhao; Bo-Ying Xu; Zheng-Ping Xu; Yong-Liang Lu

    2006-01-01

    AIM: To construct an expression plasmid encoding human wild-type midkine (MK) and enhanced green fluorescence protein (EGFP) fusion protein (MK-EGFP), and to analyze the subcellular localization of MK in different carcinoma cell lines.METHODS: Two kinds of MK coding sequences with or without signal peptide were cloned into plasmid pEGFP-N2, and the recombinant plasmids constructed were introduced into HepG2, MCF7 and DU145 cells,respectively, by transfection. With the help of laser scanning confocal microscopy, the expression and subcellular localization of MK-GFP fusion protein could be detected.RESULTS: Compared with the GFP control, in which fluorescence was detected diffusely over the entire cell body except in the nucleolus, both kinds of fusion protein MK-GFP were localized exclusively to the nucleus and accumulated in the nucleolus in the three kinds of cancer cell lines.CONCLUSION: This study reveals the specific nucleolar translocation independent of signal peptide, which may be involved in the mechanism that MK works. It provides valuable evidence for further study on the functions of MK in nucleus and its possible mechanisms, in which ribosomal RNA transcription and ribosome assembly are involved.

  13. 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.

  14. A two photon absorption laser induced fluorescence diagnostic for fusion plasmas

    Energy Technology Data Exchange (ETDEWEB)

    Magee, R. M.; Galante, M. E.; McCarren, D.; Scime, E. E. [Physics Department, West Virginia University, Morgantown, West Virginia 26506 (United States); Boivin, R. L.; Brooks, N. H.; Groebner, R. J.; Hill, D. N. [General Atomics, San Diego, California 92121 (United States); Porter, G. D. [Lawrence Livermore National Laboratory, Livermore, California 94550 (United States)

    2012-10-15

    The quality of plasma produced in a magnetic confinement fusion device is influenced to a large extent by the neutral gas surrounding the plasma. The plasma is fueled by the ionization of neutrals, and charge exchange interactions between edge neutrals and plasma ions are a sink of energy and momentum. Here we describe a diagnostic capable of measuring the spatial distribution of neutral gas in a magnetically confined fusion plasma. A high intensity (5 MW/cm{sup 2}), narrow bandwidth (0.1 cm{sup -1}) laser is injected into a hydrogen plasma to excite the Lyman {beta} transition via the simultaneous absorption of two 205 nm photons. The absorption rate, determined by measurement of subsequent Balmer {alpha} emission, is proportional to the number of particles with a given velocity. Calibration is performed in situ by filling the chamber to a known pressure of neutral krypton and exciting a transition close in wavelength to that used in hydrogen. We present details of the calibration procedure, including a technique for identifying saturation broadening, measurements of the neutral density profile in a hydrogen helicon plasma, and discuss the application of the diagnostic to plasmas in the DIII-D tokamak.

  15. Alloy development for fast induced radioactivity decay for fusion reactor applications

    International Nuclear Information System (INIS)

    The Cr-Mo ferritic (martensitic) steels and austenitic stainless steels (primarily type 316 and variations on that composition) are the leading candidates for the structural components for future fusion reactors. However, irradiation of such steels in a fusion environment produces long-lived radioactive isotopes. These isotopes lead to difficult radioactive waste disposal problems once the structure is removed from service. Such problems could be reduced by developing steels that contain only elements that produce radioactive isotopes that decay to low levels in a reasonable time (tens of years instead of hundreds or thousands of years). This report discusses the development of such steels by making elemental substitutions in the steels now under consideration. Molybdenum must be replaced in the Cr-Mo steels; nickel and molybdenum both must be replaced in the austenitic stainless steels (the nitrogen concentration must be limited, and niobium, maintained to extremely low levels). Appropriate substitutions are tungsten for molybdenum in the Cr-Mo steels and manganese for nickel in the austenitic stainless steels. Indications are that suitable ferritic steels can be developed, but development of a nickel-free austenitic stainless steel with properties similar to the Cr-Ni stainless steels appears to be much more complex

  16. Inducible cell death in plant immunity

    DEFF Research Database (Denmark)

    Hofius, Daniel; Tsitsigiannis, Dimitrios I; Jones, Jonathan D G;

    2006-01-01

    Programmed cell death (PCD) occurs during vegetative and reproductive plant growth, as typified by autumnal leaf senescence and the terminal differentiation of the endosperm of cereals which provide our major source of food. PCD also occurs in response to environmental stress and pathogen attack......, and these inducible PCD forms are intensively studied due their experimental tractability. In general, evidence exists for plant cell death pathways which have similarities to the apoptotic, autophagic and necrotic forms described in yeast and metazoans. Recent research aiming to understand these pathways...... and their molecular components in plants are reviewed here....

  17. Induced Pluripotent Stem Cells Meet Genome Editing.

    Science.gov (United States)

    Hockemeyer, Dirk; Jaenisch, Rudolf

    2016-05-01

    It is extremely rare for a single experiment to be so impactful and timely that it shapes and forecasts the experiments of the next decade. Here, we review how two such experiments-the generation of human induced pluripotent stem cells (iPSCs) and the development of CRISPR/Cas9 technology-have fundamentally reshaped our approach to biomedical research, stem cell biology, and human genetics. We will also highlight the previous knowledge that iPSC and CRISPR/Cas9 technologies were built on as this groundwork demonstrated the need for solutions and the benefits that these technologies provided and set the stage for their success. PMID:27152442

  18. Generation and characterization of induced pluripotent stem cells from Aid-deficient mice.

    Directory of Open Access Journals (Sweden)

    Ren Shimamoto

    Full Text Available It has been shown that DNA demethylation plays a pivotal role in the generation of induced pluripotent stem (iPS cells. However, the underlying mechanism of this action is still unclear. Previous reports indicated that activation-induced cytidine deaminase (Aid, also known as Aicda is involved in DNA demethylation in several developmental processes, as well as cell fusion-mediated reprogramming. Based on these reports, we hypothesized that Aid may be involved in the DNA demethylation that occurs during the generation of iPS cells. In this study, we examined the function of Aid in iPS cell generation using Aid knockout (Aid⁻/⁻ mice expressing a GFP reporter under the control of a pluripotent stem cell marker, Nanog. By introducing Oct3/4, Sox2, Klf4 and c-Myc, Nanog-GFP-positive iPS cells could be generated from the fibroblasts and primary B cells of Aid⁻/⁻ mice. Their induction efficiency was similar to that of wild-type (Aid⁺/⁺ iPS cells. The Aid⁻/⁻ iPS cells showed normal proliferation and gave rise to chimeras, indicating their capacity for self-renewal and pluripotency. A comprehensive DNA methylation analysis showed only a few differences between Aid⁺/⁺ and Aid⁻/⁻ iPS cells. These data suggest that Aid does not have crucial functions in DNA demethylation during iPS cell generation.

  19. Reciprocal t(9;22 ABL/BCR fusion proteins: leukemogenic potential and effects on B cell commitment.

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    Xiaomin Zheng

    Full Text Available BACKGROUND: t(9;22 is a balanced translocation, and the chromosome 22 breakpoints (Philadelphia chromosome--Ph+ determine formation of different fusion genes that are associated with either Ph+ acute lymphatic leukemia (Ph+ ALL or chronic myeloid leukemia (CML. The "minor" breakpoint in Ph+ ALL encodes p185(BCR/ABL from der22 and p96(ABL/BCR from der9. The "major" breakpoint in CML encodes p210(BCR/ABL and p40(ABL/BCR. Herein, we investigated the leukemogenic potential of the der9-associated p96(ABL/BCR and p40(ABL/BCR fusion proteins and their roles in the lineage commitment of hematopoietic stem cells in comparison to BCR/ABL. METHODOLOGY: All t(9;22 derived proteins were retrovirally expressed in murine hematopoietic stem cells (SL cells and human umbilical cord blood cells (UCBC. Stem cell potential was determined by replating efficiency, colony forming--spleen and competitive repopulating assays. The leukemic potential of the ABL/BCR fusion proteins was assessed by in a transduction/transplantation model. Effects on the lineage commitment and differentiation were investigated by culturing the cells under conditions driving either myeloid or lymphoid commitment. Expression of key factors of the B-cell differentiation and components of the preB-cell receptor were determined by qRT-PCR. PRINCIPAL FINDINGS: Both p96(ABL/BCR and p40(ABL/BCR increased proliferation of early progenitors and the short term stem cell capacity of SL-cells and exhibited own leukemogenic potential. Interestingly, BCR/ABL gave origin exclusively to a myeloid phenotype independently from the culture conditions whereas p96(ABL/BCR and to a minor extent p40(ABL/BCR forced the B-cell commitment of SL-cells and UCBC. CONCLUSIONS/SIGNIFICANCE: Our here presented data establish the reciprocal ABL/BCR fusion proteins as second oncogenes encoded by the t(9;22 in addition to BCR/ABL and suggest that ABL/BCR contribute to the determination of the leukemic phenotype through their

  20. DRAM1 Protects Neuroblastoma Cells from Oxygen-Glucose Deprivation/Reperfusion-Induced Injury via Autophagy

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    Mengqiang Yu

    2014-10-01

    Full Text Available DNA damage-regulated autophagy modulator protein 1 (DRAM1, a multi-pass membrane lysosomal protein, is reportedly a tumor protein p53 (TP53 target gene involved in autophagy. During cerebral ischemia/reperfusion (I/R injury, DRAM1 protein expression is increased, and autophagy is activated. However, the functional significance of DRAM1 and the relationship between DRAM1 and autophagy in brain I/R remains uncertain. The aim of this study is to investigate whether DRAM1 mediates autophagy activation in cerebral I/R injury and to explore its possible effects and mechanisms. We adopt the oxygen-glucose deprivation and reperfusion (OGD/R Neuro-2a cell model to mimic cerebral I/R conditions in vitro, and RNA interference is used to knock down DRAM1 expression in this model. Cell viability assay is performed using the LIVE/DEAD viability/cytotoxicity kit. Cell phenotypic changes are analyzed through Western blot assays. Autophagy flux is monitored through the tandem red fluorescent protein–Green fluorescent protein–microtubule associated protein 1 light chain 3 (RFP–GFP–LC3 construct. The expression levels of DRAM1 and microtubule associated protein 1 light chain 3II/I (LC3II/I are strongly up-regulated in Neuro-2a cells after OGD/R treatment and peaked at the 12 h reperfusion time point. The autophagy-specific inhibitor 3-Methyladenine (3-MA inhibits the expression of DRAM1 and LC3II/I and exacerbates OGD/R-induced cell injury. Furthermore, DRAM1 knockdown aggravates OGD/R-induced cell injury and significantly blocks autophagy through decreasing autophagosome-lysosome fusion. In conclusion, our data demonstrate that DRAM1 knockdown in Neuro-2a cells inhibits autophagy by blocking autophagosome-lysosome fusion and exacerbated OGD/R-induced cell injury. Thus, DRAM1 might constitute a new therapeutic target for I/R diseases.

  1. Uniaxial cyclic strain enhances adipose-derived stem cell fusion with skeletal myocytes

    Energy Technology Data Exchange (ETDEWEB)

    Andersen, Jens Isak; Juhl, Morten; Nielsen, Thøger; Emmersen, Jeppe; Fink, Trine; Zachar, Vladimir; Pennisi, Cristian Pablo, E-mail: cpennisi@hst.aau.dk

    2014-07-25

    Highlights: • Uniaxial cyclic tensile strain (CTS) applied to ASCs alone or in coculture with myogenic precursors. • CTS promoted the formation of a highly ordered array of parallel ASCs. • Without biochemical supplements, CTS did not support advanced myogenic differentiation of ASCs. • Mechanical stimulation of cocultures boosted fusion of ASCs with skeletal myoblasts. - Abstract: Although adult muscle tissue possesses an exceptional capacity for regeneration, in the case of large defects, the restoration to original state is not possible. A well-known source for the de novo regeneration is the adipose-derived stem cells (ASCs), which can be readily isolated and have been shown to have a broad differentiation and regenerative potential. In this work, we employed uniaxial cyclic tensile strain (CTS), to mechanically stimulate human ASCs to participate in the formation skeletal myotubes in an in vitro model of myogenesis. The application of CTS for 48 h resulted in the formation of a highly ordered array of parallel ASCs, but failed to support skeletal muscle terminal differentiation. When the same stimulation paradigm was applied to cocultures with mouse skeletal muscle myoblasts, the percentage of ASCs contributing to the formation of myotubes significantly exceeded the levels reported in the literature hitherto. In perspective, the mechanical strain may be used to increase the efficiency of incorporation of ASCs in the skeletal muscles, which could be found useful in diverse traumatic or pathologic scenarios.

  2. Modeling Electrophysiological Coupling and Fusion between Human Mesenchymal Stem Cells and Cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Joshua Mayourian

    2016-07-01

    Full Text Available Human mesenchymal stem cell (hMSC delivery has demonstrated promise in preclinical and clinical trials for myocardial infarction therapy; however, broad acceptance is hindered by limited understanding of hMSC-human cardiomyocyte (hCM interactions. To better understand the electrophysiological consequences of direct heterocellular connections between hMSCs and hCMs, three original mathematical models were developed, representing an experimentally verified triad of hMSC families with distinct functional ion channel currents. The arrhythmogenic risk of such direct electrical interactions in the setting of healthy adult myocardium was predicted by coupling and fusing these hMSC models to the published ten Tusscher midcardial hCM model. Substantial variations in action potential waveform-such as decreased action potential duration (APD and plateau height-were found when hCMs were coupled to the two hMSC models expressing functional delayed rectifier-like human ether à-go-go K+ channel 1 (hEAG1; the effects were exacerbated for fused hMSC-hCM hybrid cells. The third family of hMSCs (Type C, absent of hEAG1 activity, led to smaller single-cell action potential alterations during coupling and fusion, translating to longer tissue-level mean action potential wavelength. In a simulated 2-D monolayer of cardiac tissue, re-entry vulnerability with low (5% hMSC insertion was approximately eight-fold lower with Type C hMSCs compared to hEAG1-functional hMSCs. A 20% decrease in APD dispersion by Type C hMSCs compared to hEAG1-active hMSCs supports the claim of reduced arrhythmogenic potential of this cell type with low hMSC insertion. However, at moderate (15% and high (25% hMSC insertion, the vulnerable window increased independent of hMSC type. In summary, this study provides novel electrophysiological models of hMSCs, predicts possible arrhythmogenic effects of hMSCs when directly coupled to healthy hCMs, and proposes that isolating a subset of hMSCs absent

  3. Modeling Electrophysiological Coupling and Fusion between Human Mesenchymal Stem Cells and Cardiomyocytes.

    Science.gov (United States)

    Mayourian, Joshua; Savizky, Ruben M; Sobie, Eric A; Costa, Kevin D

    2016-07-01

    Human mesenchymal stem cell (hMSC) delivery has demonstrated promise in preclinical and clinical trials for myocardial infarction therapy; however, broad acceptance is hindered by limited understanding of hMSC-human cardiomyocyte (hCM) interactions. To better understand the electrophysiological consequences of direct heterocellular connections between hMSCs and hCMs, three original mathematical models were developed, representing an experimentally verified triad of hMSC families with distinct functional ion channel currents. The arrhythmogenic risk of such direct electrical interactions in the setting of healthy adult myocardium was predicted by coupling and fusing these hMSC models to the published ten Tusscher midcardial hCM model. Substantial variations in action potential waveform-such as decreased action potential duration (APD) and plateau height-were found when hCMs were coupled to the two hMSC models expressing functional delayed rectifier-like human ether à-go-go K+ channel 1 (hEAG1); the effects were exacerbated for fused hMSC-hCM hybrid cells. The third family of hMSCs (Type C), absent of hEAG1 activity, led to smaller single-cell action potential alterations during coupling and fusion, translating to longer tissue-level mean action potential wavelength. In a simulated 2-D monolayer of cardiac tissue, re-entry vulnerability with low (5%) hMSC insertion was approximately eight-fold lower with Type C hMSCs compared to hEAG1-functional hMSCs. A 20% decrease in APD dispersion by Type C hMSCs compared to hEAG1-active hMSCs supports the claim of reduced arrhythmogenic potential of this cell type with low hMSC insertion. However, at moderate (15%) and high (25%) hMSC insertion, the vulnerable window increased independent of hMSC type. In summary, this study provides novel electrophysiological models of hMSCs, predicts possible arrhythmogenic effects of hMSCs when directly coupled to healthy hCMs, and proposes that isolating a subset of hMSCs absent of hEAG1

  4. Agrobacterium type IV secretion system and its substrates form helical arrays around the circumference of virulence-induced cells.

    Science.gov (United States)

    Aguilar, Julieta; Zupan, John; Cameron, Todd A; Zambryski, Patricia C

    2010-02-23

    The genetic transformation of plant cells by Agrobacterium tumefaciens results from the transfer of DNA and proteins via a specific virulence (vir) -induced type IV secretion system (T4SS). To better understand T4SS function, we analyzed the localization of its structural components and substrates by deconvolution fluorescence microscopy. GFP fusions to T4SS proteins with cytoplasmic tails, VirB8 and VirD4, or cytoplasmic T4SS substrate proteins, VirD2, VirE2, and VirF, localize in a helical pattern of fluorescent foci around the perimeter of the bacterial cell. All fusion proteins were expressed at native levels of vir induction. Importantly, most fusion proteins are functional and do not exhibit dominant-negative effects on DNA transfer to plant cells. Further, GFP-VirB8 complements a virB8 deletion strain. We also detect native VirB8 localization as a helical array of foci by immunofluorescence microscopy. T4SS foci likely use an existing helical scaffold during their assembly. Indeed, the bacterial cytoskeletal component MinD colocalizes with GFP-VirB8. Helical arrays of foci are found at all times investigated between 12 and 48 h post vir induction at 19 degrees C. These data lead to a model with multiple T4SSs around the bacterial cell that likely facilitate host cell attachment and DNA transfer. In support, we find multiple T pili around vir-induced bacterial cells. PMID:20133577

  5. Genome-wide identification of genes with amplification and/or fusion in small cell lung cancer.

    Science.gov (United States)

    Iwakawa, Reika; Takenaka, Masataka; Kohno, Takashi; Shimada, Yoko; Totoki, Yasushi; Shibata, Tatsuhiro; Tsuta, Koji; Nishikawa, Ryo; Noguchi, Masayuki; Sato-Otsubo, Aiko; Ogawa, Seishi; Yokota, Jun

    2013-09-01

    To obtain a landscape of gross genetic alterations in small cell lung cancer (SCLC), genome-wide copy number analysis and whole-transcriptome sequencing were performed in 58 and 42 SCLCs, respectively. Focal amplification of known oncogene loci, MYCL1 (1p34.2), MYCN (2p24.3), and MYC (8q24.21), was frequently and mutually exclusively detected. MYCL1 and MYC were co-amplified with other regions on either the same or the different chromosome in several cases. In addition, the 9p24.1 region was identified as being amplified in SCLCs without amplification of MYC family oncogenes. Notably, expression of the KIAA1432 gene in this region was significantly higher in KIAA1432 amplified cells than in non-amplified cells, and its mRNA expression showed strong correlations with the copy numbers. Thus, KIAA1432 is a novel gene activated by amplification in SCLCs. By whole-transcriptome sequencing, a total of 60 fusion transcripts, transcribed from 95 different genes, were identified as being expressed in SCLC cells. However, no in-frame fusion transcripts were recurrently detected in ≥2 SCLCs, and genes in the amplified regions, such as PVT1 neighboring MYC and RLF in MYCL1 amplicons, were recurrently fused with genes in the same amplicons or with those in different amplicons on either the same or different chromosome. Thus, it was indicated that amplification and fusion of several genes on chromosomes 1 and 8 occur simultaneously but not sequentially through chromothripsis in the development of SCLC, and amplification rather than fusion of genes plays an important role in its development. PMID:23716474

  6. UV-Induced Cell Death in Plants

    Directory of Open Access Journals (Sweden)

    Chang Ho Kang

    2013-01-01

    Full Text Available Plants are photosynthetic organisms that depend on sunlight for energy. Plants respond to light through different photoreceptors and show photomorphogenic development. Apart from Photosynthetically Active Radiation (PAR; 400–700 nm, plants are exposed to UV light, which is comprised of UV-C (below 280 nm, UV-B (280–320 nm and UV-A (320–390 nm. The atmospheric ozone layer protects UV-C radiation from reaching earth while the UVR8 protein acts as a receptor for UV-B radiation. Low levels of UV-B exposure initiate signaling through UVR8 and induce secondary metabolite genes involved in protection against UV while higher dosages are very detrimental to plants. It has also been reported that genes involved in MAPK cascade help the plant in providing tolerance against UV radiation. The important targets of UV radiation in plant cells are DNA, lipids and proteins and also vital processes such as photosynthesis. Recent studies showed that, in response to UV radiation, mitochondria and chloroplasts produce a reactive oxygen species (ROS. Arabidopsis metacaspase-8 (AtMC8 is induced in response to oxidative stress caused by ROS, which acts downstream of the radical induced cell death (AtRCD1 gene making plants vulnerable to cell death. The studies on salicylic and jasmonic acid signaling mutants revealed that SA and JA regulate the ROS level and antagonize ROS mediated cell death. Recently, molecular studies have revealed genes involved in response to UV exposure, with respect to programmed cell death (PCD.

  7. Combinatorial synthesis and screening of cancer cell-specific nanomedicines targeted via phage fusion proteins

    Directory of Open Access Journals (Sweden)

    James W. Gillespie

    2015-06-01

    Full Text Available Active tumor targeting of nanomedicines has recently shown significant improvements in the therapeutic activity of currently existing drug delivery systems, such as liposomal doxorubicin (Doxil/Caelyx/Lipodox. Previously, we have shown that isolated pVIII major coat proteins of the fd tet filamentous phage vector, containing cancer cell-specific peptide fusions at their N terminus, can be used as active targeting ligands in a liposomal doxorubicin delivery system in vitro and in vivo. Here, we show a novel major coat protein isolation procedure in 2-propanol that allows spontaneous incorporation of the hydrophobic protein core into preformed liposomal doxorubicin with minimal damage or drug loss while still retaining the targeting ligand exposed for cell-specific targeting. Using a panel of 12 structurally unique ligands with specificity towards breast, lung, and/or pancreatic cancer, we showed the feasibility of pVIII major coat proteins to significantly increase the throughput of targeting ligand screening in a common nanomedicine core. Phage protein-modified Lipodox samples showed an average doxorubicin recovery of 82.8% across all samples with 100% of protein incorporation in the correct orientation (N-terminus exposed. Following cytotoxicity screening in a doxorubicin-sensitive breast cancer line (MCF-7, three major groups of ligands were identified. Ligands showing the most improved cytotoxicity included: DMPGTVLP, ANGRPSMT, VNGRAEAP, and ANDVYLD showing a 25-fold improvement (p < 0.05 in toxicity. Similarly DGQYLGSQ, ETYNQPYL, and GSSEQLYL ligands with specificity towards a doxorubicin-insensitive pancreatic cancer line (PANC-1 showed significant increases in toxicity (2-fold; p < 0.05. Thus, we demonstrated proof-of-concept that pVIII major coat proteins can be screened in significantly higher throughput to identify novel ligands displaying improved therapeutic activity in a desired cancer phenotype.

  8. Landscape Phage Fusion Protein-mediated Targeting of Nanomedicines Enhances their Prostate Tumor Cell Association and Cytotoxic Efficiency

    Science.gov (United States)

    Jayanna, P.K.; Bedi, D; Gillespie, J.W.; DeInnocentes, P.; Wang, T; Torchilin, V.P; Bird, R.C.; Petrenko, V.A.

    2010-01-01

    Tumor-specific cytotoxicity of drugs can be enhanced by targeting them to tumor receptors using tumor-specific ligands. Phage display offers a high-throughput approach to screen for the targeting ligands. We have successfully isolated phage fusion peptides selective and specific for PC3 prostate cancer cells. Also, we have demonstrated a novel approach of targeting liposomes through tumor-specific phage fusion coat proteins, exploiting the intrinsic properties of the phage coat protein as an integral membrane protein. Here we describe the production of Rhodamine-labeled liposomes as well as doxorubicin-loaded long circulating liposomes targeted to PC3 prostate tumor cells via PC-specific phage peptides, as an extension of our previous studies. Targeting of labeled liposomes was demonstrated using fluorescence microscopy as well as flow cytometry. Targeting of doxorubicin-loaded liposomes enhanced their cytotoxic effect against PC3 cells in vitro indicating a possible therapeutic advantage. The simplicity of the approach for generating targeted liposomes coupled with the ability to rapidly obtain tumor-specific phage fusion proteins via phage display may contribute to a combinatorial system for the production of targeted liposomal therapeutics for advanced stages of prostate tumor. PMID:20138246

  9. EFFECTS OF CALM /AF10 ANTISENSES ON PRIMARY LEUKEMIC CELLS WITH CALM /AF10 FUSION TRANSCRIPTS IN VITRO

    Institute of Scientific and Technical Information of China (English)

    LIU Ge-xiu

    2005-01-01

    Objectives: To define the involvement of CALM and AF10 fusion transcripts in primary leukaemias with t(10; 11). Methods: The AF10 and CALM fusion in five t(10; 11) leukemia samples were checked by reverse transcriptase-polymerase chain reaction (RT-PCR), and effects of CALM/AF10 antisense phosphorothioate oligodeoxynucleotides (AS PS-ODNs) on chemotherapy sensitivity and apoptosis of leukemia cells in vitro were observed. Results: Five different-sized AF10-CALM products and four different-sized CALM/AF10 products were detected by RT-PCR. The chemotherapy sensitivity of leukemic cells with t(10; 11) to drugs in vitro was lower than that of leukemic cells without t(10; 11). AS PS-ODNs increased the chemotherapy sensitivity and apoptotic rate. There were 4 cases positive at 5 (mol/L concentration, a11 cases positive at 10 (mol/L and 20 (mol/L concentration, P0.05). Conclusion: The CALM and AF10 fusion transcripts are involved in the pathogenesis of haematological malignancies with t(10, 11), and is associated with a poor prognosis. AS-PS-ODNs might be useful in therapy of t(10, 11) leukemia.

  10. Contribution of N-linked glycans on HSV-2 gB to cell–cell fusion and viral entry

    Energy Technology Data Exchange (ETDEWEB)

    Luo, Sukun [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Hu, Kai [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China); He, Siyi; Wang, Ping; Zhang, Mudan; Huang, Xin [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Du, Tao [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China); Zheng, Chunfu [Soochow University, Institutes of Biology and Medical Sciences, Suzhou 215123 (China); Liu, Yalan [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China); Hu, Qinxue, E-mail: qhu@wh.iov.cn [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China); Institute for Infection and Immunity, St George' s University of London, London SW17 0RE (United Kingdom)

    2015-09-15

    HSV-2 is the major cause of genital herpes and its infection increases the risk of HIV-1 acquisition and transmission. HSV-2 glycoprotein B together with glycoproteins D, H and L are indispensable for viral entry, of which gB, as a class III fusogen, plays an essential role. HSV-2 gB has seven potential N-linked glycosylation (N-CHO) sites, but their significance has yet to be determined. For the first time, we systematically analyzed the contributions of N-linked glycans on gB to cell–cell fusion and viral entry. Our results demonstrated that, of the seven potential N-CHO sites on gB, mutation at N390, N483 or N668 decreased cell–cell fusion and viral entry, while mutation at N133 mainly affected protein expression and the production of infectious virus particles by blocking the transport of gB from the endoplasmic reticulum to Golgi. Our findings highlight the significance of N-linked glycans on HSV-2 gB expression and function. - Highlights: • N-linked glycan at N133 is important for gB intracellular trafficking and maturation. • N-linked glycans at N390, N483 and N668 on gB are necessary for optimal cell–cell fusion. • N-linked glycans at N390, N483 and N668 on gB are necessary for optimal viral entry.

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

    International Nuclear Information System (INIS)

    A high resolution MUSIC, for low and medium energy ion, has been developed. The high pulse height resolution was obtained by coupling the preamplifiers directly to the anode pads. The pulse height measurements were performed by using a 241 Am alpha source. The energy loss distribution widths measured in P-10 gas at pressures between 200 and 300 torr are in agreement with the theory of Badhwar. The achieved resolution of the chamber is closed to the statistical limit. MUSIC was used for fusion investigation by using 11 Li radioactive beam and Si and C targets. It was found to be very useful in eliminating the energy degraded and parasitic beam admixtures. It was expected that this type of chamber could be used also for isotopes of light elements identification, in Accelerator Mass Spectrometry applications. (authors)

  12. Calculation of prompt doses induced by fusion experiments in Laser MegaJoule facility

    Energy Technology Data Exchange (ETDEWEB)

    Baggio, Jacques, E-mail: jacques.baggio@projet-lmj.org [CEA/CESTA BP2 33114 Le Barp (France); Jacquet, Henri-Patrick [CEA/DIF, Bruyeres Le Chatel 91297 Arpajon Cedex (France); Schneider, Philippe; Javier, Francois [CEA/CESTA BP2 33114 Le Barp (France)

    2011-10-15

    The Laser MegaJoule (LMJ), built at CEA/CESTA near Bordeaux, France, will conduct research in the field of plasma physics and inertial confinement fusion. Some of these experiments are designed to produce during less than one nanosecond up to 10{sup 19} neutrons at 14 MeV in indirect drive mode. The 300 m long building that hosts both laser sources and target chamber was designed to confine neutron and gamma fluxes in the experiment hall during a shot. Monte Carlo simulations were performed using a TRIPOLI 4 modelling of the facility to ensure that prompt doses in occupied areas inside the facility that host employees during a shot will be sufficiently low. Although significant radiation levels inside the experiment hall, prompt doses in occupied rooms are limited to a few {mu}Sv per shot which is fully compatible with radiation level limits.

  13. Bilateral Ankle and Subtalar Joint Fusion Secondary to Guillain Barré-Induced Foot Drop.

    Science.gov (United States)

    Seidel, Jay; Mathew, Bindu; Marks, Jeffrey

    2016-01-01

    Guillain-Barré syndrome is a serious disorder that occurs when the body's immune system mistakenly attacks the peripheral nervous system. This leads to nerve inflammation and damage that can cause muscle weakness and/or paralysis, including foot drop. Therapy ranges from supportive measures, such as physical therapy, to surgical intervention. It appears from the published data that these patients, generally, have been left to their own devices, because no known cure is available, necessitating reliance on others for their daily activities and ambulation. We present a unique surgical option, with a follow-up period of 24 months, in which bilateral subtalar and ankle fusion allowed unassisted ambulation to a patient who might otherwise have never walked again. PMID:25128306

  14. Inactivation of encapsulated cells and their therapeutic effects by means of TGL triple-fusion reporter/biosafety gene.

    Science.gov (United States)

    Santos, Edorta; Larzabal, Leyre; Calvo, Alfonso; Orive, Gorka; Pedraz, José Luis; Hernández, Rosa Ma

    2013-01-01

    The immobilization of cells within alginate-poly-l-lysine-alginate (APA) microcapsules has been demonstrated to be an effective technology design for long term delivery of therapeutic products. Despite promising advances, biosafety aspects still remain to be improved. Here, we describe a complete characterization of the strategy based on TGL triple-fusion reporter gene--which codifies for Herpes Simplex virus type 1 thymidine-kinase (HSV1-TK), green fluorescent protein (GFP) and Firefly Luciferase--(SFG(NES)TGL) to inactivate encapsulated cells and their therapeutic effects. Myoblasts genetically engineered to secrete erythropoietin (EPO) were retroviraly transduced with the SFG(NES)TGL plasmid to further characterize their ganciclovir (GCV)-mediated inactivation process. GCV sensitivity of encapsulated cells was 100-fold lower when compared to cells plated onto 2D surfaces. However, the number of cells per capsule and EPO secretion decayed to less than 15% at the same time that proliferation was arrested after 14 days of GCV treatment in vitro. In vivo, ten days of GCV treatment was enough to restore the increased hematocrit levels of mice implanted with encapsulated TGL-expressing and EPO-secreting cells. Altogether, these results show that TGL triple-fusion reporter gene may be a good starting point in the search of a suitable biosafety strategy to inactivate encapsulated cells and control their therapeutic effects. PMID:23174140

  15. Production of heavy nuclei in 40Ar-induced fusion reactions

    International Nuclear Information System (INIS)

    At the velocity filter SHIP of the GSI in Darmstadt evaporation residual nuclei were detected which were produced by fusion of 40Ar projectiles in the energy range between 4.1 and 5.1 MeV/u with the target nuclei 165Ho, 169Tm, 171Yb, 174Yb, 175Lu, 176Hf, 177Hf, 178Hf, 179Hf, 180Hf, and 181Ta. For the determination of cross sections for residual nuclei after evaporation of neutrons, protons, and α-particles the transmission of SHIP was experimentally calibrated. In the theoretical calculation of the cross section for evaporation residual nuclei besides the fusion cross section above all the fission barriers according to the droplet model, the empirically determinable ground-state shell effects, and the level densities enter. The droplet model fission barriers were determined by an empirical extrapolation into the here interesting region of proton-rich nuclei. If in the evaporation calculations conventional level densities according to the one-particle model are used then the calculated cross sections exceed in measured data in the region of the spherical N=126 shell partly about several orders of magnitude. A possible explanation of this discrepancy is the contribution of collective states to the level density at low excitation energies as it was proposed by Bjoernholm at al. (1974). In the present thesis it is shown that this modification of the level density can lead to a with increasing excitation energy increasing deformation of an in the ground state spherical compound nucleus which causes a very fast evaporation of spherical shells with the excitation energy. (orig./HSI)

  16. Influence of laser induced hot electrons on the threshold for shock ignition of fusion reactions

    Science.gov (United States)

    Colaïtis, A.; Ribeyre, X.; Le Bel, E.; Duchateau, G.; Nicolaï, Ph.; Tikhonchuk, V.

    2016-07-01

    The effects of Hot Electrons (HEs) generated by the nonlinear Laser-Plasma Interaction (LPI) on the dynamics of Shock Ignition Inertial Confinement Fusion targets are investigated. The coupling between the laser beam, plasma dynamics and hot electron generation and propagation is described with a radiative hydrodynamics code using an inline model based on Paraxial Complex Geometrical Optics [Colaïtis et al., Phys. Rev. E 92, 041101 (2015)]. Two targets are considered: the pure-DT HiPER target and a CH-DT design with baseline spike powers of the order of 200-300 TW. In both cases, accounting for the LPI-generated HEs leads to non-igniting targets when using the baseline spike powers. While HEs are found to increase the ignitor shock pressure, they also preheat the bulk of the imploding shell, notably causing its expansion and contamination of the hotspot with the dense shell material before the time of shock convergence. The associated increase in hotspot mass (i) increases the ignitor shock pressure required to ignite the fusion reactions and (ii) significantly increases the power losses through Bremsstrahlung X-ray radiation, thus rapidly cooling the hotspot. These effects are less prominent for the CH-DT target where the plastic ablator shields the lower energy LPI-HE spectrum. Simulations using higher laser spike powers of 500 TW suggest that the CH-DT capsule marginally ignites, with an ignition window width significantly smaller than without LPI-HEs, and with three quarters of the baseline target yield. The latter effect arises from the relation between the shock launching time and the shell areal density, which becomes relevant in presence of a LPI-HE preheating.

  17. Effect of increase in orientational order of lipid chains and head group spacing on non steroidal anti-inflammatory drug induced membrane fusion.

    Science.gov (United States)

    Roy, Sutapa Mondal; Bansode, Amol S; Sarkar, Munna

    2010-12-21

    Membrane fusion is a key event in many biological processes. The fusion process, both in vivo and in vitro, is induced by different agents which include mainly proteins and peptides. For protein- and peptide-mediated membrane fusion, conformational reorganization serves as a driving force. Small drug molecules do not share this advantage; hence, drug induced membrane fusion occurring in absence of any other fusogenic agent and at physiologically relevant concentration of the drugs is a very rare event. To date, 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 fusion at very low drug to lipid ratio without the aid of any other fusogenic agent. In our continued effort to understand the interplay of different physical and chemical parameters of both the participating drugs and the membrane on the mechanism of this drug induced membrane fusion, we present here the effect of increase in orientational order of the lipid chains and increase in head group spacing. This is achieved by studying the effect of low concentration cholesterol (gel to fluid transition temperature, is mainly known to increase orientational order of the lipid chains and increase head group spacing. To isolate the effect of these parameters, small unilameller vesicles (SUVs) formed by dimyristoylphosphatidylcholine (DMPC) with an average diameter of 50-60 nm were used as simple model membranes. 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. Differential scanning calorimetry (DSC) was used to study the effect of drugs in the presence of cholesterol on the chain-melting temperature which reflects the fluidization effect of the hydrophobic tail region of the bilayer. Our results show contradictory effect of low concentration

  18. Chemical -induced apoptotic cell death in tomato cells : involvement of caspase-like proteases

    NARCIS (Netherlands)

    Jong, de A.J.; Hoeberichts, F.A.; Yakimova, E.T.; Maximova, E.; Woltering, E.J.

    2000-01-01

    A new system to study programmed cell death in plants is described. Tomato (Lycopersicon esculentum Mill.) suspension cells were induced to undergo programmed cell death by treatment with known inducers of apoptosis in mammalian cells. This chemical-induced cell death was accompanied by the characte

  19. Chromosome therapy. Correction of large chromosomal aberrations by inducing ring chromosomes in induced pluripotent stem cells (iPSCs).

    Science.gov (United States)

    Kim, Taehyun; Bershteyn, Marina; Wynshaw-Boris, Anthony

    2014-01-01

    The fusion of the short (p) and long (q) arms of a chromosome is referred to as a "ring chromosome." Ring chromosome disorders occur in approximately 1 in 50,000-100,000 patients. Ring chromosomes can result in birth defects, mental disabilities, and growth retardation if additional genes are deleted during the formation of the ring. Due to the severity of these large-scale aberrations affecting multiple contiguous genes, no possible therapeutic strategies for ring chromosome disorders have so far been proposed. Our recent study (Bershteyn et al.) using patient-derived fibroblast lines containing ring chromosomes, found that cellular reprogramming of these fibroblasts into induced pluripotent stem cells (iPSCs) resulted in the cell-autonomous correction of the ring chromosomal aberration via compensatory uniparental disomy (UPD). These observations have important implications for studying the mechanism of chromosomal number control and may lead to the development of effective therapies for other, more common, chromosomal aberrations.

  20. Chemotropism and Cell Fusion in Neurospora crassa Relies on the Formation of Distinct Protein Complexes by HAM-5 and a Novel Protein HAM-14.

    Science.gov (United States)

    Jonkers, Wilfried; Fischer, Monika S; Do, Hung P; Starr, Trevor L; Glass, N Louise

    2016-05-01

    In filamentous fungi, communication is essential for the formation of an interconnected, multinucleate, syncytial network, which is constructed via hyphal fusion or fusion of germinated asexual spores (germlings). Anastomosis in filamentous fungi is comparable to other somatic cell fusion events resulting in syncytia, including myoblast fusion during muscle differentiation, macrophage fusion, and fusion of trophoblasts during placental development. In Neurospora crassa, fusion of genetically identical germlings is a highly dynamic and regulated process that requires components of a MAP kinase signal transduction pathway. The kinase pathway components (NRC-1, MEK-2 and MAK-2) and the scaffold protein HAM-5 are recruited to hyphae and germling tips undergoing chemotropic interactions. The MAK-2/HAM-5 protein complex shows dynamic oscillation to hyphae/germling tips during chemotropic interactions, and which is out-of-phase to the dynamic localization of SOFT, which is a scaffold protein for components of the cell wall integrity MAP kinase pathway. In this study, we functionally characterize HAM-5 by generating ham-5 truncation constructs and show that the N-terminal half of HAM-5 was essential for function. This region is required for MAK-2 and MEK-2 interaction and for correct cellular localization of HAM-5 to "fusion puncta." The localization of HAM-5 to puncta was not perturbed in 21 different fusion mutants, nor did these puncta colocalize with components of the secretory pathway. We also identified HAM-14 as a novel member of the HAM-5/MAK-2 pathway by mining MAK-2 phosphoproteomics data. HAM-14 was essential for germling fusion, but not for hyphal fusion. Colocalization and coimmunoprecipitation data indicate that HAM-14 interacts with MAK-2 and MEK-2 and may be involved in recruiting MAK-2 (and MEK-2) to complexes containing HAM-5. PMID:27029735

  1. Fusion of liposomes with the plasma membrane of epithelial cells: Fate of incorporated lipids as followed by freeze fracture and autoradiography of plastic sections

    OpenAIRE

    Knoll, G.; Burger, K.N.J.; Bron, R.; van Meer, G.; Verkleij, A. J.

    1988-01-01

    The fusion of liposomes with the plasma membrane of influenza virus- infected monolayers of an epithelial cell line, Madin-Darby canine kidney cells (van Meer et al., 1985. Biochemistry. 24:3593-3602), has been analyzed by morphological techniques. The distribution of liposomal lipids over the apical and basolateral plasma membrane domains after fusion was assessed by autoradiography of liposomal [3H]dipalmitoylphosphatidylcholine after rapid freezing or chemical fixation and further processi...

  2. Activation-Induced Cell Death in T Cells and Autoimmunity

    Institute of Scientific and Technical Information of China (English)

    Jian Zhang; Xuemei Xu; Yong Liu

    2004-01-01

    Activation-induced cell death (AICD), which results from the interaction between Fas and Fas ligand, is responsible for maintaining tolerance to self-antigen. A defect in AICD may lead to development of autoimmunity. During the last several years, much progress has been made in understanding the mechanism(s) of AICD and its potential role in the pathogenesis of autoimmune diseases. In this review, we summarize the most recent progress on the regulation of the susceptibility of T cells to AICD and its possible involvement in autoimmune diseases.

  3. Cell signalling pathways underlying induced pluripotent stem cell reprogramming

    Institute of Scientific and Technical Information of China (English)

    Kate; Hawkins; Shona; Joy; Tristan; Mc; Kay

    2014-01-01

    Induced pluripotent stem(i PS) cells, somatic cells reprogrammed to the pluripotent state by forced expression of defined factors, represent a uniquely valuable resource for research and regenerative medicine. However, this methodology remains inefficient due to incomplete mechanistic understanding of the reprogramming process. In recent years, various groups have endeavoured to interrogate the cell signalling that governs the reprogramming process, including LIF/STAT3, BMP, PI3 K, FGF2, Wnt, TGFβ and MAPK pathways, with the aim of increasing our understanding and identifying new mechanisms of improving safety, reproducibility and efficiency. This has led to a unified model of reprogramming that consists of 3 stages: initiation, maturation and stabilisation. Initiation of reprogramming occurs in almost all cells that receive the reprogramming transgenes; most commonly Oct4, Sox2, Klf4 and c Myc, and involves a phenotypic mesenchymal-to-epithelial transition. The initiation stage is also characterised by increased proliferation and a metabolic switch from oxidative phosphorylation to glycolysis. The maturation stage is considered the major bottleneck within the process, resulting in very few "stabilisation competent" cells progressing to the final stabilisation phase. To reach this stage in both mouse and human cells, pre-i PS cells must activate endogenous expression of the core circuitry of pluripotency, comprising Oct4, Sox2, and Nanog, and thus reach a state of transgene independence. By the stabilisation stage, i PS cells generally use the same signalling networks that govern pluripotency in embryonic stem cells. These pathways differ between mouse and human cells although recent work has demonstrated that this is context dependent. As i PS cell generation technologies move forward, tools are being developed to interrogate the process in more detail, thus allowing a greater understanding of this intriguing biological phenomenon.

  4. The oncogenic fusion protein RUNX1-CBFA2T1 supports proliferation and inhibits senescence in t(8;21-positive leukaemic cells

    Directory of Open Access Journals (Sweden)

    Nordheim Alfred

    2004-08-01

    Full Text Available Abstract Background The fusion protein RUNX1-CBFA2T1 associated with t(8;21-positive acute myeloid leukaemia is a potent inhibitor of haematopoetic differentiation. The role of RUNX1-CBFA2T1 in leukaemic cell proliferation is less clear. We examined the consequences of siRNA-mediated RUNX1-CBFA2T1 depletion regarding proliferation and clonogenicity of t(8;21-positive cell lines. Methods The t(8;21-positive cell line Kasumi-1 was electroporated with RUNX1-CBFA2T1 or control siRNAs followed by analysis of proliferation, colony formation, cell cycle distribution, apoptosis and senescence. Results Electroporation of Kasumi-1 cells with RUNX1-CBFA2T1 siRNAs, but not with control siRNAs, resulted in RUNX1-CBFA2T1 suppression which lasted for at least 5 days. A single electroporation with RUNX1-CBFA2T1 siRNA severely diminished the clonogenicity of Kasumi-1 cells. Prolonged RUNX1-CBFA2T1 depletion inhibited proliferation in suspension culture and G1-S transition during the cell cycle, diminished the number of apoptotic cells, but induced cellular senescence. The addition of haematopoetic growth factors could not rescue RUNX1-CBFA2T1-depleted cells from senescence, and could only partially restore their clonogenicity. Conclusions RUNX1-CBFA2T1 supports the proliferation and expansion of t(8;21-positive leukaemic cells by preventing cellular senescence. These findings suggest a central role of RUNX1-CBFA2T1 in the maintenance of the leukaemia. Therefore, RUNX1-CBFA2T1 is a promising and leukaemia-specific target for molecularly defined therapeutic approaches.

  5. Cell Wall Polysaccharides of Candida albicans Induce Mast Cell Degranulation in the Gut

    OpenAIRE

    Sakurai, Atsuko; Yamaguchi, Natsu; Sonoyama, Kei

    2012-01-01

    We investigated Candida albicans-induced mast cell degranulation in vitro and in vivo. Cell wall fraction but not culture supernatant and cell membrane fraction prepared from hyphally grown C. albicans induced β-hexosaminidase release in RBL-2H3 cells. Cell wall mannan and soluble β-glucan fractions also induced β-hexosaminidase release. Histological examination of mouse forestomach showed that C. albicans gut colonization induces mast cell degranulation. However, intragastric administration ...

  6. Antitumor effects and radiosensitization of cytosine deaminase and thymidine kinase fusion suicide gene on colorectal carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    De-Hua Wu; Li Liu; Long-Hua Chen

    2005-01-01

    AIM: To investigate the killing effect and radiosensitization of double suicide gene mediated by adenovirus on colorectal carcinoma cells.METHODS: Colorectal carcinoma cell line SW480 was transfected with adenovirus expression vector containing cytosine deaminase (CD) and thymidine kinase (Tk) fusion gene. The expression of CD-TK fusion gene was detected by reverse transcriptase-polymerase chain reaction. The toxic effect of ganciclovir (GCV) and 5-fiuorocytosine (5FC) on infected cells was determined by MTT assay. The radiosensitization of double suicide gene was evaluated by clonogenic assay.RESULTS: After prodrugs were used, the survival rate of colorectal carcinoma cells was markedly decreased. When GCV and 5-FC were used in combination, the cytotoxicity and bystandereffect were markedly superior to a single prodrug (x2 = 30.371, P<0.01). Both GCV and 5-FC could sensitize colorectal carcinoma cells to the toxic effect of radiation, and greater radiosensitization was achieved when both prodrug were used in combination. CONCLUSION: CD-TK double suicide gene can kill and radiosensitize colorectal carcinoma cells.

  7. Intracellular delivery of cell-penetrating peptide-transcriptional factor fusion protein and its role in selective osteogenesis

    Directory of Open Access Journals (Sweden)

    Suh JS

    2014-03-01

    Full Text Available Jin Sook Suh,1,* Jue Yeon Lee,2,* Yoon Jung Choi,1 Hyung Keun You,3 Seong-Doo Hong,4 Chong Pyoung Chung,2 Yoon Jeong Park1,2 1Dental Regenerative Biotechnology, Dental Research Institute, School of Dentistry, Seoul National University, Seoul, 2Central Research Institute, Nano Intelligent Biomedical Engineering Corporation (NIBEC, Seoul, 3Department of Periodontology, College of Dentistry, Wonkwang University, Iksan, 4Department of Oral Pathology, School of Dentistry, Seoul National University, Seoul, Republic of Korea *These authors contributed equally to this work Abstract: 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

  8. Enhanced neutralization potency of botulinum neurotoxin antibodies using a red blood cell-targeting fusion protein.

    Directory of Open Access Journals (Sweden)

    Sharad P Adekar

    Full Text Available Botulinum neurotoxin (BoNT potently inhibits cholinergic signaling at the neuromuscular junction. The ideal countermeasures for BoNT exposure are monoclonal antibodies or BoNT antisera, which form BoNT-containing immune complexes that are rapidly cleared from the general circulation. Clearance of opsonized toxins may involve complement receptor-mediated immunoadherence to red blood cells (RBC in primates or to platelets in rodents. Methods of enhancing immunoadherence of BoNT-specific antibodies may increase their potency in vivo. We designed a novel fusion protein (FP to link biotinylated molecules to glycophorin A (GPA on the RBC surface. The FP consists of an scFv specific for murine GPA fused to streptavidin. FP:mAb:BoNT complexes bound specifically to the RBC surface in vitro. In a mouse model of BoNT neutralization, the FP increased the potency of single and double antibody combinations in BoNT neutralization. A combination of two antibodies with the FP gave complete neutralization of 5,000 LD50 BoNT in mice. Neutralization in vivo was dependent on biotinylation of both antibodies and correlated with a reduction of plasma BoNT levels. In a post-exposure model of intoxication, FP:mAb complexes gave complete protection from a lethal BoNT/A1 dose when administered within 2 hours of toxin exposure. In a pre-exposure prophylaxis model, mice were fully protected for 72 hours following administration of the FP:mAb complex. These results demonstrate that RBC-targeted immunoadherence through the FP is a potent enhancer of BoNT neutralization by antibodies in vivo.

  9. Neutron-induced reactions relevant for Inertial-Cofinement Fusion Experiments

    Science.gov (United States)

    Boswell, Melissa; Merrill, Frank; Rundberg, R.; Grim, Gary; Wilde, Carl; Hayes, Anna; Fowler, Malcom; Wilhelmy, Jerry

    2012-10-01

    Measuring the fluencies of both the low- & high-energy neutrons is a powerful mechanism for studying the implosion process, and the various parameters that drive inertial confinement fusion. We have developed a number of tools to measure the spectral characteristics of the NIF neutron spectrum. Most of these methods rely on exploiting the energy dependence of (n,γ), (n,2n), (n,3n) and (n,p) reactions on a variety of materials either implicitly present in the NIF implosion or through doping the target capsule or holraum. I will be discussing both prompt activation measurements, and debris activation measurements of these materials currently under development at LANL. Focusing specifically on the development of an in-situ detector to measure short-lived activation products, as well as a low-background counting facility we are developing at the Waste Isolation Pilot Plant (WIPP) to study longer-lived activation products. Furthermore, I will also be discussing several cross section measurements that are important for the interpretation of the data collected from these activation products.

  10. Immunization with FSHβ fusion protein antigen prevents bone loss in a rat ovariectomy-induced osteoporosis model

    International Nuclear Information System (INIS)

    Highlights: •A GST-FSH fusion protein was successfully expressed in E. coli. •Immunization with GST-FSH antigen can raise high-titer anti-FSH polyclonal sera. •Anti-FSH polyclonal sera can neutralize osteoclastogenic effect of FSH in vitro. •FSH immunization can prevent bone loss in a rat osteoporosis model. -- Abstract: Osteoporosis, a metabolic bone disease, threatens postmenopausal women globally. Hormone replacement therapy (HTR), especially estrogen replacement therapy (ERT), is used widely in the clinic because it has been generally accepted that postmenopausal osteoporosis is caused by estrogen deficiency. However, hypogonadal α and β estrogen receptor null mice were only mildly osteopenic, and mice with either receptor deleted had normal bone mass, indicating that estrogen may not be the only mediator that induces osteoporosis. Recently, follicle-stimulating hormone (FSH), the serum concentration of which increases from the very beginning of menopause, has been found to play a key role in postmenopausal osteoporosis by promoting osteoclastogenesis. In this article, we confirmed that exogenous FSH can enhance osteoclast differentiation in vitro and that this effect can be neutralized by either an anti-FSH monoclonal antibody or anti-FSH polyclonal sera raised by immunizing animals with a recombinant GST-FSHβ fusion protein antigen. Moreover, immunizing ovariectomized rats with the GST-FSHβ antigen does significantly prevent trabecular bone loss and thereby enhance the bone strength, indicating that a FSH-based vaccine may be a promising therapeutic strategy to slow down bone loss in postmenopausal women

  11. Immunization with FSHβ fusion protein antigen prevents bone loss in a rat ovariectomy-induced osteoporosis model

    Energy Technology Data Exchange (ETDEWEB)

    Geng, Wenxin; Yan, Xingrong; Du, Huicong; Cui, Jihong; Li, Liwen, E-mail: liven@nwu.edu.cn; Chen, Fulin, E-mail: chenfl@nwu.edu.cn

    2013-05-03

    Highlights: •A GST-FSH fusion protein was successfully expressed in E. coli. •Immunization with GST-FSH antigen can raise high-titer anti-FSH polyclonal sera. •Anti-FSH polyclonal sera can neutralize osteoclastogenic effect of FSH in vitro. •FSH immunization can prevent bone loss in a rat osteoporosis model. -- Abstract: Osteoporosis, a metabolic bone disease, threatens postmenopausal women globally. Hormone replacement therapy (HTR), especially estrogen replacement therapy (ERT), is used widely in the clinic because it has been generally accepted that postmenopausal osteoporosis is caused by estrogen deficiency. However, hypogonadal α and β estrogen receptor null mice were only mildly osteopenic, and mice with either receptor deleted had normal bone mass, indicating that estrogen may not be the only mediator that induces osteoporosis. Recently, follicle-stimulating hormone (FSH), the serum concentration of which increases from the very beginning of menopause, has been found to play a key role in postmenopausal osteoporosis by promoting osteoclastogenesis. In this article, we confirmed that exogenous FSH can enhance osteoclast differentiation in vitro and that this effect can be neutralized by either an anti-FSH monoclonal antibody or anti-FSH polyclonal sera raised by immunizing animals with a recombinant GST-FSHβ fusion protein antigen. Moreover, immunizing ovariectomized rats with the GST-FSHβ antigen does significantly prevent trabecular bone loss and thereby enhance the bone strength, indicating that a FSH-based vaccine may be a promising therapeutic strategy to slow down bone loss in postmenopausal women.

  12. Targeting of a chimeric human histone fusion mRNA to membrane-bound polysomes in HeLa cells

    International Nuclear Information System (INIS)

    The subcellular location of histone mRNA-containing polysomes may play a key role in the posttranscriptional events that mediate histone mRNA turnover following inhibition of DNA synthesis. Previously, it has been shown that histone mRNA is found primarily on free polysomes that are associated with the cytoskeleton. The authors report here the construction of an Escherichia coli pBR322 β-lactamase signal peptide-human H3 histone fusion gene. The fusion transcript is targeted to membrane-bound polysomes and remains stable following interruption of DNA replication. Relocating mRNA within the cell may provide a procedure for studying the posttranscriptional regulation of gene expression

  13. Targeting of a chimeric human histone fusion mRNA to membrane-bound polysomes in HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Zambetti, G.; Stein, J.; Stein, G.

    1987-05-01

    The subcellular location of histone mRNA-containing polysomes may play a key role in the posttranscriptional events that mediate histone mRNA turnover following inhibition of DNA synthesis. Previously, it has been shown that histone mRNA is found primarily on free polysomes that are associated with the cytoskeleton. The authors report here the construction of an Escherichia coli pBR322 ..beta..-lactamase signal peptide-human H3 histone fusion gene. The fusion transcript is targeted to membrane-bound polysomes and remains stable following interruption of DNA replication. Relocating mRNA within the cell may provide a procedure for studying the posttranscriptional regulation of gene expression.

  14. Reprogramming T cell Lymphocytes to Induced Pluripotent Stem Cells

    Science.gov (United States)

    Bared, Kalia

    The discovery of induced pluripotent stem cells (iPSC) provided a novel technology for the study of development and pharmacology and complement embryonic stem cells (ES) for cell therapy applications. Though iPSC are derived from adult tissue they are comparable to ES cells in their behavior; multi-lineage differentiation and self-renewal. This makes iPSC research appealing because they can be studied in great detail and expanded in culture broadly. Fibroblasts were the first cell type reprogrammed to an iPSC using a retrovirus vector, since then alternative cell types including lymphocytes have been used to generate iPSC. Different types of vectors have also been developed to enhance iPSC formation and quality. However, specific T lymphocyte subsets have not been shown to reprogram to a pluripotent state to date. Here, we proposed to derive iPSC from peripheral blood effector and central memory T cells, reasoning that the resultant iPSC will maintain the epigenetic memory of a T lymphocyte, including the T cell receptor (TCR) gene rearrangement. This epigenetic memory will enable the differentiation and expansion of T cell iPSC into professional T cells containing a specific TCR. These could then be used for cell therapy to target specific antigens, as well as to improve culture techniques to expand T cells in vitro. We studied different gene delivery methods to derive iPSC from different types of T lymphocytes. We assessed the viability of viral transduction using flow cytometry to detect green fluorescent marker contained in the viral construct and quantitative real time polymerase chain reaction (qRT-PCR) to detect Oct4, Klf4, Sox2, and c-Myc gene expression. Our results demonstrate that the Sendai virus construct is the most feasible platform to reprogram T lymphocytes. We anticipate that this platform will provide an efficient and safe approach to derive iPSC from different T cell subsets, including memory T cells.

  15. The Saccharomyces cerevisiae PRM1 homolog in Neurospora crassa is involved in vegetative and sexual cell fusion events but also has postfertilization functions.

    Science.gov (United States)

    Fleissner, André; Diamond, Spencer; Glass, N Louise

    2009-02-01

    Cell-cell fusion is essential for a variety of developmental steps in many eukaryotic organisms, during both fertilization and vegetative cell growth. Although the molecular mechanisms associated with intracellular membrane fusion are well characterized, the molecular mechanisms of plasma membrane merger between cells are poorly understood. In the filamentous fungus Neurospora crassa, cell fusion events occur during both vegetative and sexual stages of its life cycle, thus making it an attractive model for studying the molecular basis of cell fusion during vegetative growth vs. sexual reproduction. In the unicellular yeast Saccharomyces cerevisiae, one of the few proteins implicated in plasma membrane merger during mating is Prm1p; prm1Delta mutants show an approximately 50% reduction in mating cell fusion. Here we report on the role of the PRM1 homolog in N. crassa. N. crassa strains with deletions of a Prm1-like gene (Prm1) showed an approximately 50% reduction in both vegetative and sexual cell fusion events, suggesting that PRM1 is part of the general cell fusion machinery. However, unlike S. cerevisiae, N. crassa strains carrying a Prm1 deletion exhibited complete sterility as either a male or female mating partner, a phenotype that was not complemented in a heterokaryon with wild type (WT). Crosses with DeltaPrm1 strains were blocked early in sexual development, well before development of ascogenous hyphae. The DeltaPrm1 sexual defect in N. crassa was not suppressed by mutations in Sad-1, which is required for meiotic silencing of unpaired DNA (MSUD). However, mutations in Sad-1 increased the number of progeny obtained in crosses with a DeltaPrm1 (Prm1-gfp) complemented strain. These data indicate multiple roles for PRM1 during sexual development.

  16. Decoupling internalization, acidification and phagosomal-endosomal/lysosomal fusion during phagocytosis of InlA coated beads in epithelial cells.

    Directory of Open Access Journals (Sweden)

    Craig D Blanchette

    Full Text Available BACKGROUND: Phagocytosis has been extensively examined in 'professional' phagocytic cells using pH sensitive dyes. However, in many of the previous studies, a separation between the end of internalization, beginning of acidification and completion of phagosomal-endosomal/lysosomal fusion was not clearly established. In addition, very little work has been done to systematically examine phagosomal maturation in 'non-professional' phagocytic cells. Therefore, in this study, we developed a simple method to measure and decouple particle internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in Madin-Darby Canine Kidney (MDCK and Caco-2 epithelial cells. METHODOLOGY/PRINCIPAL FINDINGS: Our method was developed using a pathogen mimetic system consisting of polystyrene beads coated with Internalin A (InlA, a membrane surface protein from Listeria monocytogenes known to trigger receptor-mediated phagocytosis. We were able to independently measure the rates of internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in epithelial cells by combining the InlA-coated beads (InlA-beads with antibody quenching, a pH sensitive dye and an endosomal/lysosomal dye. By performing these independent measurements under identical experimental conditions, we were able to decouple the three processes and establish time scales for each. In a separate set of experiments, we exploited the phagosomal acidification process to demonstrate an additional, real-time method for tracking bead binding, internalization and phagosomal acidification. CONCLUSIONS/SIGNIFICANCE: Using this method, we found that the time scales for internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion ranged from 23-32 min, 3-4 min and 74-120 min, respectively, for MDCK and Caco-2 epithelial cells. Both the static and real-time methods developed here are expected to be readily and broadly applicable, as they simply

  17. Human retinal pigment epithelial cell-induced apoptosis in activated T cells

    DEFF Research Database (Denmark)

    Jørgensen, A; Wiencke, A K; la Cour, M;

    1998-01-01

    induced apoptosis in several activated T-cell populations and T-cell lines, including T-cell antigen receptor (TCR)-CD3-negative T-cell lines. In contrast, RPE cells induced little or no apoptosis in resting peripheral T cells. Major histocompatibility complex (MHC) class II monoclonal antibodies, which...

  18. Binase induces apoptosis of transformed myeloid cells and does not induce T-cell immune response.

    Science.gov (United States)

    Ilinskaya, Olga N; Zelenikhin, Pavel V; Petrushanko, Irina Yu; Mitkevich, Vladimir A; Prassolov, Vladimir S; Makarov, Alexander A

    2007-10-01

    Microbial RNases along with such animal RNases as onconase and BS-RNase are a promising basis for developing new antitumor drugs. We have shown that the Bacillus intermedius RNase (binase) induces selective apoptosis of transformed myeloid cells. It attacks artificially expressing activated c-Kit myeloid progenitor FDC cells and chronic myelogenous leukemia cells K562. Binase did not induce apoptosis in leukocytes of healthy donors and in normal myeloid progenitor cells. The inability of binase to initiate expression of activation markers CD69 and IFN-gamma in CD4+ and CD8+ T-lymphocytes testifies that enzyme is devoid of superantigenic properties. Altogether, these results demonstrate that binase possesses therapeutic opportunities for treatment of genotyped human neoplasms expressing activated kit.

  19. Intradermal injection of an anti-Langerin-HIVGag fusion vaccine targets epidermal Langerhans cells in nonhuman primates and can be tracked in vivo.

    Science.gov (United States)

    Salabert, Nina; Todorova, Biliana; Martinon, Frédéric; Boisgard, Raphaël; Zurawski, Gerard; Zurawski, Sandra; Dereuddre-Bosquet, Nathalie; Cosma, Antonio; Kortulewski, Thierry; Banchereau, Jacques; Levy, Yves; Le Grand, Roger; Chapon, Catherine

    2016-03-01

    The development of new immunization strategies requires a better understanding of early molecular and cellular events occurring at the site of injection. The skin is particularly rich in immune cells and represents an attractive site for vaccine administration. Here, we specifically targeted vaccine antigens to epidermal Langerhans cells (LCs) using a fusion protein composed of HIV antigens and a monoclonal antibody targeting Langerin. We developed a fluorescence imaging approach to visualize, in vivo, the vaccine-targeted cells. Studies were performed in nonhuman primates (NHPs) because of their relevance as a model to assess human vaccines. We directly demonstrated that in NHPs, intradermally injected anti-Langerin-HIVGag specifically targets epidermal LCs and induces rapid changes in the LC network, including LC activation and migration out of the epidermis. Vaccine targeting of LCs significantly improved anti-HIV immune response without requirement of an adjuvant. Although the co-injection of the TLR-7/8 synthetic ligand, R-848 (resiquimod), with the vaccine, did not enhance significantly the antibody response, it stimulated recruitment of HLA-DR+ inflammatory cells to the site of immunization. This study allowed us to characterize the dynamics of early local events following the injection of a vaccine-targeted epidermal LCs and R-848. PMID:26678013

  20. Analysis of cytoskeleton dynamics and cell migration in drosophila ovaries using GFP-actin and E-cadherin-GFP fusion molecules

    Science.gov (United States)

    Verkhusha, Vladyslav V.; Tsukita, Shoichiro; Oda, Hiroki

    1999-06-01

    Coordination of cell migration and adhesion is essential for movement of tissues during morphogenesis. During Drosophila oogenesis so called border cells (BCs) break from an anterior epithelium of egg chamber, acquire a mesenchymal-like morphology, and migrate posteriorly between nurse cells to oocyte. The confocal microscopic observation of BCs has revealed well-developed forepart lamellipodium stained with Drosophila E-cadherin (DE-cadherin), PS2 integrin, cytoplasmic myosin and F-actin. To investigate mechanism of BC migration in vivo we have constructed a DE-cadherin-GFP and a GFP-actin fusion proteins and induced their expression BCs utilizing the UAS/GAL4 system. The DE-cadherin-GFP signal as well as immunostaining of PS2 integrin visualized a track of migrating BCs providing an evidence that adhesive molecules are pulled out and left behind on the surface of nurse cells. Our data suggest that two distinct adhesive systems, DE-cadherins and PS2 integrins simultaneously mediate the migration of BCs. Release of adhesive contacts in the tail region is a rate- limited event in BC migration. The spatial-temporal sequence of actin-based events visualized by the GFP-actin suggest a treadmilling model for actin behavior in BC lamellipodium. BC migration can be considered as simultaneous reiterating processes of lamellipodium extension and adhesive attachment, cytoskeletal contraction, and rear detachment.

  1. Mass asymmetry of fusion and fission processes induced in heavy ion reaction

    International Nuclear Information System (INIS)

    Simple parametrisations of nuclear shapes able to describe the essential features of the fission and fusion processes were introduced. The Myers-Swiatecki (1967) liquid drop model and its generalisations: the Krape-Nix (1974) finite range of nuclear forces model and the Krappe-Nix-Sierk (1979) folded Yukawa-plus-exponential model, were extended for the nuclear systems with different charge densities. Some information concerning the interaction barrier (Esub(I)) the energy release (Q) and the fission barrier (Esub(b)) trends of variation with mass and charge asymmetry were obtained from the energy at infinite separation distance between ions (or fission fragments), at the touching point and at the fused spherical system. Good agreement with experimental data was obtained and an empirical relationship for the estimation of the interaction barriers was derived. The reaction parteners, for which it is important to take into account the charge density difference, were identified and the orders of magnitude of the errors on Esub(I), Q and Esub(b) made if this difference is ignored, were estimated. The nonaxiality of the nuclear shape at the first saddle point was demonstrated on a simple model allowing to decrease the computer running time by 3 orders of magnitude. The asymmetric spheroidal oscillator, introduced by the author, can be used to study the fission process. The fission theory was successfully applied to compute the Q-values and the life-times of the alpha decay. Good agreement (within +-0.8 orders of magnitude) of the theoretical half-lives with experimental ones, over a range of 24 orders of magnitude was obtained. This is a strong argument that the alpha decay could be considered a fission process with very high mass asymmetry and charge density asymmetry. (author)

  2. The elementary fusion modalities of osteoclasts

    DEFF Research Database (Denmark)

    Søe, Kent; Hobolt-Pedersen, Anne-Sofie; Delaisse, Jean-Marie

    2015-01-01

    The last step of the osteoclast differentiation process is cell fusion. Most efforts to understand the fusion mechanism have focused on the identification of molecules involved in the fusion process. Surprisingly, the basic fusion modalities, which are well known for fusion of other cell types......, are not known for the osteoclast. Here we show that osteoclast fusion partners are characterized by differences in mobility, nuclearity, and differentiation level. Our demonstration was based on time-laps videos of human osteoclast preparations from three donors where 656 fusion events were analyzed. Fusions...

  3. Low pH Is Required for Avian Sarcoma and Leukosis Virus Env-Induced Hemifusion and Fusion Pore Formation but Not for Pore Growth

    OpenAIRE

    Melikyan, G. B.; Barnard, R. J. O.; Markosyan, R M; Young, J. A. T.; Cohen, F S

    2004-01-01

    Binding of avian sarcoma and leukosis virus (ASLV) to its cognate receptor on the cell surface causes conformational changes in its envelope protein (Env). It is currently debated whether low pH is required for ASLV infection. To elucidate the role of low pH, we studied the association between ASLV subgroup B (ASLV-B) and liposomes and fusion between effector cells expressing Env from ASLV-A and ASLV-B and target cells expressing cognate receptors. Neither EnvA nor EnvB promoted cell-cell fus...

  4. Investigation of the role of break-up processes on the fusion of 16O induced reactions

    International Nuclear Information System (INIS)

    An experiment was carried out to explore heavy ion incomplete fusion reaction dynamics, within the framework of the break-up fusion model, at energies near and above the Coulomb barrier. Excitation functions for several radionuclides produced via xn, pxn, and αxn channels were measured in the 16O+181Ta system at energies of ≅76-100 MeV. The experimental excitation functions were compared with those calculated using the theoretical model code PACE4. It was observed that excitation functions of xn/pxn channels are in good agreement with theoretical predictions. However, a significant enhancement in the measured excitation functions of α-emitting channels was observed and attributed to the incomplete fusion processes. The incomplete fusion fraction (FICF) that gives the relative importance of complete and incomplete fusion processes was found to increase with energy. The results are discussed in terms of α-cluster structure of the projectile on various fusion reactions.

  5. Fusion of CT and PET data: method and clinical relevance for planning laser-induced thermotherapy of liver metastases; Fusion von CT- und PET-Daten: Methode und klinische Bedeutung am Beispiel der Planung der laserinduzierten Thermotherapie von Lebermetastasen

    Energy Technology Data Exchange (ETDEWEB)

    Hosten, N.; Kreissig, R.; Puls, R.; Amthauer, H.; Beier, J.; Rohlfing, T.; Stroszczynski, C.; Herbel, A.; Lemke, A.J.; Felix, R. [Humboldt-Universitaet, Berlin (Germany). Strahlenklinik und Poliklinik

    2000-07-01

    Aim: Cathether-placement in liver metastases is difficult when the lesions are not visible on plain images. We evaluated the use of image fusion using CT and PET data, providing information on anatomy and liver lesions, respectively. Methods: Plain CT of the liver and whole body FDG-PET were performed in 28 patients with colorectal carcinoma in preparation for laser-induced thermotherapy. Fusions of image data and 2D-visualisation were performed and evaluated with regard to quality of the registration, number of detected lesions and influence of the procedure on laser-induced thermotherapy. Image fusion was performed using an algorithm which was developed by our group. Results: 84 focal liver lesions were visible on CT, 107 on PET images. This means that CT guided catheter placement would have to be performed 'blindly' in 23 lesions. In 6 patients previously unknown extra-hepatic tumor deposits were seen on PET images (local recurrence, 2 extrahepatic metastases, 2 second primaries). Conclusion: Hot spots from metastases, which are difficult to locate on PET images alone, may be identified on images generated by fusion of CT and PET data. These images facilitate an interventional approach to liver lesions which are not visible on plain CT. As PET allows for whole body screening, it helps identifying patients who would not profit from laser-induced thermotherapy. (orig.) [German] Ziel: Bei der CT-gesteuerten Punktion von Leberherden ist der fehlende Laesionsnachweis in der nativen CT oft hinderlich. Es wurde untersucht, ob die Bildfusion PET + CT weiterhilft. Methoden: Native Leber-CT und Ganzkoerper-FDG-PET wurden bei 28 Patienten mit kolorektalen Karzinomen durchgefuehrt, die zur LITT bei Lebermetastasen zugewiesen wurden. Die Datensaetze wurden fusioniert und hinsichtlich Qualitaet der Registrierung, Anzahl nachgewiesener Laesionen und Einfluss der fusionierten Bilder auf die Therapie ausgewertet. Ein selbstentwickelter Algorithmus wurde fuer die Fusion

  6. The enlarged lysosomes in beigej cells result from decreased lysosome fission and not increased lysosome fusion

    OpenAIRE

    Durchfort, Nina; Verhoef, Shane; Vaughn, Michael B.; Shrestha, Rishna; Adam, Dieter; Kaplan, Jerry; Ward, Diane McVey

    2011-01-01

    Chediak-Higashi Syndrome is an autosomal recessive disorder that affects vesicle morphology. The Chs1/Lyst protein is a member of the BEACH family of proteins. The absence of Chs1/Lyst gives rise to enlarged lysosomes. Lysosome size is regulated by a balance between vesicle fusion and fission and can be reversibly altered by acidifying the cytoplasm using Acetate Ringer’s or by incubating with the drug vacuolin-1. We took advantage of these procedures to determine rates of lysosome fusion and...

  7. Dynamin and endocytosis are required for the fusion of osteoclasts and myoblasts

    OpenAIRE

    Shin, Nah-Young; Choi, Hyewon; Neff, Lynn; Wu, Yumei; Saito, Hiroaki; Ferguson, Shawn M.; De Camilli, Pietro; Baron, Roland

    2014-01-01

    Cell–cell fusion is an evolutionarily conserved process that leads to the formation of multinucleated myofibers, syncytiotrophoblasts and osteoclasts, allowing their respective functions. Although cell–cell fusion requires the presence of fusogenic membrane proteins and actin-dependent cytoskeletal reorganization, the precise machinery allowing cells to fuse is still poorly understood. Using an inducible knockout mouse model to generate dynamin 1– and 2–deficient primary osteoclast precursors...

  8. The Preliminary Experimental Study of Induced Differentiation of Embryonic Stem Cells into Corneal Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    Ling Yu; Jian Ge; Zhichong Wang; Bing Huang; Keming Yu; Chongde Long; Xigu Chen

    2001-01-01

    Purpose:To study preliminarily induced differentiation of embryonic stem cells intocorneal epithelial cells in vitro.Methods: Murine embryonic stem cells were co-cultured with Rabbit limbal cornealepithelial cells in Transwell system to induce differentiation. Mophological andimmunohistochemical examination were implemented.Results: The induced cells from embryonic stem cells have an epithelial appearance.The cells formed a network and were confluent into film gradually after beingco-cultured with rabbit limbal corneal epithelial cells for 24 ~ 96 hours. The cells rangedmosaic structure and localized together with clear rim. Most of the cells showedpolygonal appearance. Transmission electron microscope showed lots of microvilli on thesurface of induced cells and tight junctions between them. These epithelial-like cellsexpressed the corneal epithelial cell specific marker cytokeratin3/cytokeratinl2.Conclusion: The potential mechanism of the differentiation of murine embryonic stemcells into corneal epithelial cells induced by limbal corneal epithelial cell-derivedinducing activity is to be further verified.

  9. Laser-induced mobilization of dust produced during fusion reactors operation

    International Nuclear Information System (INIS)

    During tokamak operation, plasma-wall interactions lead to material erosion process and dusts production. These dusts are mainly composed by carbon and tungsten, with sizes ranging from 10 nm to 100 μm. For safety reasons and to guarantee an optimum reactor functioning, the dusts have to be kept in reasonable quantity. The dusts mobilization is a first step to collect them, and the laser is a promising technique for this application. To optimize the cleaning, physical mechanisms responsible for dust ejection induced by laser have been identified. Some particles, such as aggregates, are directly ablated by the laser. The metal droplets are ejected intact by an electrostatic force, induced by the photoelectrons. We also characterized the particles ejection to choose an appropriate collection device. (author)

  10. Tumorigenic risk of human induced pluripotent stem cell explants cultured on mouse SNL76/7 feeder cells

    International Nuclear Information System (INIS)

    Highlights: • hiPS cell explants formed malignant tumors when SNL76/7 feeder cells were used. • Multi type tumors developed by interaction of SNL76/7 feeder cells with hiPS cells. • Tumorigenic risk occurs by co-culture of hiPS cells with SNL76/7 feeder cells. - Abstract: The potential for tumor formation from transplanted human induced pluripotent stem cell (hiPSC) derivatives represents a high risk in their application to regenerative medicine. We examined the genetic origin and characteristics of tumors, that were formed when 13 hiPSC lines, established by ourselves, and 201B7 hiPSC from Kyoto University were transplanted into severe combined immune-deficient (SCID) mice. Though teratomas formed in 58% of mice, five angiosarcomas, one malignant solitary fibrous tumor and one undifferentiated pleomorphic sarcoma formed in the remaining mice. Three malignant cell lines were established from the tumors, which were derived from mitomycin C (MMC)-treated SNL76/7 (MMC-SNL) feeder cells, as tumor development from fusion cells between MMC-SNL and hiPSCs was negative by genetic analysis. While parent SNL76/7 cells produced malignant tumors, neither MMC-SNL nor MMC-treated mouse embryo fibroblast (MEF) produced malignant tumors. When MMC-SNL feeder cells were co-cultured with hiPSCs, growing cell lines were generated, that expressed genes similar to the parent SNL76/7 cells. Thus, hiPSCs grown on MMC-SNL feeder cells have a high risk of generating feeder-derived malignant tumors. The possible mechanism(s) of growth restoration and the formation of multiple tumor types are discussed with respect of the interactions between MMC-SNL and hiPSC

  11. Tumorigenic risk of human induced pluripotent stem cell explants cultured on mouse SNL76/7 feeder cells

    Energy Technology Data Exchange (ETDEWEB)

    Kamada, Mizuna; Mitsui, Youji, E-mail: y-mitsui8310@hb.tp1.jp; Kumazaki, Tsutomu; Kawahara, Yuta; Matsuo, Taira; Takahashi, Tomoko, E-mail: t-takahashi@kph.bunri-u.ac.jp

    2014-10-24

    Highlights: • hiPS cell explants formed malignant tumors when SNL76/7 feeder cells were used. • Multi type tumors developed by interaction of SNL76/7 feeder cells with hiPS cells. • Tumorigenic risk occurs by co-culture of hiPS cells with SNL76/7 feeder cells. - Abstract: The potential for tumor formation from transplanted human induced pluripotent stem cell (hiPSC) derivatives represents a high risk in their application to regenerative medicine. We examined the genetic origin and characteristics of tumors, that were formed when 13 hiPSC lines, established by ourselves, and 201B7 hiPSC from Kyoto University were transplanted into severe combined immune-deficient (SCID) mice. Though teratomas formed in 58% of mice, five angiosarcomas, one malignant solitary fibrous tumor and one undifferentiated pleomorphic sarcoma formed in the remaining mice. Three malignant cell lines were established from the tumors, which were derived from mitomycin C (MMC)-treated SNL76/7 (MMC-SNL) feeder cells, as tumor development from fusion cells between MMC-SNL and hiPSCs was negative by genetic analysis. While parent SNL76/7 cells produced malignant tumors, neither MMC-SNL nor MMC-treated mouse embryo fibroblast (MEF) produced malignant tumors. When MMC-SNL feeder cells were co-cultured with hiPSCs, growing cell lines were generated, that expressed genes similar to the parent SNL76/7 cells. Thus, hiPSCs grown on MMC-SNL feeder cells have a high risk of generating feeder-derived malignant tumors. The possible mechanism(s) of growth restoration and the formation of multiple tumor types are discussed with respect of the interactions between MMC-SNL and hiPSC.

  12. Multinucleation and cell dysfunction induced by amorphous silica nanoparticles in an L-02 human hepatic cell line

    Directory of Open Access Journals (Sweden)

    Wang W

    2013-09-01

    Full Text Available Wen Wang,1–3,* Yang Li,1–3,* Xiaomei Liu,3 Minghua Jin,3 Haiying Du,3 Ying Liu,3 Peili Huang,1,2 Xianqing Zhou,1,2 Lan Yuan,4 Zhiwei Sun1–3 1School of Public Health, Capital Medical University, Beijing, 2Beijing Key Laboratory of Environmental Toxicology, Capital Medical University, Beijing, 3School of Public Health, Jilin University, Changchun, Jilin, 4Medical and Healthy Analysis Centre, Peking University, Beijing, People's Republic of China *These authors contributed equally to this work Abstract: Silica nanoparticles (SNPs are one of the most important nanomaterials, and have been widely used in a variety of fields. Therefore, their effects on human health and the environment have been addressed in a number of studies. In this work, the effects of amorphous SNPs were investigated with regard to multinucleation in L-02 human hepatic cells. Our results show that L-02 cells had an abnormally high incidence of multinucleation upon exposure to silica, that increased in a dose-dependent manner. Propidium iodide staining showed that multinucleated cells were arrested in G2/M phase of the cell cycle. Increased multinucleation in L-02 cells was associated with increased generation of cellular reactive oxygen species and mitochondrial damage on flow cytometry and confocal microscopy, which might have led to failure of cytokinesis in these cells. Further, SNPs inhibited cell growth and induced apoptosis in exposed cells. Taken together, our findings demonstrate that multinucleation in L-02 human hepatic cells might be a failure to undergo cytokinesis or cell fusion in response to SNPs, and the increase in cellular reactive oxygen species could be responsible for the apoptosis seen in both mononuclear cells and multinucleated cells. Keywords: silica nanoparticles, human hepatic cell L-02, multinucleation, cell cycle, cell dysfunction, apoptosis

  13. IL-35 is a novel cytokine with therapeutic effects against collagen-induced arthritis through the expansion of regulatory T cells and suppression of Th17 cells.

    Science.gov (United States)

    Niedbala, Wanda; Wei, Xiao-Qing; Cai, Beilei; Hueber, Axel J; Leung, Bernard P; McInnes, Iain B; Liew, Foo Y

    2007-11-01

    Epstein-Barr virus-induced gene 3 (EBI3) and the p35 subunit of IL-12 have been reported to form a heterodimeric hematopoietin in human and mouse. We have constructed a heterodimeric protein covalently linking EBI3 and p35, to form a novel cytokine which we now call IL-35. The Fc fusion protein of IL-35 induced proliferation of murine CD4(+)CD25(+) and CD4(+)CD25(-) T cells when stimulated with immobilized anti-CD3 and anti-CD28 antibodies in vitro. The IL-35-expanded CD4(+)CD25(+) T cell population expressed Foxp3 and produced elevated levels of IL-10, whereas the IL-35-induced CD4(+)CD25(-) T cells produced IFN-gamma but not IL-4. The in vitro expanded CD4(+)CD25(+) T cells retained their suppressive functions against CD4(+)CD25(-) effector cells. Furthermore, when cultured with soluble anti-CD3 antibody and antigen-presenting cells, IL-35 suppressed the proliferation of CD4(+)CD25(-) effector cells. Moreover, IL-35 inhibited the differentiation of Th17 cells in vitro. In vivo, IL-35 effectively attenuated established collagen-induced arthritis in mice, with concomitant suppression of IL-17 production but enhanced IFN-gamma synthesis. Thus, IL-35 is a novel anti-inflammatory cytokine suppressing the immune response through the expansion of regulatory T cells and suppression of Th17 cell development.

  14. Tat-APE1/ref-1 protein inhibits TNF-alpha-induced endothelial cell activation.

    Science.gov (United States)

    Song, Yun Jeong; Lee, Ji Young; Joo, Hee Kyoung; Kim, Hyo Shin; Lee, Sang Ki; Lee, Kwon Ho; Cho, Chung-Hyun; Park, Jin Bong; Jeon, Byeong Hwa

    2008-03-28

    Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/ref-1) is a multifunctional protein involved both in DNA base excision repair and redox regulation. In this study we evaluated the protective role of Tat-mediated APE1/ref-1 transduction on the tumor necrosis factor (TNF)-alpha-activated endothelial activation in cultured human umbilical vein endothelial cells. To construct Tat-APE1/ref-1 fusion protein, human full length of APE1/ref-1 was fused with Tat-protein transduction domain. Purified Tat-APE1/ref-1 fusion protein efficiently transduced cultured endothelial cells in a dose-dependent manner and reached maximum expression at 1h after incubation. Transduced Tat-APE1/ref-1 showed inhibitory activity on the TNF-alpha-induced monocyte adhesion and vascular cell adhesion molecule-1 expression in cultured endothelial cells. These results suggest Tat-APE1/ref-1 might be useful to reduce vascular endothelial activation or vascular inflammatory disorders.

  15. Mutations in the DI-DII Linker of Human Parainfluenza Virus Type 3 Fusion Protein Result in Diminished Fusion Activity.

    Directory of Open Access Journals (Sweden)

    Wenyan Xie

    Full Text Available Human parainfluenza virus type 3 (HPIV3 can cause severe respiratory tract diseases in infants and young children, but no licensed vaccines or antiviral agents are currently available for treatment. Fusing the viral and target cell membranes is a prerequisite for its entry into host cells and is directly mediated by the fusion (F protein. Although several domains of F are known to have important effects on regulating the membrane fusion activity, the roles of the DI-DII linker (residues 369-374 of the HPIV3 F protein in the fusogenicity still remains ill-defined. To facilitate our understanding of the role of this domain might play in F-induced cell-cell fusion, nine single mutations were engineered into this domain by site-directed mutagenesis. A vaccinia virus-T7 RNA polymerase transient expression system was employed to express the wild-type or mutated F proteins. These mutants were analyzed for membrane fusion activity, cell surface expression, and interaction between F and HN protein. Each of the mutated F proteins in this domain has a cell surface expression level similar to that of wild-type F. All of them resulted in a significant reduction in fusogenic activity in all steps of membrane fusion. Furthermore, all these fusion-deficient mutants reduced the amount of the HN-F complexes at the cell surface. Together, the results of our work suggest that this region has an important effect on the fusogenic activity of F.

  16. Jamming dynamics of stretch-induced surfactant release by alveolar type II cells.

    Science.gov (United States)

    Majumdar, Arnab; Arold, Stephen P; Bartolák-Suki, Erzsébet; Parameswaran, Harikrishnan; Suki, Béla

    2012-03-01

    Secretion of pulmonary surfactant by alveolar epithelial type II cells is vital for the reduction of interfacial surface tension, thus preventing lung collapse. To study secretion dynamics, rat alveolar epithelial type II cells were cultured on elastic membranes and cyclically stretched. The amounts of phosphatidylcholine, the primary lipid component of surfactant, inside and outside the cells, were measured using radiolabeled choline. During and immediately after stretch, cells secreted less surfactant than unstretched cells; however, stretched cells secreted significantly more surfactant than unstretched cells after an extended lag period. We developed a model based on the hypothesis that stretching leads to jamming of surfactant traffic escaping the cell, similar to vehicular traffic jams. In the model, stretch increases surfactant transport from the interior to the exterior of the cell. This transport is mediated by a surface layer with a finite capacity due to the limited number of fusion pores through which secretion occurs. When the amount of surfactant in the surface layer approaches this capacity, interference among lamellar bodies carrying surfactant reduces the rate of secretion, effectively creating a jam. When the stretch stops, the jam takes an extended time to clear, and subsequently the amount of secreted surfactant increases. We solved the model analytically and show that its dynamics are consistent with experimental observations, implying that surfactant secretion is a fundamentally nonlinear process with memory representing collective behavior at the level of single cells. Our results thus highlight the importance of a jamming dynamics in stretch-induced cellular secretory processes. PMID:22033531

  17. New model for cardiomyocyte sheet transplantation using avirus-cell fusion technique

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    AIM To facilitate close contacts between transplantedcardiomyocytes and host skeletal muscle using cellfusion mediated by hemagglutinating virus of Japanenvelope (HVJ-E) and tissue maceration.METHODS: Cardiomyocytes (1.5 × 106) from fetalrats were first cultured. After proliferation, some cellswere used for fusion with adult muscle fibers usingHVJ-E. Other cells were used to create cardiomyocytesheets (area: about 3.5 cm2 including 2.1 × 106cells), which were then treated with Nile blue, separated,and transplanted between the latissimusdorsi and intercostal muscles of adult rats with fourcombinations of HVJ-E and/or NaOH maceration:G1: HVJ-E(+), NaOH(+), Cardiomyocytes(+); G2:HVJ-E(-), NaOH(+), Cardiomyocytes(+); G3: HVJ-E(+),NaOH(-), Cardiomyocytes(+); G4: HVJ-E(-), NaOH(-),Cardiomyocytes(-). At 1 and 2 wk after transplantation,the four groups were compared by detection of beatingdomains, motion images using moving target analysissoftware, action potentials, gene expression of MLC-2v and Mesp1 by reverse transcription-polymerasechain reaction, hematoxylin-eosin staining, and immunostainingfor cardiac troponin and skeletal myosin.RESULTS: In vitro cardiomyocytes were fused withskeletal muscle fibers using HVJ-E. Cardiomyocytesheets remained in the primary transplanted sites for2 wk. Although beating domains were detected inG1, G2, and G3 rats, G1 rats prevailed in the number,size, motion image amplitudes, and action potentialcompared with G2 and G3 rats. Close contacts wereonly found in G1 rats. At 1 wk after transplantation,the cardiomyocyte sheets showed adhesion at variouspoints to the myoblast layer in the latissimus dorsimuscle. At 2 wk after transplantation, close contactswere seen over a broad area. Part of the skeletalmuscle sarcoplasma seemed to project into themyocardiocyte plasma and some nuclei appeared toshare both sarcoplasmas.CONCLUSION: The present results

  18. Single-Copy Green Fluorescent Protein Gene Fusions Allow Accurate Measurement of Salmonella Gene Expression In Vitro and during Infection of Mammalian Cells

    OpenAIRE

    Hautefort, Isabelle; Proença, Maria José; Hinton, Jay C. D.

    2003-01-01

    We developed a reliable and flexible green fluorescent protein (GFP)-based system for measuring gene expression in individual bacterial cells. Until now, most systems have relied upon plasmid-borne gfp gene fusions, risking problems associated with plasmid instability. We show that a recently developed GFP variant, GFP+, is suitable for assessing bacterial gene expression. Various gfp+ transcriptional fusions were constructed and integrated as single copies into the chromosome of Salmonella e...

  19. CD36 is required for myoblast fusion during myogenic differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Park, Seung-Yoon [Department of Biochemistry, College of Medicine, Dongguk University and Medical Institute of Dongguk University, Gyeongju 780-714 (Korea, Republic of); Yun, Youngeun [Department of Biochemistry and Cell Biology, Cell and Matrix Research Institute, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Kim, In-San, E-mail: iskim@knu.ac.kr [Department of Biochemistry and Cell Biology, Cell and Matrix Research Institute, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Biomedical Research Institute, Korea Institute Science and Technology, Seoul (Korea, Republic of)

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer CD36 expression was induced during myogenic differentiation. Black-Right-Pointing-Pointer CD36 expression was localized in multinucleated myotubes. Black-Right-Pointing-Pointer The expression of myogenic markers is attenuated in CD36 knockdown C2C12 cells. Black-Right-Pointing-Pointer Knockdown of CD36 significantly inhibited myotube formation during differentiation. -- Abstract: Recently, CD36 has been found to be involved in the cytokine-induced fusion of macrophage. Myoblast fusion to form multinucleated myotubes is required for myogenesis and muscle regeneration. Because a search of gene expression database revealed the attenuation of CD36 expression in the muscles of muscular dystrophy patients, the possibility that CD36 could be required for myoblast fusion was investigated. CD36 expression was markedly up-regulated during myoblast differentiation and localized in multinucleated myotubes. Knockdown of endogenous CD36 significantly decreased the expression of myogenic markers as well as myotube formation. These results support the notion that CD36 plays an important role in cell fusion during myogenic differentiation. Our finding will aid the elucidation of the common mechanism governing cell-to-cell fusion in various fusion models.

  20. Organelle acidification negatively regulates vacuole membrane fusion in vivo

    Science.gov (United States)

    Desfougères, Yann; Vavassori, Stefano; Rompf, Maria; Gerasimaite, Ruta; Mayer, Andreas

    2016-01-01

    The V-ATPase is a proton pump consisting of a membrane-integral V0 sector and a peripheral V1 sector, which carries the ATPase activity. In vitro studies of yeast vacuole fusion and evidence from worms, flies, zebrafish and mice suggested that V0 interacts with the SNARE machinery for membrane fusion, that it promotes the induction of hemifusion and that this activity requires physical presence of V0 rather than its proton pump activity. A recent in vivo study in yeast has challenged these interpretations, concluding that fusion required solely lumenal acidification but not the V0 sector itself. Here, we identify the reasons for this discrepancy and reconcile it. We find that acute pharmacological or physiological inhibition of V-ATPase pump activity de-acidifies the vacuole lumen in living yeast cells within minutes. Time-lapse microscopy revealed that de-acidification induces vacuole fusion rather than inhibiting it. Cells expressing mutated V0 subunits that maintain vacuolar acidity were blocked in this fusion. Thus, proton pump activity of the V-ATPase negatively regulates vacuole fusion in vivo. Vacuole fusion in vivo does, however, require physical presence of a fusion-competent V0 sector. PMID:27363625

  1. Artesunate induces AIF-dependent apoptosis in A549 cells

    Science.gov (United States)

    Zhou, Chen-juan; Chen, Tong-Sheng

    2012-03-01

    Artesunate (ART), a semi-synthetic derivative of the sesquiterpene artemisinin extracted from the Chinese herb Artemisia annua, exerts a broad spectrum of clinical activity against human cancers. It has been shown that ART induces cancer cells death through apoptosis pathway. This study investigated whether ART treatment induced reactive oxygen species (ROS)-dependent cell death in the apoptosis fashion in human lung adenocarconoma A549 cell line and the proapoptotic protein apoptosis inducing factor (AIF) is involved in ART-induced apoptosis. Cells treated with ART exhibited typical apoptotic morphology as chromatin condensation, margination and shrunken nucleus. ART treatment also induced a loss of mitochondrial membrane potential and AIF release from mitochondria. Silencing AIF can remarkable attenuated ART-induced apoptosis. Collectively, ART induces apoptosis by caspase-independent intrinsic pathway in A549 cells.

  2. EMP Fusion

    OpenAIRE

    KUNTAY, Isık

    2010-01-01

    This paper introduces a novel fusion scheme, called EMP Fusion, which has the promise of achieving breakeven and realizing commercial fusion power. The method is based on harnessing the power of an electromagnetic pulse generated by the now well-developed flux compression technology. The electromagnetic pulse acts as a means of both heating up the plasma and confining the plasma, eliminating intermediate steps. The EMP Fusion device is simpler compared to other fusion devices and this reduces...

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    Osteolytic lesions are a hallmark of multiple myeloma. They are due to the hyperactivity of bone resorbing osteoclasts and hypoactivity of bone forming osteoblasts, in response to neighbouring myeloma cells. This study identified a structure that deeply affects this response, because of its impact...... on the physical organisation of the myeloma cell microenvironment. The proximity between myeloma cells and osteoclasts or osteoblasts was shown to be conditioned by the recently discovered layer of flat cells that separates the osteoclasts and osteoblasts from the bone marrow, by forming a canopy over bone......, this disruption and increased proximity and density of myeloma cells coincides with key myeloma-induced bone events, such as osteolytic lesions, impaired bone formation despite increased bone resorption, and fusion of myeloma cells with osteoclasts thereby forming myeloma-osteoclast hybrid cells. These findings...

  4. Sensitization of radiation-induced cell death by genistein

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Tae Rim; Kim, In Gyu [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2010-03-15

    A number of epidemiological studies as well as biological experiments, showed that genistein, one of the isoflavone, prevents prostate cancer occurrence. In this study, we showed that genistein inhibited the cell proliferation of human promyeoltic leukemia HL-60 cells and induced G2/M phase arrest. In addition, combination of genistein treatment and {gamma}-irradiation displayed synergistic effect in apoptotic cell death of HL-60 cells. This means that the repair of genistein-induced DNA damage was hindered by {gamma}-irradiation and thus cell death was increased. In conclusion, genistein is one of the important chemicals that sensitize radiation-induced cell death.

  5. Abrasion-resistant solgel antireflective films with a high laser-induced damage threshold for inertial confinement fusion

    Science.gov (United States)

    Xu, Yao; Zhang, Lei; Wu, Dong; Sun, Yu Han; Huang, Zu Xing; Jiang, Xiao Dong; Wei, Xiao Feng; Li, Zhi Hong; Dong, Bao Zhong; Wu, Zhong Hua

    2005-09-01

    To prepare abrasion-resistant antireflective (AR) films for inertial confinement fusion, four solgel routes have been investigated on polysiloxane-modified and polyvinylalcohol- (PVA-) modified SiO2 sols. As confirmed with a transmissive electron microscope, different fractal structure characteristics of the modified SiO2 particles are disclosed by small-angle x-ray scattering technology. And it is these special fractal characteristics that determine the performance of AR films on the level of internal microstructure. A 29Si magic angle spinning and nuclear magnetic resonance study has been successfully applied in explaining the fractal microstructure and its relation to the laser-induced damage threshold (LIDT) of AR films. The films modified by PVA120000 or acetic acid-catalyzed polysiloxane have higher LIDTs than those films modified by PVA16000 or hydrochloride acid-catalyzed polysiloxane. The films from PVA-modified SiO2 sols have a stronger abrasion resistance but lower antireflection than those films from polysiloxane-modified SiO2 sols. In addition, the films from polysiloxane-modified SiO2 sols can possess high transmittance and high LIDT if the polysiloxane synthesis condit