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Sample records for cell fusion induced

  1. Optically-Induced Cell Fusion on Cell Pairing Microstructures

    NASA Astrophysics Data System (ADS)

    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. Optically-Induced Cell Fusion on Cell Pairing Microstructures

    PubMed Central

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

    2016-01-01

    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. PMID:26912054

  3. Femtosecond laser-induced fusion of nonadherent cells and two-cell porcine embryos

    NASA Astrophysics Data System (ADS)

    Kuetemeyer, Kai; Lucas-Hahn, Andrea; Petersen, Bjoern; Niemann, Heiner; Heisterkamp, Alexander

    2011-08-01

    Cell fusion is a fundamental biological process that can be artificially induced by different methods. Although femtosecond (fs) lasers have been successfully employed for cell fusion over the past few years, the underlying mechanisms are still unknown. In our experimental study, we investigated the correlation between fs laser-induced cell fusion and membrane perforation, and the influence of laser parameters on the fusion efficiency of nonadherent HL-60 cells. We found that shorter exposure times resulted in higher fusion efficiencies with a maximum of 21% at 10 ms and 100 mJ/cm2 (190 mW). Successful cell fusion was indicated by the formation of a long-lasting vapor bubble in the irradiated area with an average diameter much larger than in cell perforation experiments. With this knowledge, we demonstrated, for the first time, the fusion of very large parthenogenetic two-cell porcine embryos with high efficiencies of 55% at 20 ms and 360 mJ/cm2 (670 mW). Long-term viability of fused embryos was proven by successful development up to the blastocyst stage in 70% of cases with no significant difference to controls. In contrast to previous studies, our results indicate that fs laser-induced cell fusion occurs when the membrane pore size exceeds a critical value, preventing immediate membrane resealing.

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

    SciTech Connect

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

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  6. Cell-Cell Fusion Induced by Measles Virus Amplifies the Type I Interferon Response▿ †

    PubMed Central

    Herschke, F.; Plumet, S.; Duhen, T.; Azocar, O.; Druelle, J.; Laine, D.; Wild, T. F.; Rabourdin-Combe, C.; Gerlier, D.; Valentin, H.

    2007-01-01

    Measles virus (MeV) infection is characterized by the formation of multinuclear giant cells (MGC). We report that beta interferon (IFN-β) production is amplified in vitro by the formation of virus-induced MGC derived from human epithelial cells or mature conventional dendritic cells. Both fusion and IFN-β response amplification were inhibited in a dose-dependent way by a fusion-inhibitory peptide after MeV infection of epithelial cells. This effect was observed at both low and high multiplicities of infection. While in the absence of virus replication, the cell-cell fusion mediated by MeV H/F glycoproteins did not activate any IFN-α/β production, an amplified IFN-β response was observed when H/F-induced MGC were infected with a nonfusogenic recombinant chimerical virus. Time lapse microscopy studies revealed that MeV-infected MGC from epithelial cells have a highly dynamic behavior and an unexpected long life span. Following cell-cell fusion, both of the RIG-I and IFN-β gene deficiencies were trans complemented to induce IFN-β production. Production of IFN-β and IFN-α was also observed in MeV-infected immature dendritic cells (iDC) and mature dendritic cells (mDC). In contrast to iDC, MeV infection of mDC induced MGC, which produced enhanced amounts of IFN-α/β. The amplification of IFN-β production was associated with a sustained nuclear localization of IFN regulatory factor 3 (IRF-3) in MeV-induced MGC derived from both epithelial cells and mDC, while the IRF-7 up-regulation was poorly sensitive to the fusion process. Therefore, MeV-induced cell-cell fusion amplifies IFN-α/β production in infected cells, and this indicates that MGC contribute to the antiviral immune response. PMID:17898060

  7. Regulation of HSV glycoprotein induced cascade of events governing cell-cell fusion.

    PubMed

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

    2016-09-14

    Receptor dependent HSV-induced fusion requires glycoproteins gD, gH/gL, and gB. Our current model posits that during fusion receptor-activated conformational changes in gD activate gH/gL, which subsequently triggers transformation of the pre-fusion form of gB into a fusogenic state. To examine the role of each glycoprotein in receptor dependent cell-cell fusion we took advantage of our discovery that fusion by wild type HSV-2 glycoproteins occurs twice as fast as that achieved by HSV-1 glycoproteins. By sequentially swapping each glycoprotein between the two serotypes, we established that fusion speed was governed by gH/gL, with gH being the main contributor. While the mutant forms of gB fuse at distinct rates that are dictated by their molecular structure, these restrictions can be overcome by gH2/gL2, thereby enhancing their activity. We also found that deregulated forms of gD1 and gH2/gL2 can alter the fusogenic potential of gB, promoting cell fusion in the absence of a cellular receptor and that deregulated forms of gB can drive the fusion machinery to even higher levels. Low pH enhanced fusion by affecting the structure of both gB and gH/gL mutants. Together, our data highlight the complexity of the fusion machinery, the impact of the activation state of each glycoprotein on the fusion process and the critical role of gH/gL in regulating HSV induced fusion.

  8. [Molecular Mechanism of Glycoprotein-induced Cell-Cell Fusion of Herpesviruses].

    PubMed

    Feng, Daishen; Jia, Renyong

    2016-01-01

    Herpesviridae is a large family comprising linear, double-stranded DNA viruses. Herpesviridae contains three subfamilies: α-, β- and γ-herpesviruses. The glycoproteins gB, gH and gL of each subfamily form the "core fusion function" in cell-cell fusion. Other herpesviruses also need additional glycoproteins to promote fusion, such as gD of the Herpes simplex virus, gp42 of the Epstein-Barr virus, and gO or UL128-131 of the Human cytomegalovirus. In contrast, glycoproteins gM or gM/gN of herpesvirus inhibit fusion. We describe the molecular mechanisms of glycoprotein-induced fusion and entry of herpesviruses. It will be helpful to further study the pathogenic mechanism of herpesvirus.

  9. Polyethylene glycol-induced fusion of two-cell mouse embryo blastomeres

    SciTech Connect

    Spindle, A.

    1981-01-01

    Polyethylene glycol (PEG) was found to be an effective fusion-inducing agent for early mouse embryo blastomeres. A brief exposure of zona-intact 2-cell embryos to 40% PEG induced fusion of blastomeres in > 80% of embryos, and the treatment did not interfere with subsequent development of embryos to the blastocyst stage.

  10. Genetic studies of cell fusion induced by herpes simplex virus type 1

    SciTech Connect

    Read, G.S.; Person, S.; Keller, P.M.

    1980-07-01

    Eight cell fusion-causing syn mutants were isolated from the KOS strain of herpes simplex virus type 1. Unlike the wild-type virus, the mutants produced plaques containing multinucleated cells, or syncytia. Fusion kinetics curves were established with a Coulter Counter assay for the mutants and wild-type virus in single infections of human embryonic lung (HEL) cells, for the mutants and wild-type virus in mixed infections (dominance test), and for pairs of mutants in mixed infection and proceeded with an exponential decrease in the number of small single cells. At some later time that was characteristic of the mutant, there was a significant reduction in the rate of fusion for all but possibly one of the mutants. Although the wild-type virus did not produce syncytial plaques, it did induce a small amount of fusion that stopped abruptly about 2 h after it started. These data are consistent with the hypothesis that both mutants and wild type induce an active fusion inducer and that the activity of this inducer is subsequently inhibited. The extent of fusion is apparently determined by the length of the interval during which the fusion inducer is active. That fusion is actively inhibited in wild-type infections is indicated by the observation that syn mutant-infected cells fused more readily with uninfected cells than with wild type-infected cells.

  11. Fusion induced by a class II viral fusion protein, semliki forest virus E1, is dependent on the voltage of the target cell.

    PubMed

    Markosyan, Ruben M; Kielian, Margaret; Cohen, Fredric S

    2007-10-01

    Cells expressing the low pH-triggered class II viral fusion protein E1 of Semliki Forest virus (SFV) were fused to target cells. Fusion was monitored by electrical capacitance and aqueous dye measurements. Electrical voltage-clamp measurements showed that SFV E1-induced cell-cell fusion occurred quickly after acidification for a trans-negative potential across the target membrane (i.e., negative potential inside the target cell) but that a trans-positive potential eliminated all fusion. Use of an ionophore to control potentials for a large population of cells confirmed the dependence of fusion on voltage polarity. In contrast, fusion induced by the class I fusion proteins of human immunodeficiency virus, avian sarcoma leukosis virus, and influenza virus was independent of the voltage polarity across the target cell. Initial pore size and pore growth were also independent of voltage polarity for the class I proteins. An intermediate of SFV E1-induced fusion was created by transient acidification at low temperature. Membranes were hemifused at this intermediate state, and raising the temperature at neutral pH allowed full fusion to occur. Capacitance measurements showed that maintaining a trans-positive potential definitely blocked fusion at steps following the creation of the hemifusion intermediate and may have inhibited fusion at prior steps. It is proposed that the trans-negative voltage across the endosomal membrane facilitates fusion after low-pH-induced conformational changes of SFV E1 have occurred.

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

    SciTech Connect

    Nakanishi-Matsui, Mayumi 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.

  13. Proteolysis of ankyrin and of band 3 protein in chemically induced cell fusion. Ca2+ is not mandatory for fusion.

    PubMed Central

    Lang, R D; Wickenden, C; Wynne, J; Lucy, J A

    1984-01-01

    Human erythrocytes were fused by incubation with 0.5-2 mM-chlorpromazine hydrochloride at pH 6.8-7.6. Fusogenic preparations of chlorpromazine were cloudy suspensions of microdroplets, and below pH 6.8 chlorpromazine gave clear solutions that were inactive. Unlike control cells, the lateral mobility of the intramembranous particles of the PF-fracture face of chlorpromazine-treated cells was relatively unrestricted, since the particles were partly clustered at 37 degrees C and they exhibited extensive cold-induced clustering. Ca2+ stimulated fusion, but fusion was only very weakly inhibited by EGTA (10 mM) and by N-ethylmaleimide (50 mM); pretreatment of the cells with Tos-Lys-CH2Cl (7-amino-1-chloro-3-L-tosylamidoheptan-2-one) (7.5 mM) markedly inhibited fusion. Changes in the membrane proteins of erythrocytes fused by chlorpromazine, before and after treatment with chymotrypsin to remove band 3 protein, were investigated. The several observations made indicate that the Ca2+-insensitive component of fusion is associated with degradation of ankyrin (band 2.1 protein) to band 2.3-2.6 proteins and to smaller polypeptides by a serine proteinase that is inhibited by Tos-Lys-CH2Cl, and that the component of fusion inhibited by EGTA and N-ethylmaleimide is associated with degradation of band 3 protein to band 4.5 protein by a Ca2+-activated cysteine proteinase. Proteolysis of ankyrin appeared to be sufficient to permit the chlorpromazine-induced fusion of human erythrocytes, but fusion occurred more rapidly when band 3 protein was also degraded in the presence of Ca2+. Since other cells have structures comparable with the spectrin-actin skeleton of the erythrocyte membrane, the observations reported may be relevant to the initiation of naturally occurring fusion reactions in biomembranes. It is also suggested that, should polypeptides with fusogenic properties be produced from integral and skeletal membrane proteins by endogenous proteolysis, their formation would provide

  14. Antibodies to CD9, a tetraspan transmembrane protein, inhibit canine distemper virus-induced cell-cell fusion but not virus-cell fusion.

    PubMed

    Schmid, E; Zurbriggen, A; Gassen, U; Rima, B; ter Meulen, V; Schneider-Schaulies, J

    2000-08-01

    Canine distemper virus (CDV) causes a life-threatening disease in several carnivores including domestic dogs. Recently, we identified a molecule, CD9, a member of the tetraspan transmembrane protein family, which facilitates, and antibodies to which inhibit, the infection of tissue culture cells with CDV (strain Onderstepoort). Here we describe that an anti-CD9 monoclonal antibody (MAb K41) did not interfere with binding of CDV to cells and uptake of virus. In addition, in single-step growth experiments, MAb K41 did not induce differences in the levels of viral mRNA and proteins. However, the virus release of syncytium-forming strains of CDV, the virus-induced cell-cell fusion in lytically infected cultures, and the cell-cell fusion of uninfected with persistently CDV-infected HeLa cells were strongly inhibited by MAb K41. These data indicate that anti-CD9 antibodies selectively block virus-induced cell-cell fusion, whereas virus-cell fusion is not affected.

  15. Human papillomavirus 16 E5 induces bi-nucleated cell formation by cell-cell fusion

    SciTech Connect

    Hu Lulin; Plafker, Kendra; Vorozhko, Valeriya; Zuna, Rosemary E.; Hanigan, Marie H.; Gorbsky, Gary J.; Plafker, Scott M.; Angeletti, Peter C.; Ceresa, Brian P.

    2009-02-05

    Human papillomaviruses (HPV) 16 is a DNA virus encoding three oncogenes - E5, E6, and E7. The E6 and E7 proteins have well-established roles as inhibitors of tumor suppression, but the contribution of E5 to malignant transformation is controversial. Using spontaneously immortalized human keratinocytes (HaCaT cells), we demonstrate that expression of HPV16 E5 is necessary and sufficient for the formation of bi-nucleated cells, a common characteristic of precancerous cervical lesions. Expression of E5 from non-carcinogenic HPV6b does not produce bi-nucleate cells. Video microscopy and biochemical analyses reveal that bi-nucleates arise through cell-cell fusion. Although most E5-induced bi-nucleates fail to propagate, co-expression of HPV16 E6/E7 enhances the proliferation of these cells. Expression of HPV16 E6/E7 also increases bi-nucleated cell colony formation. These findings identify a new role for HPV16 E5 and support a model in which complementary roles of the HPV16 oncogenes lead to the induction of carcinogenesis.

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

    SciTech Connect

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

    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.

  17. Evidence of cell fusion in carcinogen-induced mice gastric carcinoma.

    PubMed

    Yan, Yongjia; Hsu, Yiling; He, Xianghui; Lu, Ning; Wei, Wei; Zhang, Zhixiang; Zhu, Liwei

    2015-07-01

    The role of bone marrow-derived cells in gastric cancer formation was not fully understood. In this study, bone marrow from female green fluorescent protein transgenic mice was transplanted into male wild-type mice to generate sex-mismatched chimeric mice. The chimeric mice were treated with carcinogen to induce gastric cancer. At time of sacrifice, 18.2 % (2/11) of mice showed severe dysplasia and 25 % (3/12) of mice successfully induced with cancer. Fluorescence in situ hybridization results showed that bone marrow-derived cells participated in renewal of gastric mucosa and cell fusion was observed in both precancerous lesions and adenocarcinoma, but no sign of fusion was observed in squamous cell carcinoma. Our findings suggest that bone marrow-derived cells participate in renewal of gastric mucosa during chronic damage and might have acquired the phenotype of gastric epithelial cells through cell fusion. Fusion between gastric epithelial cells and bone marrow-derived cells was involved in increased carcinogenesis.

  18. Clustering and Mobility of HIV-1 Env at Viral Assembly Sites Predict Its Propensity To Induce Cell-Cell Fusion

    PubMed Central

    Roy, Nathan H.; Chan, Jany; Lambelé, Marie

    2013-01-01

    HIV-1 Env mediates virus attachment to and fusion with target cell membranes, and yet, while Env is still situated at the plasma membrane of the producer cell and before its incorporation into newly formed particles, Env already interacts with the viral receptor CD4 on target cells, thus enabling the formation of transient cell contacts that facilitate the transmission of viral particles. During this first encounter with the receptor, Env must not induce membrane fusion, as this would prevent the producer cell and the target cell from separating upon virus transmission, but how Env's fusion activity is controlled remains unclear. To gain a better understanding of the Env regulation that precedes viral transmission, we examined the nanoscale organization of Env at the surface of producer cells. Utilizing superresolution microscopy (stochastic optical reconstruction microscopy [STORM]) and fluorescence recovery after photobleaching (FRAP), we quantitatively assessed the clustering and dynamics of Env upon its arrival at the plasma membrane. We found that Gag assembly induced the aggregation of small Env clusters into larger domains and that these domains were completely immobile. Truncation of the cytoplasmic tail (CT) of Env abrogated Gag's ability to induce Env clustering and restored Env mobility at assembly sites, both of which correlated with increased Env-induced fusion of infected and uninfected cells. Hence, while Env trapping by Gag secures Env incorporation into viral particles, Env clustering and its sequestration at assembly sites likely also leads to the repression of its fusion function, and thus, by preventing the formation of syncytia, Gag helps to secure efficient transfer of viral particles to target cells. PMID:23637402

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

    PubMed Central

    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

  20. Antigen-specific CD8{sup +} T cells induced by the ubiquitin fusion degradation pathway

    SciTech Connect

    Imai, Takashi; Duan Xuefeng; Hisaeda, Hajime; Himeno, Kunisuke

    2008-01-25

    We have developed a DNA vaccine encoding a fusion protein of ubiquitin (Ub) and target proteins at the N-terminus for effective induction of antigen-specific CD8{sup +} T cells. A series of expression plasmids encoding a model antigen, ovalbumin (OVA), fused with mutated Ub, was constructed. Western blotting analyses using COS7 cells transfected with these plasmids revealed that there were three types of amino acid causing different binding capacities between Ub and OVA. Natural Ub with a C-terminal glycine readily dissociated from OVA; on the other hand, artificially mutated Ub, the C-terminal amino acid of which had been exchanged to valine or arginine, stably united with the polypeptide, while Ub with a C-terminal alanine partially dissociated. The ability of DNA vaccination to induce OVA-specific CD8{sup +} T cells closely correlated with the stability of Ub fusion to OVA. Our strategy could be used to optimize the effect of genetic vaccines on the induction of CD8{sup +} T cells.

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

    PubMed

    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

    2015-10-08

    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.

  2. The nectin-1{alpha} transmembrane domain, but not the cytoplasmic tail, influences cell fusion induced by HSV-1 glycoproteins

    SciTech Connect

    Subramanian, Ravi P.; Dunn, Jennifer E.; Geraghty, Robert J. . E-mail: rgeragh@uky.edu

    2005-09-01

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

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

    SciTech Connect

    Yue, Xiao-shan; Fujishiro, Masako; Toyoda, Masashi; Akaike, Toshihiro; Ito, Yoshihiro

    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. Monoclonal antibody production by receptor-mediated electrically induced cell fusion

    NASA Astrophysics Data System (ADS)

    Lo, Mathew M. S.; Tsong, Tian Yow; Conrad, Mary K.; Strittmatter, Stephen M.; Hester, Lynda D.; Snyder, Solomon H.

    1984-08-01

    Fusion of myeloma cells and B lymphocytes to form hybridomas which produce monoclonal antibodies has been a major advance1-3, but the poor efficiency and randomness of viral or polyethylene glycol fusion techniques generally gives poor yields of specific, high affinity antibodies. High voltage electrical fields with dielectrophoresis to ensure cell alignment can fuse a limited number of cells under direct microscopic examination4-9, but it is not possible to identify B-cells destined to secrete relevant antibodies. However, B-cells express, on their surface, antigen receptor immunoglobulins of the same antigenic specificity as the secreted antibodies. Binding of antigen to surface immunoglobulins stimulates proliferation and differentiation of B-cells into plasma cells. Here we report the use of the selective, high affinity interaction of antigen with surface immunoglobulins on B-cells to facilitate a close adherence to myeloma cells. The antigen, covalently conjugated to avidin, binds to the surface immunoglobulins on B-cells. This B-cell-antigen-avidin complex binds to biotin covalently attached to the surface of myeloma cells. An intense electric field across a bulk cell suspension then produces selective fusion of cells in contact, that is, of myeloma cells with B-cells which make the appropriate antibody. We have used this technique with several antigens, and all resultant hybridomas secrete appropriate antibodies with very high affinity.

  5. Regulation of cell-cell fusion by nanotopography

    NASA Astrophysics Data System (ADS)

    Padmanabhan, Jagannath; Augelli, Michael J.; Cheung, Bettina; Kinser, Emily R.; Cleary, Barnett; Kumar, Priyanka; Wang, Renhao; Sawyer, Andrew J.; Li, Rui; Schwarz, Udo D.; Schroers, Jan; Kyriakides, Themis R.

    2016-09-01

    Cell-cell fusion is fundamental to a multitude of biological processes ranging from cell differentiation and embryogenesis to cancer metastasis and biomaterial-tissue interactions. Fusogenic cells are exposed to biochemical and biophysical factors, which could potentially alter cell behavior. While biochemical inducers of fusion such as cytokines and kinases have been identified, little is known about the biophysical regulation of cell-cell fusion. Here, we designed experiments to examine cell-cell fusion using bulk metallic glass (BMG) nanorod arrays with varying biophysical cues, i.e. nanotopography and stiffness. Through independent variation of stiffness and topography, we found that nanotopography constitutes the primary biophysical cue that can override biochemical signals to attenuate fusion. Specifically, nanotopography restricts cytoskeletal remodeling-associated signaling, which leads to reduced fusion. This finding expands our fundamental understanding of the nanoscale biophysical regulation of cell fusion and can be exploited in biomaterials design to induce desirable biomaterial-tissue interactions.

  6. Regulation of cell-cell fusion by nanotopography

    PubMed Central

    Padmanabhan, Jagannath; Augelli, Michael J.; Cheung, Bettina; Kinser, Emily R.; Cleary, Barnett; Kumar, Priyanka; Wang, Renhao; Sawyer, Andrew J.; Li, Rui; Schwarz, Udo D.; Schroers, Jan; Kyriakides, Themis R.

    2016-01-01

    Cell-cell fusion is fundamental to a multitude of biological processes ranging from cell differentiation and embryogenesis to cancer metastasis and biomaterial-tissue interactions. Fusogenic cells are exposed to biochemical and biophysical factors, which could potentially alter cell behavior. While biochemical inducers of fusion such as cytokines and kinases have been identified, little is known about the biophysical regulation of cell-cell fusion. Here, we designed experiments to examine cell-cell fusion using bulk metallic glass (BMG) nanorod arrays with varying biophysical cues, i.e. nanotopography and stiffness. Through independent variation of stiffness and topography, we found that nanotopography constitutes the primary biophysical cue that can override biochemical signals to attenuate fusion. Specifically, nanotopography restricts cytoskeletal remodeling-associated signaling, which leads to reduced fusion. This finding expands our fundamental understanding of the nanoscale biophysical regulation of cell fusion and can be exploited in biomaterials design to induce desirable biomaterial-tissue interactions. PMID:27615159

  7. Vaccination with TAT-antigen fusion protein induces protective, CD8(+) T cell-mediated immunity against Leishmania major.

    PubMed

    Kronenberg, Katharina; Brosch, Sven; Butsch, Florian; Tada, Yayoi; Shibagaki, Naotaka; Udey, Mark C; von Stebut, Esther

    2010-11-01

    In murine leishmaniasis, healing is mediated by IFN-γ-producing CD4(+) and CD8(+) T cells. Thus, an efficacious vaccine should induce Th1 and Tc1 cells. Dendritic cells (DCs) pulsed with exogenous proteins primarily induce strong CD4-dependent immunity; induction of CD8 responses has proven to be difficult. We evaluated the immunogenicity of fusion proteins comprising the protein transduction domain of HIV-1 TAT and the Leishmania antigen LACK (Leishmania homolog of receptors for activated C kinase), as TAT-fusion proteins facilitate major histocompatibility complex class I-dependent antigen presentation. In vitro, TAT-LACK-pulsed DCs induced stronger proliferation of Leishmania-specific CD8(+) T cells compared with DCs incubated with LACK alone. Vaccination with TAT-LACK-pulsed DCs or fusion proteins plus adjuvant in vivo significantly improved disease outcome in Leishmania major-infected mice and was superior to vaccination with DCs treated with LACK alone. Vaccination with DC+TAT-LACK resulted in stronger proliferation of CD8(+) T cells when compared with immunization with DC+LACK. Upon depletion of CD4(+) or CD8(+) T cells, TAT-LACK-mediated protection was lost. TAT-LACK-pulsed IL-12p40-deficient DCs did not promote protection in vivo. In summary, these data show that TAT-fusion proteins are superior in activating Leishmania-specific Tc1 cells when compared with antigen alone and suggest that IL-12-dependent preferential induction of antigen-specific CD8(+) cells promotes significant protection against this important human pathogen.

  8. Cell fusion and nuclear fusion in plants.

    PubMed

    Maruyama, Daisuke; Ohtsu, Mina; Higashiyama, Tetsuya

    2016-12-01

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

  9. Mechanical tension drives cell membrane fusion.

    PubMed

    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

    2015-03-09

    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 invasive fusion partner. MyoII acts as a mechanosensor, which directs its force-induced recruitment to the fusion site, and the mechanosensory response of MyoII is amplified by chemical signaling initiated by cell adhesion molecules. The accumulated MyoII, in turn, increases cortical tension and promotes fusion pore formation. We propose that the protrusive and resisting forces from fusion partners put the fusogenic synapse under high mechanical tension, which helps to overcome energy barriers for membrane apposition and drives cell membrane fusion.

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

    PubMed

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

    2012-03-01

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

  11. A tuberculosis vaccine based on phosphoantigens and fusion proteins induces distinct gammadelta and alphabeta T cell responses in primates.

    PubMed

    Cendron, Delphine; Ingoure, Sophie; Martino, Angelo; Casetti, Rita; Horand, Françoise; Romagné, François; Sicard, Hélène; Fournié, Jean-Jacques; Poccia, Fabrizio

    2007-02-01

    Phosphoantigens are mycobacterial non-peptide antigens that might enhance the immunogenicity of current subunit candidate vaccines for tuberculosis. However, their testing requires monkeys, the only animal models suitable for gammadelta T cell responses to mycobacteria. Thus here, the immunogenicity of 6-kDa early secretory antigenic target-mycolyl transferase complex antigen 85B (ESAT-6-Ag85B) (H-1 hybrid) fusion protein associated or not to a synthetic phosphoantigen was compared by a prime-boost regimen of two groups of eight cynomolgus. Although phosphoantigen activated immediately a strong release of systemic Th1 cytokines (IL-2, IL-6, IFN-gamma, TNF-alpha), it further anergized blood gammadelta T lymphocytes selectively. By contrast, the hybrid H-1 induced only memory alphabeta T cell responses, regardless of phosphoantigen. These latter essentially comprised cytotoxic T lymphocytes specific for Ag85B (on average + 430 cells/million PBMC) and few IFN-gamma-secreting cells (+ 40 cells/million PBMC, equally specific for ESAT-6 and for Ag85B). Hence, in macaques, a prime-boost with the H-1/phosphoantigen subunit combination induces two waves of immune responses, successively by gammadelta T and alphabeta T lymphocytes.

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

    SciTech Connect

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

    2008-01-25

    The ubiquitin-proteasome system (UPS) plays an indispensable role in inducing MHC class I-restricted CD8{sup +} T cells. In this study, we exploited UPS to induce CD8{sup +} 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-{gamma}-producing CD8{sup +} T cells compared with those from the latter group of mice.

  13. Blockade of CD26-mediated T cell costimulation with soluble caveolin-1-Ig fusion protein induces anergy in CD4{sup +}T cells

    SciTech Connect

    Ohnuma, Kei; Uchiyama, Masahiko; Hatano, Ryo; Takasawa, Wataru; Endo, Yuko; Dang, Nam H.; Morimoto, Chikao

    2009-08-21

    CD26 binds to caveolin-1 in antigen-presenting cells (APC), and that ligation of CD26 by caveolin-1 induces T cell proliferation in a TCR/CD3-dependent manner. We report herein the effects of CD26-caveolin-1 costimulatory blockade by fusion protein caveolin-1-Ig (Cav-Ig). Soluble Cav-Ig inhibits T cell proliferation and cytokine production in response to recall antigen, or allogeneic APC. Our data hence suggest that blocking of CD26-associated signaling by soluble Cav-Ig may be an effective approach as immunosuppressive therapy.

  14. Adenoviral Vectors Armed with Cell Fusion-Inducing Proteins as Anti-Cancer Agents

    PubMed Central

    Del Papa, Joshua; Parks, Robin J.

    2017-01-01

    Cancer is a devastating disease that affects millions of patients every year, and causes an enormous economic burden on the health care system and emotional burden on affected families. The first line of defense against solid tumors is usually extraction of the tumor, when possible, by surgical methods. In cases where solid tumors can not be safely removed, chemotherapy is often the first line of treatment. As metastatic cancers often become vigorously resistant to treatments, the development of novel, more potent and selective anti-cancer strategies is of great importance. Adenovirus (Ad) is the most commonly used virus in cancer clinical trials, however, regardless of the nature of the Ad-based therapeutic, complete responses to treatment remain rare. A number of pre-clinical studies have shown that, for all vector systems, viral spread throughout the tumor mass can be a major limiting factor for complete tumor elimination. By expressing exogenous cell-fusion proteins, many groups have shown improved spread of Ad-based vectors. This review summarizes the research done to examine the potency of Ad vectors expressing fusogenic proteins as anti-cancer therapeutics. PMID:28106842

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

    SciTech Connect

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

    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.

  16. Virus-like particle-induced fusion from without in tissue culture cells: role of outer-layer proteins VP4 and VP7.

    PubMed Central

    Gilbert, J M; Greenberg, H B

    1997-01-01

    We recently described an assay that measures fusion from without induced in tissue culture cells by rotavirus, a nonenveloped, triple-protein-layered member of the Reoviridae family (M. M. Falconer, J. M. Gilbert, A. M. Roper, H. B. Greenberg, and J. S. Gavora, J. Virol. 69:5582-5591, 1995). The conditions required for syncytium formation are similar to those for viral penetration of the plasma membrane during the course of viral infection of host cells, as the presence of the outer-layer proteins VP4 and VP7 and the cleavage of VP4 are required. Here we present evidence that virus-like particles (VLPs) produced in Spodoptera frugiperda Sf-9 cells from recombinant baculoviruses expressing the four structural proteins of rotavirus can induce cell-cell fusion to the same extent as native rotavirus. This VLP-mediated fusion activity was dependent on trypsinization of VP4, and the strain-specific phenotype of individual VP4 molecules was retained in the syncytium assay similar to what has been seen with reassortant rotaviruses. We show that intact rotavirus and VLPs induce syncytia with cells that are permissive to rotavirus infection whereas nonpermissive cells are refractory to syncytium formation. This finding further supports our hypothesis that the syncytium assay accurately reflects very early events involved in viral infection and specifically the events related to viral entry into the cell. Our results also demonstrate that neither viral replication nor rotavirus proteins other than VP2, VP6, VP4, and VP7 are required for fusion and that both VP4 and VP7 are essential. The combination of a cell-cell fusion assay and the availability of recombinant VLPs will permit us to dissect the mechanisms of rotavirus penetration into host cells. PMID:9151849

  17. Reprogramming of Somatic Cells Towards Pluripotency by Cell Fusion.

    PubMed

    Malinowski, Andrzej R; Fisher, Amanda G

    2016-01-01

    Pluripotent reprogramming can be dominantly induced in a somatic nucleus upon fusion with a pluripotent cell such as embryonic stem (ES) cell. Cell fusion between ES cells and somatic cells results in the formation of heterokaryons, in which the somatic nuclei begin to acquire features of the pluripotent partner. The generation of interspecies heterokaryons between mouse ES- and human somatic cells allows an experimenter to distinguish the nuclear events occurring specifically within the reprogrammed nucleus. Therefore, cell fusion provides a simple and rapid approach to look at the early nuclear events underlying pluripotent reprogramming. Here, we describe a polyethylene glycol (PEG)-mediated cell fusion protocol to generate interspecies heterokaryons and intraspecies hybrids between ES cells and B lymphocytes or fibroblasts.

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

    SciTech Connect

    Schnabl, Bernd Brandl, Katharina; Fink, Marina; Gross, Philipp; Taura, Kojiro; Gaebele, Erwin; Hellerbrand, Claus; Falk, Werner

    2008-10-17

    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 NF{kappa}B 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. Insights into the mechanism of cell death induced by saporin delivered into cancer cells by an antibody fusion protein targeting the transferrin receptor 1

    PubMed Central

    Daniels-Wells, Tracy R.; Helguera, Gustavo; Rodríguez, José A.; Leoh, Lai Sum; Erb, Michael A.; Diamante, Graciel; Casero, David; Pellegrini, Matteo; Martínez-Maza, Otoniel; Penichet, Manuel L.

    2012-01-01

    We previously developed an antibody-avidin fusion protein (ch128.1Av) that targets the human transferrin receptor 1 (TfR1) and exhibits direct cytotoxicity against malignant B cells in an iron-dependent manner. ch128.1Av is also a delivery system and its conjugation with biotinylated saporin (b-SO6), a plant ribosome-inactivating toxin, results in a dramatic iron-independent cytotoxicity, both in malignant cells that are sensitive or resistant to ch128.1Av alone, in which the toxin effectively inhibits protein synthesis and triggers caspase activation. We have now found that the ch128.1Av/b-SO6 complex induces a transcriptional response consistent with oxidative stress and DNA damage, a response that is not observed with ch128.1Av alone. Furthermore, we show that the antioxidant N-acetylcysteine partially blocks saporin-induced apoptosis suggesting that oxidative stress contributes to DNA damage and ultimately saporin-induced cell death. Interestingly, the toxin was detected in nuclear extracts by immunoblotting, suggesting the possibility that saporin might induce direct DNA damage. However, confocal microscopy did not show a clear and consistent pattern of intranuclear localization. Finally, using the long-term culture-initiating cell assay we found that ch128.1Av/b-SO6 is not toxic to normal human hematopoietic stem cells suggesting that this critical cell population would be preserved in therapeutic interventions using this immunotoxin. PMID:23085102

  20. Insights into the mechanism of cell death induced by saporin delivered into cancer cells by an antibody fusion protein targeting the transferrin receptor 1.

    PubMed

    Daniels-Wells, Tracy R; Helguera, Gustavo; Rodríguez, José A; Leoh, Lai Sum; Erb, Michael A; Diamante, Graciel; Casero, David; Pellegrini, Matteo; Martínez-Maza, Otoniel; Penichet, Manuel L

    2013-02-01

    We previously developed an antibody-avidin fusion protein (ch128.1Av) that targets the human transferrin receptor 1 (TfR1) and exhibits direct cytotoxicity against malignant B cells in an iron-dependent manner. ch128.1Av is also a delivery system and its conjugation with biotinylated saporin (b-SO6), a plant ribosome-inactivating toxin, results in a dramatic iron-independent cytotoxicity, both in malignant cells that are sensitive or resistant to ch128.1Av alone, in which the toxin effectively inhibits protein synthesis and triggers caspase activation. We have now found that the ch128.1Av/b-SO6 complex induces a transcriptional response consistent with oxidative stress and DNA damage, a response that is not observed with ch128.1Av alone. Furthermore, we show that the antioxidant N-acetylcysteine partially blocks saporin-induced apoptosis suggesting that oxidative stress contributes to DNA damage and ultimately saporin-induced cell death. Interestingly, the toxin was detected in nuclear extracts by immunoblotting, suggesting the possibility that saporin might induce direct DNA damage. However, confocal microscopy did not show a clear and consistent pattern of intranuclear localization. Finally, using the long-term culture-initiating cell assay we found that ch128.1Av/b-SO6 is not toxic to normal human hematopoietic stem cells suggesting that this critical cell population would be preserved in therapeutic interventions using this immunotoxin.

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

    PubMed

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

    2012-03-01

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

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

    PubMed Central

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

    2000-01-01

    We have reported that fusions of murine dendritic cells (DCs) and murine carcinoma cells reverse unresponsiveness to tumor-associated antigens and induce the rejection of established metastases. In the present study, fusions were generated with primary human breast carcinoma cells and autologous DCs. Fusion cells coexpressed tumor-associated antigens and DC-derived costimulatory molecules. The fusion cells also retained the functional potency of DCs and stimulated autologous T cell proliferation. Significantly, the results show that autologous T cells are primed by the fusion cells to induce MHC class I-dependent lysis of autologous breast tumor cells. These findings demonstrate that fusions of human breast cancer cells and DCs activate T cell responses against autologous tumors. PMID:10688917

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

    PubMed

    Paquette, Stéphane G; Banner, David; Chi, Le Thi Bao; Leόn, Alberto J; Xu, Luoling; Ran, Longsi; Huang, Stephen S H; Farooqui, Amber; Kelvin, David J; Kelvin, Alyson A

    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.

  4. EP300-ZNF384 fusion gene product up-regulates GATA3 gene expression and induces hematopoietic stem cell gene expression signature in B-cell precursor acute lymphoblastic leukemia cells.

    PubMed

    Yaguchi, Akinori; Ishibashi, Takeshi; Terada, Kazuki; Ueno-Yokohata, Hitomi; Saito, Yuya; Fujimura, Junya; Shimizu, Toshiaki; Ohki, Kentaro; Manabe, Atsushi; Kiyokawa, Nobutaka

    2017-04-04

    ZNF384-related fusion genes are associated with a distinct subgroup of B-cell precursor acute lymphoblastic leukemias in childhood, with a frequency of approximately 3-4%. We previously identified a novel EP300-ZNF384 fusion gene. Patients with the ZNF384-related fusion gene exhibit a hematopoietic stem cell (HSC) gene expression signature and characteristic immunophenotype with negative or low expression of CD10 and aberrant expression of myeloid antigens, such as CD33 and CD13. However, the molecular basis of this pathogenesis remains completely unknown. In the present study, we examined the biological effects of EP300-ZNF384 expression induced by retrovirus-mediated gene transduction in an REH B-cell precursor acute lymphoblastic leukemia cell line, and observed the acquisition of the HSC gene expression signature and an up-regulation of GATA3 gene expression, as assessed by microarray analysis. In contrast, the gene expression profile induced by wild-type ZNF384 in REH cells was significantly different from that by EP300-ZNF384 expression. Together with the results of reporter assays, which revealed the enhancement of GATA3-promoter activity by EP300-ZNF384 expression, these findings suggest that EP300-ZNF384 mediates GATA3 gene expression and may be involved in the acquisition of the HSC gene expression signature and characteristic immunophenotype in B-cell precursor acute lymphoblastic leukemia cells.

  5. The Dark Side of Cell Fusion

    PubMed Central

    Bastida-Ruiz, Daniel; Van Hoesen, Kylie; Cohen, Marie

    2016-01-01

    Cell fusion is a physiological cellular process essential for fertilization, viral entry, muscle differentiation and placental development, among others. In this review, we will highlight the different cancer cell-cell fusions and the advantages obtained by these fusions. We will specially focus on the acquisition of metastatic features by cancer cells after fusion with bone marrow-derived cells. The mechanism by which cancer cells fuse with other cells has been poorly studied thus far, but the presence in several cancer cells of syncytin, a trophoblastic fusogen, leads us to a cancer cell fusion mechanism similar to the one used by the trophoblasts. The mechanism by which cancer cells perform the cell fusion could be an interesting target for cancer therapy. PMID:27136533

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

    SciTech Connect

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

  7. Measurement of membrane fusion activity from viral membrane fusion proteins based on a fusion-dependent promoter induction system in insect cells

    PubMed Central

    Slack, J. M.; Blissard, G. W.

    2013-01-01

    Summary A number of viral membrane fusion proteins can be expressed alone on the surface of host cells, then triggered to induce cell-to-cell fusion or syncytium formation. Although rapid and easily observed, syncytium formation is not easily quantified and differences in fusion activity are not easily distinguished or measured. To address this problem, we developed a rapid and quantitative cell-to-cell fusion system that is useful for comparative analysis and may be suitable for high throughput screening. In this system, expression of a reporter protein, the enhanced green fluorescent protein (EGFP), is dependent on cell-to-cell fusion. Spodoptera frugiperda (Sf9) insect cells expressing a chimeric Lac Repressor-IE1 protein were fused to Sf9 cells containing an EGFP reporter construct under the control of a responsive lac operator containing promoter. Membrane fusion efficiency was measured from the resulting EGFP fluorescence activity. Sf9 cells expressing the Orgyia pseudotsugata Multicapsid Nucleopolyhedrovirus (OpMNPV) GP64 envelope fusion protein were used as a model to test this fusion assay. Subtle changes in fusion activities of GP64 proteins containing single amino acid substitutions in a putative membrane fusion domain were distinguished, and decreases in EGFP fluorescence corresponded to decreases in the hydrophobicity in the small putative membrane fusion domain. PMID:11562545

  8. Cytoplasmic calcium increase via fusion with inactivated Sendai virus induces apoptosis in human multiple myeloma cells by downregulation of c-Myc oncogene

    PubMed Central

    Jiang, Yingzhe; Saga, Kotaro; Miyamoto, Yasuhide; Kaneda, Yasufumi

    2016-01-01

    Because the emergence of drug resistance is a major limitation of current treatments for multiple myeloma (MM), it is necessary to continuously develop novel anticancer strategies. Here, using an inactivated Sendai virus (Hemagglutinating Virus of Japan; HVJ) envelope (HVJ-E), we discovered that increase of cytoplasmic Ca2+ by virus-cell fusion significantly induced apoptosis against human MM cells but not peripheral blood mononuclear cells from healthy donors. Interaction of F protein of HVJ-E with MM cells increased intracellular Ca2+ level of MMs by the induction of Ca2+ efflux from endoplasmic reticulum but not influx from extracellular region. The elevation of the Ca2+ cytoplasmic level induced SMAD1/5/8 phosphorylation and translocation into the nucleus, and SMAD1/5/8 and SMAD4 complex suppressed c-Myc transcription. Meanwhile, HVJ-E decreases S62 phosphorylation of c-Myc and promotes c-Myc protein degradation. Thus, HVJ-E-induced cell death of MM resulted from suppression of c-Myc by both destabilization of c-Myc protein and downregulation of c-Myc transcription. This study indicates that HVJ-E will be a promising tool for MM therapy. PMID:27145280

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

    PubMed Central

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

    2015-01-01

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

  10. 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

    SciTech Connect

    Ishikawa, Mayuko; Ogihara, Yusuke; Miura, Masahiko

    2009-11-20

    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.

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

    PubMed

    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.

  12. A Molluscum contagiosum fusion protein inhibits CCL1-induced chemotaxis of cells expressing CCR8 and penetrates human neonatal foreskins: clinical applications proposed.

    PubMed

    Paslin, David A; Reykjalin, Erik; Tsadik, Elias; Schour, Lionel; Lucas, Alexander

    2015-04-01

    Inflammation in atopic dermatitis is mediated in part by the chemokine CCL1 and its receptor, CCR8. Recombinant Molluscum contagiosum viral protein (rMC148p), a cc-chemokine homolog, inhibits CCL1-induced chemotaxis of cells expressing CCR8. rMC148p was prepared using the baculovirus/Sf9 insect cell expression system. The recombinant MC148 fusion protein (rMC148fp), rMC148-TAT-6xHis, was similarly prepared by adding base sequences onto the PCR primers to fuse TAT and 6xHis to rMC148p at the carboxyl terminus. rMC148fp retains the capacity of rMC148p to inhibit CCL1-induced chemotaxis. Furthermore, unlike rMC148p, topically applied rMC148fp penetrates stratum corneum of human neonatal foreskins and concentrates along the basal and lower spinous cell layers of the epidermis. rMC148fp may be a safe and effective agent in the treatment of atopic dermatitis and other CCR8-mediated disorders.

  13. Ca-dependent Nonsecretory Vesicle Fusion in a Secretory Cell

    PubMed Central

    Wang, Tzu-Ming; Hilgemann, Donald W.

    2008-01-01

    We have compared Ca-dependent exocytosis in excised giant membrane patches and in whole-cell patch clamp with emphasis on the rat secretory cell line, RBL. Stable patches of 2–4 pF are easily excised from RBL cells after partially disrupting actin cytoskeleton with latrunculin A. Membrane fusion is triggered by switching the patch to a cytoplasmic solution containing 100–200 μM free Ca. Capacitance and amperometric recording show that large secretory granules (SGs) containing serotonin are mostly lost from patches. Small vesicles that are retained (non-SGs) do not release serotonin or other substances detected by amperometry, although their fusion is reduced by tetanus toxin light chain. Non-SG fusion is unaffected by N-ethylmaleimide, phosphatidylinositol-4,5-bis-phosphate (PI(4,5)P2) ligands, such as neomycin, a PI-transfer protein that can remove PI from membranes, the PI(3)-kinase inhibitor LY294002 and PI(4,5)P2, PI(3)P, and PI(4)P antibodies. In patch recordings, but not whole-cell recordings, fusion can be strongly reduced by ATP removal and by the nonspecific PI-kinase inhibitors wortmannin and adenosine. In whole-cell recording, non-SG fusion is strongly reduced by osmotically induced cell swelling, and subsequent recovery after shrinkage is then inhibited by wortmannin. Thus, membrane stretch that occurs during patch formation may be a major cause of differences between excised patch and whole-cell fusion responses. Regarding Ca sensors for non-SG fusion, fusion remains robust in synaptotagmin (Syt) VII−/− mouse embryonic fibroblasts (MEFs), as well as in PLCδ1, PLC δ1/δ4, and PLCγ1−/− MEFs. Thus, Syt VII and several PLCs are not required. Furthermore, the Ca dependence of non-SG fusion reflects a lower Ca affinity (KD ∼71 μM) than expected for these C2 domain–containing proteins. In summary, we find that non-SG membrane fusion behaves and is regulated substantially differently from SG fusion, and we have identified an ATP

  14. Optical imaging of cell fusion and fusion proteins in Caenorhabditis elegans.

    PubMed

    Ems, Star; Mohler, William A

    2008-01-01

    Cell fusion is a very dynamic process in which the entire membrane and cellular contents of two or more cells merge into one. Strategies developed to understand the component processes that make up a full fusion event require imaging to be performed over a range of space and time scales. These strategies must cover detection of nanometer-sized pores, monitoring cytoplasmic diffusion and the dynamic localization of proteins that induce fusion competence, and three-dimensional reconstruction of multinucleated cells. Caenorhabditis elegans' small size, predictable development, and transparent body make this organism optimal for microscopic investigations. In this chapter, focus is placed on light microscopy techniques that have been used thus far to study developmental fusion events in C. elegans and the insights that have been gained from them. There is also a general overview of the developmental timing of the cell fusion events. Additionally, several protocols are described for preparing both fixed and live specimens at various developmental stages of C. elegans for examination via optical microscopy.

  15. The conserved Cockayne syndrome B-piggyBac fusion protein (CSB-PGBD3) affects DNA repair and induces both interferon-like and innate antiviral responses in CSB-null cells

    PubMed Central

    Bailey, Arnold D.; Gray, Lucas T.; Pavelitz, Thomas; Newman, John C.; Horibata, Katsuyoshi; Tanaka, Kiyoji; Weiner, Alan M.

    2012-01-01

    Cockayne syndrome is a segmental progeria most often caused by mutations in the CSB gene encoding a SWI/SNF-like ATPase required for transcription-coupled DNA repair (TCR). Over 43 Mya before marmosets diverged from humans, a piggyBac3 (PGBD3) transposable element integrated into intron 5 of the CSB gene. As a result, primate CSB genes now generate both CSB protein and a conserved CSB-PGBD3 fusion protein in which the first 5 exons of CSB are alternatively spliced to the PGBD3 transposase. Using a host cell reactivation assay, we show that the fusion protein inhibits TCR of oxidative damage but facilitates TCR of UV damage. We also show by microarray analysis that expression of the fusion protein alone in CSB-null UV-sensitive syndrome (UVSS) cells induces an interferon-like response that resembles both the innate antiviral response and the prolonged interferon response normally maintained by unphosphorylated STAT1 (U-STAT1); moreover, as might be expected based on conservation of the fusion protein, this potentially cytotoxic interferon-like response is largely reversed by coexpression of functional CSB protein. Interestingly, expression of CSB and the CSB-PGBD3 fusion protein together, but neither alone, upregulates the insulin growth factor binding protein IGFBP5 and downregulates IGFBP7, suggesting that the fusion protein may also confer a metabolic advantage, perhaps in the presence of DNA damage. Finally, we show that the fusion protein binds in vitro to members of a dispersed family of 900 internally deleted piggyBac elements known as MER85s, providing a potential mechanism by which the fusion protein could exert widespread effects on gene expression. Our data suggest that the CSB-PGBD3 fusion protein is important in both health and disease, and could play a role in Cockayne syndrome. PMID:22483866

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

    PubMed Central

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

    2016-01-01

    Magnetoreception in animals illustrates the interaction of biological systems with the geomagnetic field (geoMF). However, there are few studies that identified the impact of high magnetic field (MF) exposure from Magnetic Resonance Imaging (MRI) scanners (>100,000 times of geoMF) on specific biological targets. Here, we investigated the effects of a 14 Tesla MRI scanner on zebrafish larvae. All zebrafish larvae aligned parallel to the B0 field, i.e. the static MF, in the MRI scanner. The two otoliths (ear stones) in the otic vesicles of zebrafish larvae older than 24 hours post fertilization (hpf) fused together after the high MF exposure as short as 2 hours, yielding a single-otolith phenotype with aberrant swimming behavior. The otolith fusion was blocked in zebrafish larvae under anesthesia or embedded in agarose. Hair cells may play an important role on the MF-induced otolith fusion. This work provided direct evidence to show that high MF interacts with the otic vesicle of zebrafish larvae and causes otolith fusion in an “all-or-none” manner. The MF-induced otolith fusion may facilitate the searching for MF sensors using genetically amenable vertebrate animal models, such as zebrafish. PMID:27063288

  17. Amiodarone-induced T-U fusion.

    PubMed

    Omar, Hesham R

    2012-11-01

    Amiodarone is a widely used antiarrythmic drug for various atrial and ventricular arrhythmias. It has the potential to cause prolongation of the QT interval, which, in turn, can increases the incidence of torsade de pointes. Amiodarone is also one of the causes of prominent U waves. The presented case exemplifies the phenomenon of amiodarone-induced T-U fusion and QT prolongation. Other causes of QT prolongation as electrolyte abnormalities or administration of other drugs that prolong the QT interval were excluded. Awareness of this phenomenon and method of calculation of QT interval in this scenario is of utmost importance.

  18. DNA fusion-gene vaccination in patients with prostate cancer induces high-frequency CD8(+) T-cell responses and increases PSA doubling time.

    PubMed

    Chudley, Lindsey; McCann, Katy; Mander, Ann; Tjelle, Torunn; Campos-Perez, Juan; Godeseth, Rosemary; Creak, Antonia; Dobbyn, James; Johnson, Bernadette; Bass, Paul; Heath, Catherine; Kerr, Paul; Mathiesen, Iacob; Dearnaley, David; Stevenson, Freda; Ottensmeier, Christian

    2012-11-01

    We report on the immunogenicity and clinical effects in a phase I/II dose escalation trial of a DNA fusion vaccine in patients with prostate cancer. The vaccine encodes a domain (DOM) from fragment C of tetanus toxin linked to an HLA-A2-binding epitope from prostate-specific membrane antigen (PSMA), PSMA(27-35). We evaluated the effect of intramuscular vaccination without or with electroporation (EP) on vaccine potency. Thirty-two HLA-A2(+) patients were vaccinated and monitored for immune and clinical responses for a follow-up period of 72 weeks. At week 24, cross-over to the immunologically more effective delivery modality was permitted; this was shown to be with EP based on early antibody data, and subsequently, 13/15 patients crossed to the +EP arm. Thirty-two HLA-A2(-) control patients were assessed for time to next treatment and overall survival. Vaccination was safe and well tolerated. The vaccine induced DOM-specific CD4(+) and PSMA(27)-specific CD8(+) T cells, which were detectable at significant levels above baseline at the end of the study (p = 0.0223 and p = 0.00248, respectively). Of 30 patients, 29 had a measurable CD4(+) T-cell response and PSMA(27)-specific CD8(+) T cells were detected in 16/30 patients, with or without EP. At week 24, before cross-over, both delivery methods led to increased CD4(+) and CD8(+) vaccine-specific T cells with a trend to a greater effect with EP. PSA doubling time increased significantly from 11.97 months pre-treatment to 16.82 months over the 72-week follow-up (p = 0.0417), with no clear differential effect of EP. The high frequency of immunological responses to DOM-PSMA(27) vaccination and the clinical effects are sufficiently promising to warrant further, randomized testing.

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

    PubMed

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

    2014-07-01

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

  20. Cell Fusion as a Cause of Prostate Cancer Metastases

    DTIC Science & Technology

    2011-04-01

    Fusion as a Cause of Prostate Cancer Metastases 2 14. ABSTRACT The main goal of the study funded by this grant was to test a hypothesis that cell...24 4 INTRODUCTION The main goal of this study was to test a...PC3 cultured in normal medium were used as a control (B). Some of the cells in which expression of EGFP was induced are indicated by arrows

  1. Prolonged interval between fusion and activation impairs embryonic development by inducing chromosome scattering and nuclear aneuploidy in pig somatic cell nuclear transfer.

    PubMed

    You, Jinyoung; Song, Kilyoung; Lee, Eunsong

    2010-01-01

    The aim of the present study was to examine the effect of various intervals between electrofusion and activation (FA interval) on the nuclear remodelling and development of somatic cell nuclear transfer (SCNT) embryos in pigs. Reconstructed oocytes were activated at 0 (simultaneous fusion and activation; SFA), 1, 2 and 3 h (delayed activation) after electrofusion; these groups were designated as DA1, DA2 and DA3, respectively. When oocyte nuclear status was examined at 0.5, 1, 2 and 3 h after electrofusion, the incidence of chromosome scattering was increased (P < 0.01) as the FA interval was extended (0.0%, 12.0%, 77.3% and 78.0%, respectively). Extending the FA interval led to an increase (P < 0.01) in the percentage of oocytes containing multiple (>or=3) pseudopronuclei (PPN) (0.0% of SFA; 5.3% of DA1; 21.7% of DA2; and 33.5% of DA3). The development of SCNT embryos to the blastocyst stage was decreased (P < 0.05) in DA2 (5.7%) and DA3 (5.0%) compared with SFA (18.1%) and DA1 (19.5%). Our results demonstrate that extending the FA interval impairs the development of SCNT pig embryos by inducing chromosome scattering and the formation of multiple PPN, which may result in increased nuclear aneuploidy.

  2. Embryonic stem cell-somatic cell fusion and postfusion enucleation.

    PubMed

    Sumer, Huseyin; Verma, Paul J

    2015-01-01

    Embryonic stem (ES) cells are able to reprogram somatic cells following cell fusion. The resulting cell hybrids have been shown to have similar properties to pluripotent cells. It has also been shown that transcriptional changes can occur in a heterokaryon, without nuclear hybridization. However it is unclear whether these changes can be sustained following removal of the dominant ES nucleus. In this chapter, methods are described for the cell fusion of mouse tetraploid ES cells with somatic cells and enrichment of the resulting heterokaryons. We next describe the conditions for the differential removal of the ES cell nucleus, allowing for the recovery of somatic cells.

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

    PubMed

    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.

  4. Khz (fusion product of Ganoderma lucidum and Polyporus umbellatus mycelia) induces apoptosis in human colon carcinoma HCT116 cells, accompanied by an increase in reactive oxygen species, activation of caspase 3, and increased intracellular Ca²⁺.

    PubMed

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

    2015-03-01

    Khz (a fusion mycelium of Ganoderma lucidum and Polyporus umbellatus mycelia) is isolated from ganoderic acid and P. umbellatus and it exerts antiproliferative effects against malignant cells. However, no previous study has reported the inhibitory effects of Khz on the growth of human colon cancer cells. In the present study, we found that Khz suppressed cell division and induced apoptosis in HCT116 cells. Khz cytotoxicity was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Khz reduced cell viability and mitochondrial membrane potential levels and it also induced disruption of the mitochondrial membrane potential and increased calcium concentration and reactive oxygen species generation. Khz increased caspase 3, PARP, caspase 7, and caspase 9 levels, but reduced Bcl-2 protein levels. Flow cytometry showed that the percentage of HCT116 cells in the sub-G1 phase of the cell cycle increased in response to Khz treatment.

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

    NASA Astrophysics Data System (ADS)

    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.

  6. Studies on the fusion peptide of a paramyxovirus fusion glycoprotein: roles of conserved residues in cell fusion.

    PubMed Central

    Horvath, C M; Lamb, R A

    1992-01-01

    The role of residues in the conserved hydrophobic N-terminal fusion peptide of the paramyxovirus fusion (F) protein in causing cell-cell fusion was examined. Mutations were introduced into the cDNA encoding the simian virus 5 (SV5) F protein, the altered F proteins were expressed by using an eukaryotic vector, and their ability to mediate syncytium formation was determined. The mutant F proteins contained both single- and multiple-amino-acid substitutions, and they exhibited a variety of intracellular transport properties and fusion phenotypes. The data indicate that many substitutions in the conserved amino acids of the simian virus 5 F fusion peptide can be tolerated without loss of biological activity. Mutant F proteins which were not transported to the cell surface did not cause cell-cell fusion, but all of the mutants which were transported to the cell surface were fusion competent, exhibiting fusion properties similar to or better than those of the wild-type F protein. Mutant F proteins containing glycine-to-alanine substitutions had altered intracellular transport characteristics, yet they exhibited a great increase in fusion activity. The potential structural implications of this substitution and the possible importance of these glycine residues in maintaining appropriate levels of fusion activity are discussed. Images PMID:1548771

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

    PubMed

    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.

  8. Mechanism of membrane fusion induced by vesicular stomatitis virus G protein.

    PubMed

    Kim, Irene S; Jenni, Simon; Stanifer, Megan L; Roth, Eatai; Whelan, Sean P J; van Oijen, Antoine M; Harrison, Stephen C

    2017-01-03

    The glycoproteins (G proteins) of vesicular stomatitis virus (VSV) and related rhabdoviruses (e.g., rabies virus) mediate both cell attachment and membrane fusion. The reversibility of their fusogenic conformational transitions differentiates them from many other low-pH-induced viral fusion proteins. We report single-virion fusion experiments, using methods developed in previous publications to probe fusion of influenza and West Nile viruses. We show that a three-stage model fits VSV single-particle fusion kinetics: (i) reversible, pH-dependent, G-protein conformational change from the known prefusion conformation to an extended, monomeric intermediate; (ii) reversible trimerization and clustering of the G-protein fusion loops, leading to an extended intermediate that inserts the fusion loops into the target-cell membrane; and (iii) folding back of a cluster of extended trimers into their postfusion conformations, bringing together the viral and cellular membranes. From simulations of the kinetic data, we conclude that the critical number of G-protein trimers required to overcome membrane resistance is 3 to 5, within a contact zone between the virus and the target membrane of 30 to 50 trimers. This sequence of conformational events is similar to those shown to describe fusion by influenza virus hemagglutinin (a "class I" fusogen) and West Nile virus envelope protein ("class II"). Our study of VSV now extends this description to "class III" viral fusion proteins, showing that reversibility of the low-pH-induced transition and architectural differences in the fusion proteins themselves do not change the basic mechanism by which they catalyze membrane fusion.

  9. Mechanism of membrane fusion induced by vesicular stomatitis virus G protein

    PubMed Central

    Kim, Irene S.; Jenni, Simon; Stanifer, Megan L.; Roth, Eatai; Whelan, Sean P. J.; van Oijen, Antoine M.; Harrison, Stephen C.

    2017-01-01

    The glycoproteins (G proteins) of vesicular stomatitis virus (VSV) and related rhabdoviruses (e.g., rabies virus) mediate both cell attachment and membrane fusion. The reversibility of their fusogenic conformational transitions differentiates them from many other low-pH-induced viral fusion proteins. We report single-virion fusion experiments, using methods developed in previous publications to probe fusion of influenza and West Nile viruses. We show that a three-stage model fits VSV single-particle fusion kinetics: (i) reversible, pH-dependent, G-protein conformational change from the known prefusion conformation to an extended, monomeric intermediate; (ii) reversible trimerization and clustering of the G-protein fusion loops, leading to an extended intermediate that inserts the fusion loops into the target-cell membrane; and (iii) folding back of a cluster of extended trimers into their postfusion conformations, bringing together the viral and cellular membranes. From simulations of the kinetic data, we conclude that the critical number of G-protein trimers required to overcome membrane resistance is 3 to 5, within a contact zone between the virus and the target membrane of 30 to 50 trimers. This sequence of conformational events is similar to those shown to describe fusion by influenza virus hemagglutinin (a “class I” fusogen) and West Nile virus envelope protein (“class II”). Our study of VSV now extends this description to “class III” viral fusion proteins, showing that reversibility of the low-pH-induced transition and architectural differences in the fusion proteins themselves do not change the basic mechanism by which they catalyze membrane fusion. PMID:27974607

  10. Rapamycin induces the fusion-type independent downregulation of the EWS/FLI-1 proteins and inhibits Ewing's sarcoma cell proliferation.

    PubMed

    Mateo-Lozano, Silvia; Tirado, Oscar M; Notario, Vicente

    2003-12-18

    Ewing's sarcoma (ES) is the prototype of a family of tumors (ESFT) of neuroectodermal origin formed by small, round cells with limited neural differentiation, which arise most frequently within bones in children or adolescents. The proliferation of ESFT cells is highly dependent on the establishment of, and signaling through several growth factor-mediated autocrine loops. The mammalian target of rapamycin (mTOR) is a central regulator of translation and cell proliferation, involved in the cellular response to various nutritional, stress and mitogenic effectors. As mTOR has recently been associated with certain human cancers, we investigated the possibility that mTOR played a role in the regulation of ES cell proliferation. Results showed that ES cell lines carrying EWS/FLI-1 alleles of different types expressed different levels of total and phosphorylated mTOR protein. We demonstrate that rapamycin, an mTOR inhibitor, efficiently blocked the proliferation of all cell lines by promoting cell cycle arrest at the G1 phase. This was paralleled by the downregulation of the levels of the EWS/FLI-1 proteins, regardless of their fusion type, and the concomitant restoration of the expression of the TGF-beta type 2 receptor (TGFbeta RII), which is known to be repressed by several EWS-ETS fusion proteins. The expression of a rapamycin-resistant mTOR construct prevented both the proliferation blockade and the EWS/FLI-1 downregulation. These data demonstrate that mTOR signaling plays a central role in ES cell pathobiology and strongly suggest that the use of rapamycin as a cytostatic agent may be an efficient tool for the treatment of ES patients.

  11. Incomplete cytokinesis and re-fusion of small mononucleated Hodgkin cells lead to giant multinucleated Reed–Sternberg cells

    PubMed Central

    Rengstl, Benjamin; Newrzela, Sebastian; Heinrich, Tim; Weiser, Christian; Thalheimer, Frederic B.; Schmid, Frederike; Warner, Kathrin; Hartmann, Sylvia; Schroeder, Timm; Küppers, Ralf; Rieger, Michael A.; Hansmann, Martin-Leo

    2013-01-01

    Multinucleated Reed–Sternberg (RS) cells are pathognomonic for classical Hodgkin lymphoma (HL), and their presence is essential for diagnosis. How these giant tumor cells develop is controversial, however. It has been postulated that RS cells arise from mononucleated Hodgkin cells via endomitosis. Conversely, continuous single-cell tracking of HL cell lines by long-term time-lapse microscopy has identified cell fusion as the main route of RS cell formation. In contrast to growth-induced formation of giant Hodgkin cells, fusion of small mononuclear cells followed by a size increase gives rise to giant RS cells. Of note, fusion of cells originating from the same ancestor, termed re-fusion, is seen nearly exclusively. In the majority of cases, re-fusion of daughter cells is preceded by incomplete cytokinesis, as demonstrated by microtubule bonds among the cells. We confirm at the level of individual tracked cells that giant Hodgkin and RS cells have little proliferative capacity, further supporting small mononuclear Hodgkin cells as the proliferative compartment of the HL tumor clone. In addition, sister cells show a shared propensity for re-fusion, providing evidence of early RS cell fate commitment. Thus, RS cell generation is related neither to cell fusion of unrelated Hodgkin cells nor to endomitosis, but rather is mediated by re-fusion of daughter cells that underwent mitosis. This surprising finding supports the existence of a unique mechanism for the generation of multinuclear RS cells that may have implications beyond HL, given that RS-like cells are frequently observed in several other lymphoproliferative diseases as well. PMID:24302766

  12. Inflammation and Proliferation Act Together to Mediate Intestinal Cell Fusion

    PubMed Central

    Swain, John R.; Wong, Melissa H.

    2009-01-01

    Cell fusion between circulating bone marrow-derived cells (BMDCs) and non-hematopoietic cells is well documented in various tissues and has recently been suggested to occur in response to injury. Here we illustrate that inflammation within the intestine enhanced the level of BMDC fusion with intestinal progenitors. To identify important microenvironmental factors mediating intestinal epithelial cell fusion, we performed bone marrow transplantation into mouse models of inflammation and stimulated epithelial proliferation. Interestingly, in a non-injury model or in instances where inflammation was suppressed, an appreciable baseline level of fusion persisted. This suggests that additional mediators of cell fusion exist. A rigorous temporal analysis of early post-transplantation cellular dynamics revealed that GFP-expressing donor cells first trafficked to the intestine coincident with a striking increase in epithelial proliferation, advocating for a required fusogenic state of the host partner. Directly supporting this hypothesis, induction of augmented epithelial proliferation resulted in a significant increase in intestinal cell fusion. Here we report that intestinal inflammation and epithelial proliferation act together to promote cell fusion. While the physiologic impact of cell fusion is not yet known, the increased incidence in an inflammatory and proliferative microenvironment suggests a potential role for cell fusion in mediating the progression of intestinal inflammatory diseases and cancer. PMID:19657387

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

    PubMed Central

    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

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

    PubMed

    Maruyama, Daisuke; Völz, Ronny; Takeuchi, Hidenori; Mori, Toshiyuki; Igawa, Tomoko; Kurihara, Daisuke; Kawashima, Tomokazu; Ueda, Minako; Ito, Masaki; Umeda, Masaaki; Nishikawa, Shuh-Ichi; Groß-Hardt, Rita; Higashiyama, Tetsuya

    2015-05-07

    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.

  15. Phenylarsine oxide-induced increase in alveolar macrophage surface receptors: evidence for fusion of internal receptor pools with the cell surface

    PubMed Central

    1985-01-01

    Rabbit alveolar macrophages which were treated at 0 degrees C with phenylarsine oxide and then incubated at 37 degrees C for 10 min exhibited a two- to threefold increase in surface receptor activity for macroglobulin.protease complexes, diferric transferrin, and mannose- terminal glycoproteins. Analysis of the concentration-dependence of ligand binding indicated that changes in ligand-binding activity were due to changes in receptor number rather than alterations in ligand- receptor affinity. Surface receptor number could also be increased by treatment of cells with three other sulfhydryl reagents, N- ethylmaleimide, p-chloromercurobenzoate, and iodoacetic acid. The increase in receptor activity was maximal after 10 min and decreased over the next hour. This decrease in cell-associated receptor activity was due to the release of large membrane vesicles which demonstrated a uniform buoyant density by isopycnic sucrose gradient centrifugation. Treatment of cells with phenylarsine oxide did not decrease the cellular content of lactate dehydrogenase or beta-galactosidase, indicating that cell integrity was maintained and lysosomal enzyme release did not occur. Our studies indicate that phenylarsine oxide treatment in the presence of extracellular Ca2+ results in the fusion of receptor-containing vesicles with the cell surface. PMID:2409094

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

    SciTech Connect

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

    2014-10-15

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

  17. Recombinant Bivalent Fusion Protein rVE Induces CD4+ and CD8+ T-Cell Mediated Memory Immune Response for Protection Against Yersinia enterocolitica Infection

    PubMed Central

    Singh, Amit K.; Kingston, Joseph J.; Gupta, Shishir K.; Batra, Harsh V.

    2015-01-01

    Studies investigating the correlates of immune protection against Yersinia infection have established that both humoral and cell mediated immune responses are required for the comprehensive protection. In our previous study, we established that the bivalent fusion protein (rVE) comprising immunologically active regions of Y. pestis LcrV (100–270 aa) and YopE (50–213 aa) proteins conferred complete passive and active protection against lethal Y. enterocolitica 8081 challenge. In the present study, cohort of BALB/c mice immunized with rVE or its component proteins rV, rE were assessed for cell mediated immune responses and memory immune protection against Y. enterocolitica 8081. rVE immunization resulted in extensive proliferation of both CD4 and CD8 T cell subsets; significantly high antibody titer with balanced IgG1: IgG2a/IgG2b isotypes (1:1 ratio) and up-regulation of both Th1 (TNF-α, IFN-γ, IL-2, and IL-12) and Th2 (IL-4) cytokines. On the other hand, rV immunization resulted in Th2 biased IgG response (11:1 ratio) and proliferation of CD4+ T-cell; rE group of mice exhibited considerably lower serum antibody titer with predominant Th1 response (1:3 ratio) and CD8+ T-cell proliferation. Comprehensive protection with superior survival (100%) was observed among rVE immunized mice when compared to the significantly lower survival rates among rE (37.5%) and rV (25%) groups when IP challenged with Y. enterocolitica 8081 after 120 days of immunization. Findings in this and our earlier studies define the bivalent fusion protein rVE as a potent candidate vaccine molecule with the capability to concurrently stimulate humoral and cell mediated immune responses and a proof of concept for developing efficient subunit vaccines against Gram negative facultative intracellular bacterial pathogens. PMID:26733956

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

    SciTech Connect

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

    2010-08-15

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

  19. Synergistic inhibition in cell-cell fusion mediated by the matrix and nucleocapsid protein of canine distemper virus.

    PubMed

    Wiener, Dominique; Plattet, Philippe; Cherpillod, Pascal; Zipperle, Ljerka; Doherr, Marcus G; Vandevelde, Marc; Zurbriggen, Andreas

    2007-11-01

    Canine distemper virus (CDV) causes a chronic, demyelinating, progressive or relapsing neurological disease in dogs, because CDV persists in the CNS. Persistence of virulent CDV, such as the A75/17 strain has been reproduced in cell cultures where it is associated with a non-cytolytic infection with very limited cell-cell fusion. This is in sharp contrast to attenuated CDV infection in cell cultures, such as the Onderstepoort (OP) CDV strain, which produces extensive fusion activity and cytolysis. Fusion efficiency may be determined by the structure of the viral fusion protein per se but also by its interaction with other structural proteins of CDV. This was studied by combining genes derived from persistent and non-persistent CDV strains in transient transfection experiments. It was found that fusion efficiency was markedly attenuated by the structure of the fusion protein of the neurovirulent A75/17-CDV. Moreover, we showed that the interaction of the surface glycoproteins with the M protein of the persistent strain greatly influenced fusion activity. Site directed mutagenesis showed that the c-terminus of the M protein is of particular importance in this respect. Interestingly, although the nucleocapsid protein alone did not affect F/H-induced cell-cell fusion, maximal inhibition occurred when the latter was added to combined glycoproteins with matrix protein. Thus, the present study suggests that very limited fusogenicity in virulent CDV infection, which favours persistence by limiting cell destruction involves complex interactions between all viral structural proteins.

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

    PubMed

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

    2014-10-15

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

  1. Kinetic Modeling of Laser-Induced Fusion

    DTIC Science & Technology

    2007-09-01

    Thermal neutrons are of considerable interest to the Department of Defense and for commercial applications. Unlike high- energy photons, neutrons easily...develop a compact generator for thermal neutrons with large enough flux. The limited availability of radio-isotopes, combined with the relatively...Deuterium-Tritium (D-T) fusion, which generates Alpha particles and fast neutrons . In these sources, Deuterium ions are accelerated to about 130 keV and hit

  2. High-Frequency Gravitational Wave Induced Nuclear Fusion

    SciTech Connect

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

    2007-01-30

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

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

    SciTech Connect

    Mukai, Atsushi; Hashimoto, Naohiro

    2008-01-15

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

  4. Different activities of the reovirus FAST proteins and influenza hemagglutinin in cell-cell fusion assays and in response to membrane curvature agents

    SciTech Connect

    Clancy, Eileen K.; Barry, Chris; Ciechonska, Marta; Duncan, Roy

    2010-02-05

    The reovirus fusion-associated small transmembrane (FAST) proteins evolved to induce cell-cell, rather than virus-cell, membrane fusion. It is unclear whether the FAST protein fusion reaction proceeds in the same manner as the enveloped virus fusion proteins. We now show that fluorescence-based cell-cell and cell-RBC hemifusion assays are unsuited for detecting lipid mixing in the absence of content mixing during FAST protein-mediated membrane fusion. Furthermore, membrane curvature agents that inhibit hemifusion or promote pore formation mediated by influenza hemagglutinin had no effect on p14-induced cell-cell fusion, even under conditions of limiting p14 concentrations. Standard assays used to detect fusion intermediates induced by enveloped virus fusion proteins are therefore not applicable to the FAST proteins. These results suggest the possibility that the nature of the fusion intermediates or the mechanisms used to transit through the various stages of the fusion reaction may differ between these distinct classes of viral fusogens.

  5. Fusion of Ubiquitin to HIV Gag Impairs Human Monocyte-Derived Dendritic Cell Maturation and Reduces Ability to Induce Gag T Cell Responses

    PubMed Central

    Herath, Shanthi; Benlahrech, Adel; Papagatsias, Timos; Athanasopoulos, Takis; Bouzeboudjen, Zineb; Hervouet, Catherine; Klavinskis, Linda; Meiser, Andrea; Kelleher, Peter; Dickson, George; Patterson, Steven

    2014-01-01

    The efficient induction of CD8 T cell immunity is dependent on the processing and presentation of antigen on MHC class I molecules by professional antigen presenting cells (APC). To develop an improved T cell vaccine for HIV we investigated whether fusing the ubiquitin gene to the N terminus of the HIV gag gene enhanced targeting to the proteasome resulting in better CD8 T cell responses. Human monocyte derived dendritic cells (moDC), transduced with adenovirus vectors carrying either ubiquitinated or non-ubiquitinated gag transgene constructs, were co-cultured with autologous naïve T cells and T cell responses were measured after several weekly cycles of stimulation. Despite targeting of the ubiquitin gag transgene protein to the proteasome, ubiquitination did not increase CD8 T cell immune responses and in some cases diminished responses to gag peptides. There were no marked differences in cytokines produced from ubiquitinated and non-ubiquitinated gag stimulated cultures or in the expression of inhibitory molecules on expanded T cells. However, the ability of moDC transduced with ubiquitinated gag gene to upregulate co-stimulatory molecules was reduced, whilst no difference in moDC maturation was observed with a control ubiquitinated and non-ubiquitinated MART gene. Furthermore moDC transduced with ubiquitinated gag produced more IL-10 than transduction with unmodified gag. Thus failure of gag ubiquitination to enhance CD8 responses may be caused by suppression of moDC maturation. These results indicate that when designing a successful vaccine strategy to target a particular cell population, attention must also be given to the effect of the vaccine on APCs. PMID:24505475

  6. Acute myeloid leukemia fusion proteins deregulate genes involved in stem cell maintenance and DNA repair

    PubMed Central

    Alcalay, Myriam; Meani, Natalia; Gelmetti, Vania; Fantozzi, Anna; Fagioli, Marta; Orleth, Annette; Riganelli, Daniela; Sebastiani, Carla; Cappelli, Enrico; Casciari, Cristina; Sciurpi, Maria Teresa; Mariano, Angela Rosa; Minardi, Simone Paolo; Luzi, Lucilla; Muller, Heiko; Di Fiore, Pier Paolo; Frosina, Guido; Pelicci, Pier Giuseppe

    2003-01-01

    Acute myelogenous leukemias (AMLs) are genetically heterogeneous and characterized by chromosomal rearrangements that produce fusion proteins with aberrant transcriptional regulatory activities. Expression of AML fusion proteins in transgenic mice increases the risk of myeloid leukemias, suggesting that they induce a preleukemic state. The underlying molecular and biological mechanisms are, however, unknown. To address this issue, we performed a systematic analysis of fusion protein transcriptional targets. We expressed AML1/ETO, PML/RAR, and PLZF/RAR in U937 hemopoietic precursor cells and measured global gene expression using oligonucleotide chips. We identified 1,555 genes regulated concordantly by at least two fusion proteins that were further validated in patient samples and finally classified according to available functional information. Strikingly, we found that AML fusion proteins induce genes involved in the maintenance of the stem cell phenotype and repress DNA repair genes, mainly of the base excision repair pathway. Functional studies confirmed that ectopic expression of fusion proteins constitutively activates pathways leading to increased stem cell renewal (e.g., the Jagged1/Notch pathway) and provokes accumulation of DNA damage. We propose that expansion of the stem cell compartment and induction of a mutator phenotype are relevant features underlying the leukemic potential of AML-associated fusion proteins. PMID:14660751

  7. Oral administration of a non-absorbable plant cell-expressed recombinant anti-TNF fusion protein induces immunomodulatory effects and alleviates nonalcoholic steatohepatitis

    PubMed Central

    Ilan, Yaron; Ben Ya'acov, Ami; Shabbat, Yehudit; Gingis-Velitski, Svetlana; Almon, Einat; Shaaltiel, Yoseph

    2016-01-01

    AIM To evaluate the immunomodulatory effect of oral administration of PRX-106 in the high-fat diet model. METHODS For 22 wk, C57BL/6 HFD-fed mice received daily oral treatments with BY-2 cells expressing recombinant anti-tumor necrosis factor alpha fusion protein (PRX-106). Mice were followed for serum liver enzyme and triglyceride levels, liver histology and intrahepatic and systemic FACS. RESULTS The orally administered non-absorbable PRX-106 was biologically active. Altered distribution of CD4+CD25+FoxP3+ between the liver and spleen and an increase in the intrasplenic-to-intrahepatic CD4+CD25+FoxP3+ ratio and a decrease in the intrasplenic-to-intrahepatic CD8+CD25+FoxP3+ ratio were observed. An increase in intrahepatic NKT cells and a decrease in the intrasplenic-to-intrahepatic NKT ratio were noted. Assessment of the CD4-to-CD8 ratios showed sequestration of CD8+ lymphocytes in the liver. These effects were associated with a decrease in serum triglyceride levels, decrease in the aspartate aminotransferase levels, serum glucose levels, and HOMA-IR score. A decrease in hepatic triglycerides content was observed in the high dose-treated mice. CONCLUSION Orally administered PRX-106 shows biological activity and exerts an immunomodulatory effect, alleviating liver damage. The data suggest that PRX-106 may provide an oral immunotherapy for nonalcoholic steatohepatitis. PMID:27818591

  8. Cell fusion: biological perspectives and potential for regenerative medicine.

    PubMed

    Alvarez-Dolado, Manuel

    2007-01-01

    Cell fusion has emerged as a powerful subject of debate in the last few years. Adult stem cell plasticity and the search for mechanisms to explain this process have led to the "rediscovery" of cell fusion. In nature, cell fusion is a normal process involved in sexual reproduction, tissue formation, and immune response. The recent observation that bone marrow derived cells fuse with several cell types introduces new and provocative questions. In this review, I shall recapitulate what is known about cell fusion and discuss its more controversial aspects. I shall highlight the most exciting open questions; its biological potential; pros and cons; and their implications on stem cell plasticity, regenerative medicine, and development.

  9. A formin-nucleated actin aster concentrates cell wall hydrolases for cell fusion in fission yeast

    PubMed Central

    Dudin, Omaya; Bendezú, Felipe O.; Groux, Raphael; Laroche, Thierry; Seitz, Arne

    2015-01-01

    Cell–cell fusion is essential for fertilization. For fusion of walled cells, the cell wall must be degraded at a precise location but maintained in surrounding regions to protect against lysis. In fission yeast cells, the formin Fus1, which nucleates linear actin filaments, is essential for this process. In this paper, we show that this formin organizes a specific actin structure—the actin fusion focus. Structured illumination microscopy and live-cell imaging of Fus1, actin, and type V myosins revealed an aster of actin filaments whose barbed ends are focalized near the plasma membrane. Focalization requires Fus1 and type V myosins and happens asynchronously always in the M cell first. Type V myosins are essential for fusion and concentrate cell wall hydrolases, but not cell wall synthases, at the fusion focus. Thus, the fusion focus focalizes cell wall dissolution within a broader cell wall synthesis zone to shift from cell growth to cell fusion. PMID:25825517

  10. Virus-cell fusion as a trigger of innate immunity dependent on the adaptor STING

    PubMed Central

    Holm, Christian K; Jensen, Søren B; Jakobsen, Martin R; Cheshenko, Natalia; Horan, Kristy A; Moeller, Hanne B; Gonzalez-Dosal, Regina; Rasmussen, Simon B; Christensen, Maria H.; Yarovinsky, Timur O; Rixon, Frazer J; Herold, Betsy C; Fitzgerald, Katherine A; Paludan, Søren R

    2012-01-01

    The innate immune system senses infection by detecting evolutionarily conserved molecules essential for microbial survival or abnormal location of molecules. Here we demonstrate the existence of a novel innate detection mechanism, which is induced by fusion between viral envelopes and target cells. Virus-cell fusion specifically stimulated a type I interferon (IFN) response with expression of IFN-stimulated genes (ISGs), in vivo recruitment of leukocytes, and potentiation of Toll-like receptor 7 and 9 signaling. The fusion dependent response was dependent on stimulator of interferon genes (STING) but independent of DNA, RNA and viral capsid. We suggest that membrane fusion is sensed as a danger signal with potential implications for defense against enveloped viruses and various conditions of giant cell formation. PMID:22706339

  11. Dendritic-tumor fusion cells in cancer immunotherapy.

    PubMed

    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.

  12. In AtT20 and HeLa cells brefeldin A induces the fusion of tubular endosomes and changes their distribution and some of their endocytic properties

    PubMed Central

    1992-01-01

    We have studied the effects of brefeldin A (BFA) on the tubular endosomes in AtT20 and HeLa cells (Tooze, J., and M. Hollinshead. 1991. J. Cell Biol. 115:635-653) by electron microscopy of cells labeled with three endocytic tracers, HRP, BSA-gold, and transferrin conjugated to HRP, and by immunofluorescence microscopy. For the latter we used antibodies specific for transferrin receptor, and, in the case of AtT20 cells, also antibodies specific for synaptophysin. In HeLa cells BFA at concentrations ranging from 1 micrograms to 10 micrograms/ml causes the dispersed patches of network of preexisting tubular early endosomes to be incorporated within 5 min into tubules approximately 50 nm in diameter but up to 40-50 microns long. These long, straight tubular endosomes are aligned along microtubules; they branch relatively infrequently to form an open network or reticulum extending from the cell periphery to the microtubule organizing center (MTOC). As the incubation with BFA is prolonged beyond 5 min, a steady state is reached in which many tubules are located in a dense network enclosing the centrioles, with branches extending in a more open network to the periphery. This effect of BFA, which is fully reversed within 15-30 min of washing out, is inhibited by pre-incubating the cells with sodium azide and 2-deoxy-D-glucose. In AtT20 cells BFA at 5 micrograms/ml or above causes the same sorts of changes, preexisting tubular endosomes are recruited into a more continuous endosomal network, and there is a massive accumulation of this network around the MTOC. Maintenance of the BFA-induced endosomal reticulum in both cell types is dependent upon the integrity of microtubules. In AtT20 cells BFA at 1 microgram/ml has no detectable effect on the early endosomal system but the Golgi stacks are converted to clusters of tubules and vesicles that remain in the region of the MTOC during prolonged incubations. Therefore, the Golgi apparatus in these cells is more sensitive to BFA

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

    EPA Science Inventory

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

  14. Inhibition of HIV-1 Env-Mediated Cell-Cell Fusion by Lectins, Peptide T-20, and Neutralizing Antibodies

    PubMed Central

    Yee, Michael; Konopka, Krystyna; Balzarini, Jan; Düzgüneş, Nejat

    2011-01-01

    Background: Broadly cross-reactive, neutralizing human monoclonal antibodies, including 2F5, 2G12, 4E10 and IgG1 b12, can inhibit HIV-1 infection in vitro at very low concentrations. We examined the ability of these antibodies to inhibit cell-cell fusion between Clone69TRevEnv cells induced to express the viral envelope proteins, gp120/gp41 (Env), and highly CD4-positive SupT1 cells. The cells were loaded with green and red-orange cytoplasmic fluorophores, and fusion was monitored by fluorescence microscopy. Results: Cell-cell fusion was inhibited completely by the carbohydrate binding proteins (CBPs), Hippeastrum hybrid (Amaryllis) agglutinin (HHA), and Galanthus nivalis (Snowdrop) agglutinin (GNA), and by the peptide, T-20, at relatively low concentrations. Anti-gp120 and anti-gp41 antibodies, at concentrations much higher than those required for neutralization, were not particularly effective in inhibiting fusion. Monoclonal antibodies b12, m14 IgG and 2G12 had moderate inhibitory activity; the IC50 of 2G12 was about 80 µg/ml. Antibodies 4E10 and 2F5 had no inhibitory activity at the concentrations tested. Conclusions: These observations raise concerns about the ability of neutralizing antibodies to inhibit the spread of viral genetic material from infected cells to uninfected cells via cell-cell fusion. The interaction of gp120/gp41 with cell membrane CD4 may be different in cell-cell and virus-cell membrane fusion reactions, and may explain the differential effects of antibodies in these two systems. The fluorescence assay described here may be useful in high throughput screening of potential HIV fusion inhibitors. PMID:21660189

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

    PubMed Central

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

    2016-01-01

    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. PMID:27420051

  16. Proteolytic Cleavage of the Fusion Protein but Not Membrane Fusion Is Required for Measles Virus-Induced Immunosuppression In Vitro

    PubMed Central

    Weidmann, Armin; Maisner, Andrea; Garten, Wolfgang; Seufert, Marion; ter Meulen, Volker; Schneider-Schaulies, Sibylle

    2000-01-01

    Immunosuppression induced by measles virus (MV) is associated with unresponsiveness of peripheral blood lymphocytes (PBL) to mitogenic stimulation ex vivo and in vitro. In mixed lymphocyte cultures and in an experimental animal model, the expression of the MV glycoproteins on the surface of UV-inactivated MV particles, MV-infected cells, or cells transfected to coexpress the MV fusion (F) and the hemagglutinin (H) proteins was found to be necessary and sufficient for this phenomenon. We now show that MV fusion-inhibitory peptides do not interfere with the induction of immunosuppression in vitro, indicating that MV F-H-mediated fusion is essentially not involved in this process. Proteolytic cleavage of MV F0 protein by cellular proteases, such as furin, into the F1-F2 subunits is, however, an absolute requirement, since (i) the inhibitory activity of MV-infected BJAB cells was significantly impaired in the presence of a furin-inhibitory peptide and (ii) cells expressing or viruses containing uncleaved F0 proteins revealed a strongly reduced inhibitory activity which was improved following trypsin treatment. The low inhibitory activity of effector structures containing mainly F0 proteins was not due to an impaired F0-H interaction, since both surface expression and cocapping efficiencies were similar to those found with the authentic MV F and H proteins. These results indicate that the fusogenic activity of the MV F-H complexes can be uncoupled from their immunosuppressive activity and that the immunosuppressive domains of these proteins are exposed only after proteolytic activation of the MV F0 protein. PMID:10644371

  17. β-MSCs: successful fusion of MSCs with β-cells results in a β-cell like phenotype

    PubMed Central

    Azizi, Zahra; Lange, Claudia; Paroni, Federico; Ardestani, Amin; Meyer, Anke; Wu, Yonghua; Zander, Axel R.

    2016-01-01

    Bone marrow mesenchymal stromal cells (MSC) have anti-inflammatory, anti-apoptotic and immunosuppressive properties and are a potent source for cell therapy. Cell fusion has been proposed for rapid generation of functional new reprogrammed cells. In this study, we aimed to establish a fusion protocol of bone marrow−derived human MSCs with the rat beta-cell line (INS-1E) as well as human isolated pancreatic islets in order to generate insulin producing beta-MSCs as a cell-based treatment for diabetes. Human eGFP+ puromycin+ MSCs were co-cultured with either stably mCherry-expressing rat INS-1E cells or human dispersed islet cells and treated with phytohemagglutinin (PHA-P) and polyethylene glycol (PEG) to induce fusion. MSCs and fused cells were selected by puromycin treatment. With an improved fusion protocol, 29.8 ± 2.9% of all MSCs were β-MSC heterokaryons based on double positivity for mCherry and eGFP. After fusion and puromycin selection, human NKX6.1 and insulin as well as rat Neurod1, Nkx2.2, MafA, Pdx1 and Ins1 mRNA were highly elevated in fused human MSC/INS-1E cells, compared to the mixed control population. Such induction of beta-cell markers was confirmed in fused human MSC/human dispersed islet cells, which showed elevated NEUROD1, NKX2.2, MAFA, PDX1 and insulin mRNA compared to the mixed control. Fused cells had higher insulin content and improved insulin secretion compared to the mixed control and insulin positive beta-MSCs also expressed nuclear PDX1. We established a protocol for fusion of human MSCs and beta cells, which resulted in a beta cell like phenotype. This could be a novel tool for cell-based therapies of diabetes. PMID:27374092

  18. Vaccination with Dendritic Cell Myeloma Fusions in Conjuction with Stem Cell Transplantation and PD-1 Blockade

    DTIC Science & Technology

    2015-07-01

    Award Number: W81XWH-09-1-0296 TITLE: Vaccination with Dendritic Cell Myeloma Fusions in Conjuction with Stem Cell Transplantation and PD-1...Addendum 3. DATES COVERED (From - To) 1May2014 - 30Apr2015 4. TITLE AND SUBTITLE Vaccination with Dendritic Cell Myeloma Fusions in Conjuction with Stem...anti-PD1 antibody (CT-011) alone (Cohort 1) and in conjunction with a dendritic cell/myeloma fusion cell vaccine (Cohort 2) following autologous

  19. Reptilian reovirus utilizes a small type III protein with an external myristylated amino terminus to mediate cell-cell fusion.

    PubMed

    Corcoran, Jennifer A; Duncan, Roy

    2004-04-01

    Reptilian reovirus is one of a limited number of nonenveloped viruses that are capable of inducing cell-cell fusion. A small, hydrophobic, basic, 125-amino-acid fusion protein encoded by the first open reading frame of a bicistronic viral mRNA is responsible for this fusion activity. Sequence comparisons to previously characterized reovirus fusion proteins indicated that p14 represents a new member of the fusion-associated small transmembrane (FAST) protein family. Topological analysis revealed that p14 is a representative of a minor subset of integral membrane proteins, the type III proteins N(exoplasmic)/C(cytoplasmic) (N(exo)/C(cyt)), that lack a cleavable signal sequence and use an internal reverse signal-anchor sequence to direct membrane insertion and protein topology. This topology results in the unexpected, cotranslational translocation of the essential myristylated N-terminal domain of p14 across the cell membrane. The topology and structural motifs present in this novel reovirus membrane fusion protein further accentuate the diversity and unusual properties of the FAST protein family and clearly indicate that the FAST proteins represent a third distinct class of viral membrane fusion proteins.

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

    PubMed

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

    2017-01-01

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

  1. Recombinant fusion protein of cholera toxin B subunit with YVAD secreted by Lactobacillus casei inhibits lipopolysaccharide-induced caspase-1 activation and subsequent IL-1 beta secretion in Caco-2 cells

    PubMed Central

    2014-01-01

    Background Lactobacillus species are used as bacterial vectors to deliver functional peptides to the intestine because they are delivered live to the intestine, colonize the mucosal surface, and continue to produce the desired protein. Previously, we generated a recombinant Lactobacillus casei secreting the cholera toxin B subunit (CTB), which can translocate into intestinal epithelial cells (IECs) through GM1 ganglioside. Recombinant fusion proteins of CTB with functional peptides have been used as carriers for the delivery of these peptides to IECs because of the high cell permeation capacity of recombinant CTB (rCTB). However, there have been no reports of rCTB fused with peptides expressed or secreted by Lactobacillus species. In this study, we constructed L. casei secreting a recombinant fusion protein of CTB with YVAD (rCTB–YVAD). YVAD is a tetrapeptide (tyrosine–valine–alanine–aspartic acid) that specifically inhibits caspase-1, which catalyzes the production of interleukin (IL)-1β, an inflammatory cytokine, from its inactive precursor. Here, we examined whether rCTB–YVAD secreted by L. casei binds to GM1 ganglioside and inhibits caspase-1 activation in Caco-2 cells used as a model of IECs. Results We constructed the rCTB–YVAD secretion vector pSCTB–YVAD by modifying the rCTB secretion vector pSCTB. L. casei secreting rCTB–YVAD was generated by transformation with pSCTB–YVAD. Both the culture supernatant of pSCTB–YVAD-transformed L. casei and purified rCTB–YVAD bound to GM1 ganglioside, as did the culture supernatant of pSCTB-transformed L. casei and purified rCTB. Interestingly, although both purified rCTB–YVAD and rCTB translocated into Caco-2 cells, regardless of lipopolysaccharide (LPS), only purified rCTB–YVAD but not rCTB inhibited LPS-induced caspase-1 activation and subsequent IL-1β secretion in Caco-2 cells, without affecting cell viability. Conclusions The rCTB protein fused to a functional peptide secreted by L. casei

  2. Effect of lateral mobility of fluorescent probes in lipid mixing assays of cell fusion.

    PubMed Central

    Huang, S K; Cheng, M; Hui, S W

    1990-01-01

    Monolayers of human erythrocytes, immobilized on a cover slip, were induced to fuse by polyethylene glycol (mol wt 8,000). The mobility of fluorescent probes, 1-oleoyl-2-[12-[(7-nitro-2,1,3-benzoxadizol-4-yl)amino]dodecanoyl] phosphatidyl-choline (C12-NBD-PC), from labeled cells to unlabeled cells was monitored by video-enhanced fluorescence microscopy. A dequenching curve was obtained from the measurement of fluorescence intensities of pairs of fused cells over time. The dequenching curve and the curve obtained from macroscopic measurements of a cell monolayer (described in the preceding article) were compared and discussed. The slow probe transfer rate between a pair of fused cells was explained by a diffusion model based on membrane area conservation and the geometry of the fusion lumen. An equivalent lumen between two fused cells, thought to be the main rate limitation of probe mobility after fusion, was calculated to be approximately 130 nm in diameter. Lumens of 75 nm in diameter were observed by electron microscopy. Thus, the rate of macroscopic fluorescence dequenching depends not only upon the fusion efficiency, but also upon the number of simultaneous fusion partners, the geometry of their contact points, and the lateral mobility of the fluorescent probes through these points. The relative fusion efficiency can be derived only from the saturation dequenching values. Images FIGURE 3 FIGURE 5 FIGURE 7 PMID:2291938

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

    EPA Science Inventory

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

  4. Spatial focalization of pheromone/MAPK signaling triggers commitment to cell-cell fusion.

    PubMed

    Dudin, Omaya; Merlini, Laura; Martin, Sophie G

    2016-10-01

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

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

    SciTech Connect

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

    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.

  6. Isolation of gene fusions (soi::lacZ) inducible by oxidative stress in Escherichia coli.

    PubMed Central

    Kogoma, T; Farr, S B; Joyce, K M; Natvig, D O

    1988-01-01

    Mu dX phage was used to isolate three gene fusions to the lacZ gene (soi::lacZ; soi for superoxide radical inducible) that were induced by treatment with superoxide radical anion generators such as paraquat and plumbagin. The induction of beta-galactosidase in these fusion strains with the superoxide radical generating agents required aerobic metabolism. Hyperoxygenation (i.e., bubbling of cultures with oxygen gas) also induced the fusions. On the other hand, hydrogen peroxide did not induce the fusions at concentrations that are known to invoke an adaptive response. Introduction of oxyR, htpR, or recA mutations did not affect the induction. Two of the fusion strains exhibited increased sensitivity to paraquat but not to hydrogen peroxide. The third fusion strain showed no increased sensitivity to either agent. All three fusions were located in the 45- to 61-min region of the Escherichia coli chromosome. PMID:2838846

  7. Cell Therapy to Obtain Spinal Fusion

    DTIC Science & Technology

    2009-07-01

    creates controlled flexion and extension in a mouse spine, so that spine fusions could be assessed using the same computer-assisted methods that are...reducing motion within the spine, we set up some experiments to look at flexion /tension under bending at specific angles. Briefly, three groups of...significant drop in the disc angle and in turn flexion of the spine. The reduction in flexion is consistent with what has been demonstrated in human

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

    PubMed

    Goh, Qingnian; Millay, Douglas P

    2017-02-10

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

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

    PubMed Central

    Goh, Qingnian; Millay, Douglas P

    2017-01-01

    Fusion of skeletal muscle stem/progenitor cells is required for proper development and regeneration, however the significance of this process during adult muscle hypertrophy has not been explored. In response to muscle overload after synergist ablation in mice, we show that myomaker, a muscle specific membrane protein essential for myoblast fusion, is activated mainly in muscle progenitors and not myofibers. We rendered muscle progenitors fusion-incompetent through genetic deletion of myomaker in muscle stem cells and observed a complete reduction of overload-induced hypertrophy. This blunted hypertrophic response was associated with a reduction in Akt and p70s6k signaling and protein synthesis, suggesting a link between myonuclear accretion and activation of pro-hypertrophic pathways. Furthermore, fusion-incompetent muscle exhibited increased fibrosis after muscle overload, indicating a protective role for normal stem cell activity in reducing myofiber strain associated with hypertrophy. These findings reveal an essential contribution of myomaker-mediated stem cell fusion during physiological adult muscle hypertrophy. DOI: http://dx.doi.org/10.7554/eLife.20007.001 PMID:28186492

  10. Wortmannin-induced vacuole fusion enhances amyloplast dynamics in Arabidopsis zigzag1 hypocotyls

    PubMed Central

    Alvarez, Ashley Ann; Han, Sang Won; Toyota, Masatsugu; Brillada, Carla; Zheng, Jiameng; Gilroy, Simon

    2016-01-01

    Gravitropism in Arabidopsis shoots depends on the sedimentation of amyloplasts in the endodermis, and a complex interplay between the vacuole and F-actin. Gravity response is inhibited in zigzag-1 (zig-1), a mutant allele of VTI11, which encodes a SNARE protein involved in vacuole fusion. zig-1 seedlings have fragmented vacuoles that fuse after treatment with wortmannin, an inhibitor of phosphatidylinositol 3-kinase, and underscore a role of phosphoinositides in vacuole fusion. Using live-cell imaging with a vertical stage microscope, we determined that young endodermal cells below the apical hook that are smaller than 70 μm in length are the graviperceptive cells in dark-grown hypocotyls. This result was confirmed by local wortmannin application to the top of zig-1 hypocotyls, which enhanced shoot gravitropism in zig-1 mutants. Live-cell imaging of zig-1 hypocotyl endodermal cells indicated that amyloplasts are trapped between juxtaposed vacuoles and their movement is severely restricted. Wortmannin-induced fusion of vacuoles in zig-1 seedlings increased the formation of transvacuolar strands, enhanced amyloplast sedimentation and partially suppressed the agravitropic phenotype of zig-1 seedlings. Hypergravity conditions at 10 g were not sufficient to displace amyloplasts in zig-1, suggesting the existence of a physical tether between the vacuole and amyloplasts. Our results overall suggest that vacuole membrane remodeling may be involved in regulating the association of vacuoles and amyloplasts during graviperception. PMID:27816929

  11. Cell fusion mediates dramatic alterations in the actin cytoskeleton, focal adhesions, and E-cadherin in trophoblastic cells.

    PubMed

    Ishikawa, Atsuko; Omata, Waka; Ackerman, William E; Takeshita, Toshiyuki; Vandré, Dale D; Robinson, John M

    2014-04-01

    The syncytiotrophoblast of the human placenta is a unique epithelia structure with millions of nuclei sharing a common cytoplasm. The syncytiotrophoblast forms by cell-cell fusion of cytotrophoblasts (CTB), the mononuclear precursor cells. The trophoblastic BeWo cell line has been used as a surrogate for CTB since they can be induced to fuse, and subsequently display numerous syncytiotrophoblast differentiation markers following syncytial formation. In this study, we have focused on alterations in the cell-adhesion molecule E-cadherin, actin cytoskeleton, and focal adhesions following BeWo cell fusion, since these entities may be interrelated. There was a dramatic reorganization of the distribution of E-cadherin as well as a reduction in the amount of E-cadherin following cell fusion. Reorganization of the actin cytoskeleton was also observed, which was associated with a change in the globular actin (G-actin)/filamentous actin (F-actin) ratio. Concomitantly, the morphology of focal adhesions was altered, but this occurred without a corresponding change in the levels of focal adhesion marker proteins. Thus, extensive remodeling of the actin cytoskeleton and focal adhesions accompanies cell fusion and differentiation and appears related to alterations in E-cadherin in trophoblastic cells.

  12. Fusion

    NASA Astrophysics Data System (ADS)

    Herman, Robin

    1990-10-01

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

  13. Hot cell facility design for large fusion devices

    SciTech Connect

    Barrett, R.J.; Bussell, G.T.

    1985-01-01

    Large hot cell facilities will be necessary to support the operation of large fusion devices. The supporting hot cells will be needed to serve a variety of different functions and tasks, which include reactor component maintenance, tool and maintenance equipment repair, and preparation of radioactive material for shipment and disposal. This paper discusses hot cell facility functions, requirements, and design issues and techniques. Suggested solutions and examples are given.

  14. Nanodisc-cell fusion: control of fusion pore nucleation and lifetimes by SNARE protein transmembrane domains

    PubMed Central

    Wu, Zhenyong; Auclair, Sarah M.; Bello, Oscar; Vennekate, Wensi; Dudzinski, Natasha R.; Krishnakumar, Shyam S.; Karatekin, Erdem

    2016-01-01

    The initial, nanometer-sized connection between the plasma membrane and a hormone- or neurotransmitter-filled vesicle –the fusion pore– can flicker open and closed repeatedly before dilating or resealing irreversibly. Pore dynamics determine release and vesicle recycling kinetics, but pore properties are poorly known because biochemically defined single-pore assays are lacking. We isolated single flickering pores connecting v-SNARE-reconstituted nanodiscs to cells ectopically expressing cognate, “flipped” t-SNAREs. Conductance through single, voltage-clamped fusion pores directly reported sub-millisecond pore dynamics. Pore currents fluctuated, transiently returned to baseline multiple times, and disappeared ~6 s after initial opening, as if the fusion pore fluctuated in size, flickered, and resealed. We found that interactions between v- and t-SNARE transmembrane domains (TMDs) promote, but are not essential for pore nucleation. Surprisingly, TMD modifications designed to disrupt v- and t-SNARE TMD zippering prolonged pore lifetimes dramatically. We propose that the post-fusion geometry of the proteins contribute to pore stability. PMID:27264104

  15. The Interaction of Mitochondrial Biogenesis and Fission/Fusion Mediated by PGC-1α Regulates Rotenone-Induced Dopaminergic Neurotoxicity.

    PubMed

    Peng, Kaige; Yang, Likui; Wang, Jian; Ye, Feng; Dan, Guorong; Zhao, Yuanpeng; Cai, Ying; Cui, Zhihong; Ao, Lin; Liu, Jinyi; Zou, Zhongmin; Sai, Yan; Cao, Jia

    2016-06-07

    Parkinson's disease is a common neurodegenerative disease in the elderly, and mitochondrial defects underlie the pathogenesis of PD. Impairment of mitochondrial homeostasis results in reactive oxygen species formation, which in turn can potentiate the accumulation of dysfunctional mitochondria, forming a vicious cycle in the neuron. Mitochondrial fission/fusion and biogenesis play important roles in maintaining mitochondrial homeostasis. It has been reported that PGC-1α is a powerful transcription factor that is widely involved in the regulation of mitochondrial biogenesis, oxidative stress, and other processes. Therefore, we explored mitochondrial biogenesis, mitochondrial fission/fusion, and especially PGC-1α as the key point in the signaling mechanism of their interaction in rotenone-induced dopamine neurotoxicity. The results showed that mitochondrial number and mass were reduced significantly, accompanied by alterations in proteins known to regulate mitochondrial fission/fusion (MFN2, OPA1, Drp1, and Fis1) and mitochondrial biogenesis (PGC-1α and mtTFA). Further experiments proved that inhibition of mitochondrial fission or promotion of mitochondrial fusion has protective effects in rotenone-induced neurotoxicity and also promotes mitochondrial biogenesis. By establishing cell models of PGC-1α overexpression and reduced expression, we found that PGC-1α can regulate MFN2 and Drp1 protein expression and phosphorylation to influence mitochondrial fission/fusion. In summary, it can be concluded that PGC-1α-mediated cross talk between mitochondrial biogenesis and fission/fusion contributes to rotenone-induced dopaminergic neurodegeneration.

  16. In vitro evaluation of human hybrid cell lines generated by fusion of B-lymphoblastoid cells and ex vivo tumour cells as candidate vaccines for haematological malignancies.

    PubMed

    Mohamed, Yehia S; Dunnion, Debbie; Teobald, Iryna; Walewska, Renata; Browning, Michael J

    2012-10-12

    Fusions of dendritic cells (DCs) and tumour cells have been shown to induce protective immunity to tumour challenge in animal models, and to represent a promising approach to cancer immunotherapy. The broader clinical application of this approach, however, is potentially constrained by the lack of replicative capacity and limited standardisation of fusion cell preparations. We show here that fusion of ex vivo tumour cells isolated from patients with a range of haematological malignancies with the human B-lymphoblastoid cell line (LCL), HMy2, followed by chemical selection of the hybridomas, generated stable, self-replicating human hybrid cell lines that grew continuously in tissue culture, and survived freeze/thawing cycles. The hybrid cell lines expressed HLA class I and class II molecules, and the major T-cell costimulatory molecules, CD80 and CD86. All but two of 14 hybrid cell lines generated expressed tumour-associated antigens that were not expressed by HMy2 cells, and were therefore derived from the parent tumour cells. The hybrid cell lines stimulated allogeneic T-cell proliferative responses and interferon-gamma release in vitro to a considerably greater degree than their respective parent tumour cells. The enhanced T-cell stimulation was inhibited by CTLA4-Ig fusion protein, and by blocking antibodies to MHC class I and class II molecules. Finally, all of five LCL/tumour hybrid cell lines tested induced tumour antigen-specific cytotoxic T-cell responses in vitro in PBL from healthy, HLA-A2+ individuals, as detected by HLA-A2-peptide pentamer staining and cellular cytotoxicity. These data show that stable hybrid cell lines, with enhanced immunostimulatory properties and potential for therapeutic vaccination, can be generated by in vitro fusion and chemical selection of B-LCL and ex vivo haematological tumour cells.

  17. A cell attracting composite of lumbar fusion cage.

    PubMed

    Gunay, Busra; Hasirci, Nesrin; Hasirci, Vasif

    2017-06-01

    Lumbar fusion cages are devices used in spinal fusion procedures for disorders such as spondylosis and degenerative disc diseases that may occur due to age, trauma or genetic reasons. These devices are most frequently made of metals and polymers. The mechanical properties of such devices should be comparable to the bone to avoid stress shielding. Besides, cages should interact with the cells to prevent extrusion and achieve satisfactory fusion. In this study, poly(methylmethacrylate) (PMMA) and hydroxyapatite (HAp) were compounded to create products with HAp contents up to 40% (w/w), processed by hot melt extrusion and injection molded to produce composites with maximum polymer-mineral interaction. The morphology, interaction with the plates and rate of proliferation of human osteoblast-like (HOB) cells were studied in vitro. We learned that cells interact more with HAp when the HAp content is higher than 20%. Tensile and compressive properties of PMMA were significantly increased with increasing HAp content; from an elastic modulus (E) of 2.08 to 3.92 GPa in tension, and from 349 to 562 MPa in compression. High HAp content of the samples increased the roughness from 0.69 μm for pure PMMA to 1.35 μm for 40% (w/w) HAp loaded PMMA, increased cell proliferation and as a result the cells presented filopodia indicating a satisfactory level of interaction with the cage surface. Based on mechanical and in vitro studies, a HAp content of around 30% (w/w) was found to be appropriate for good cell adhesion and satisfactory mechanical properties for use in the construction of a fusion cage. It was concluded that when PMMA and HAp were compounded at an optimal value, a cage material with adequate mechanical properties and increased cell attachment can be obtained for use in spinal fusion applications.

  18. Effects of polycystin‑1 N‑terminal fragment fusion protein on the proliferation and apoptosis of rat mesangial cells.

    PubMed

    Guan, Tianjun; Gao, Qing; Chen, Ping; Fu, Lili; Zhao, Haidan; Zou, Zhuying; Mei, Changlin

    2014-09-01

    Mesangial proliferative glomerulonephritis (MsPGN) is characterized by widespread mesangial cell proliferation and an accumulation of extracellular matrix (ECM) in the mesangial area. In a previous study we developed a polycystin‑1 N‑terminal fragment (PC‑1 NF) fusion protein that inhibits the proliferation of cyst‑lining epithelial cells in autosomal dominant polycystic kidney disease. In addition, the PC‑1 NF fusion protein arrests the cell cycle of cancer cells at the G0/G1 phase, inhibiting their proliferation. In the present study, the effect of the PC‑1 NF fusion protein on MsPGN was investigated. It was found that the PC‑1 NF fusion protein inhibited the proliferation of rat mesangial cells and induced G0/G1 phase arrest and apoptosis in vitro. PC‑1 NF fusion protein treatment also resulted in a decrease in mRNA expression levels of proliferating cell nuclear antigen, cyclin D1 and B‑cell lymphoma‑2 (Bcl‑2) and an increase in mRNA expression levels of Bcl‑2‑associated X protein (Bax) and p21Waf1. Furthermore, a decrease in Bcl‑2, c‑fos, c‑jun and protein kinase C‑α protein levels was observed, whereas Bax protein levels increased. Additionally, PC‑1 NF fusion protein induced ECM degradation and inhibited ECM expansion. The results also demonstrated that PC‑1 NF fusion protein treatment resulted in a decrease in type IV collagen and tissue inhibitor of metalloproteinase mRNA levels but an increase in matrix metalloproteinase 2 mRNA levels. In combination, these results suggest that the PC‑1 NF fusion protein inhibits proliferation, promotes apoptosis and induces ECM degradation in MsPGN rats. This study offers novel perspectives for the treatment of MsPGN.

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

    PubMed

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

    2011-03-01

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

  20. pH-dependent vesicle fusion induced by the ectodomain of the human immunodeficiency virus membrane fusion protein gp41: Two kinetically distinct processes and fully-membrane-associated gp41 with predominant β sheet fusion peptide conformation.

    PubMed

    Ratnayake, Punsisi U; Sackett, Kelly; Nethercott, Matthew J; Weliky, David P

    2015-01-01

    The gp41 protein of the Human Immunodeficiency Virus (HIV) catalyzes fusion between HIV and host cell membranes. The ~180-residue ectodomain of gp41 is outside the virion and is the most important gp41 region for membrane fusion. The ectodomain consists of an apolar fusion peptide (FP) region hypothesized to bind to the host cell membrane followed by N-heptad repeat (NHR), loop, and C-heptad repeat (CHR) regions. The present study focuses on the large gp41 ectodomain constructs "Hairpin" (HP) containing NHR+loop+CHR and "FP-Hairpin" (FP-HP) containing FP+NHR+loop+CHR. Both proteins induce rapid and extensive fusion of anionic vesicles at pH4 where the protein is positively-charged but do not induce fusion at pH7 where the protein is negatively charged. This observation, along with lack of fusion of neutral vesicles at either pH supports the significance of attractive protein/membrane electrostatics in fusion. There are two kinetically distinct fusion processes at pH4: (1) a faster ~100 ms⁻¹ process with rate strongly positively correlated with vesicle charge; and (2) a slower ~5 ms⁻¹ process with extent strongly inversely correlated with this charge. The slower process may be more physiologically relevant because HIV/host cell fusion occurs at physiologic pH with gp41 restricted to the narrow region between the two membranes. Previous solid-state NMR (SSNMR) of membrane-associated FP-HP has supported protein oligomers with FP's in an intermolecular antiparallel sheet. There was an additional population of molecules with α helical FPs and the samples likely contained a mixture of membrane-bound and -unbound proteins. For the present study, samples were prepared with fully membrane-bound FP-HP and subsequent SSNMR showed dominant β FP conformation at both low and neutral pH. SSNMR also showed close contact of the FP with the lipid headgroups at both low and neutral pH whereas the NHR+CHR regions had contact at low pH and were more distant at neutral p

  1. [Metapneumovirus expands the understanding of Paramyxovirus cell fusion--a review].

    PubMed

    Liu, Xiaoyu; Zhang, Xiaodong; Wei, Yongwei

    2014-04-04

    For most viruses in Paramyxoviridae, cell fusion requires both attachment protein and fusion protein. The attachment protein is responsible for the binding to its cognate receptors, while the interaction between fusion protein and attachment protein triggers the fusion protein which is responsible for the fusion. However, the Metapneumovirus fusion in Pneumovirinae subfamily displayed different mechanism where the attachment protein is not required. The cell fusion is accomplished by fusion protein alone without the help of the attachment protein. Recent studies indicate that low pH is required for cell fusion promoted by some hMPV strains. The fusion protein of aMPV type A is highly fusogenic, whereas that of type B is low. The original fusion models for Paramyxovirus cannot explain the phenomenon above. The mechanism to regulate the cell fusion of Metapneumovirus is poorly understood. It is becoming a hot spot for the study of cell fusion triggered by Paramyxovirus where it enlarged the traditional scope of Paramyxovirus fusion. In this review, we discuss the new achievements and advances in the understanding of cell fusion triggered by Metapneumovirus.

  2. Cell fusion in the brain: two cells forward, one cell back.

    PubMed

    Kemp, Kevin; Wilkins, Alastair; Scolding, Neil

    2014-11-01

    Adult stem cell populations, notably those which reside in the bone marrow, have been shown to contribute to several neuronal cell types in the rodent and human brain. The observation that circulating bone marrow cells can migrate into the central nervous system and fuse with, in particular, cerebellar Purkinje cells has suggested, at least in part, a potential mechanism behind this process. Experimentally, the incidence of cell fusion in the brain is enhanced with age, radiation exposure, inflammation, chemotherapeutic drugs and even selective damage to the neurons themselves. The presence of cell fusion, shown by detection of increased bi-nucleated neurons, has also been described in a variety of human central nervous system diseases, including both multiple sclerosis and Alzheimer's disease. Accumulating evidence is therefore raising new questions into the biological significance of cell fusion, with the possibility that it represents an important means of cell-mediated neuroprotection or rescue of highly complex neurons that cannot be replaced in adult life. Here, we discuss the evidence behind this phenomenon in the rodent and human brain, with a focus on the subsequent research investigating the physiological mechanisms of cell fusion underlying this process. We also highlight how these studies offer new insights into endogenous neuronal repair, opening new exciting avenues for potential therapeutic interventions against neurodegeneration and brain injury.

  3. Bone resorptive activity of human peripheral blood mononuclear cells after fusion with polyethylene glycol.

    PubMed

    Manrique, Edwin; Castillo, Luz M; Lazala, Oswaldo; Guerrero, Carlos A; Acosta, Orlando

    2017-03-01

    The bone remodeling process occurs through bone formation by osteoblasts and bone resorption by osteoclasts, a process involving the contribution of endocrine and nervous systems. The mechanisms associated to differentiation and proliferation of osteoclasts and osteoblasts are considered a potential therapeutic target for treating some erosive bone diseases. The aim of the present study is to explore the feasibility of generating active osteoclast-like cells from peripheral blood mononuclear cells (PBMCs) following polyethylene glycol (PEG)-induced fusion. PEG-fused PBMCs showed TRAP(+)-multinucleated cells and bone resorption activity, and were also positive for osteoclast markers such as carbonic anhydrase II, calcitonin receptor, vacuolar ATPase, and cathepsin K, when examined by reverse transcription-polymerase chain reaction, immunochemistry and Western blotting. TRAP expression and bone resorptive activity were higher in whole PEG-fused PBMCs than in separated T lymphocytes, B lymphocytes or monocytes. Both TRAP expression and bone resorptive activity were also higher in osteogenesis imperfecta patients compared to PEG-fused PBMCs from healthy individuals. PEG-induced fusion was more efficient in inducing TRAP and bone resorptive activities than macrophage colony-stimulating factor or dexamethasone treatment. Bone resorptive activity of PEG-fused PMBCs was inhibited by bisphosphonates. Evidence is provided that the use of PEG-based cell fusion is a straightforward and amenable method for studying human osteoclast differentiation and testing new therapeutic strategies.

  4. Drug Delivery via Cell Membrane Fusion Using Lipopeptide Modified Liposomes

    PubMed Central

    2016-01-01

    Efficient delivery of drugs to living cells is still a major challenge. Currently, most methods rely on the endocytotic pathway resulting in low delivery efficiency due to limited endosomal escape and/or degradation in lysosomes. Here, we report a new method for direct drug delivery into the cytosol of live cells in vitro and invivo utilizing targeted membrane fusion between liposomes and live cells. A pair of complementary coiled-coil lipopeptides was embedded in the lipid bilayer of liposomes and cell membranes respectively, resulting in targeted membrane fusion with concomitant release of liposome encapsulated cargo including fluorescent dyes and the cytotoxic drug doxorubicin. Using a wide spectrum of endocytosis inhibitors and endosome trackers, we demonstrate that the major site of cargo release is at the plasma membrane. This method thus allows for the quick and efficient delivery of drugs and is expected to have many invitro, ex vivo, and invivo applications. PMID:27725960

  5. TALEN-Induced Translocations in Human Cells.

    PubMed

    Piganeau, Marion; Renouf, Benjamin; Ghezraoui, Hind; Brunet, Erika

    2016-01-01

    Induction of chromosomal translocations in human cells is of a great interest to study tumorigenesis and genome instability. Here, we explain in detail a method to induce translocations using the transcription activator-like effector nucleases (TALENs). We describe how to detect translocation formation by PCR, calculate translocation frequency by 96-well PCR screen, and analyze breakpoint junctions. When inducing cancer translocations, it is also possible to detect the fusion gene by FISH analysis or western blot.

  6. RAB-5- and DYNAMIN-1-Mediated Endocytosis of EFF-1 Fusogen Controls Cell-Cell Fusion

    PubMed Central

    Smurova, Ksenia; Podbilewicz, Benjamin

    2016-01-01

    Summary Cell-cell fusion plays essential roles during fertilization and organogenesis. Previous studies in C. elegans led to the identification of the eukaryotic fusion protein (EFF-1 fusogen), which has structural homology to class II viral fusogens. Transcriptional repression of EFF-1 ensures correct fusion fates, and overexpression of EFF-1 results in embryonic lethality. EFF-1 must be expressed on the surface of both fusing cells; however, little is known regarding how cells regulate EFF-1 surface exposure. Here, we report that EFF-1 is actively removed from the plasma membrane of epidermal cells by dynamin- and RAB-5-dependent endocytosis and accumulates in early endosomes. EFF-1 was transiently localized to apical domains of fusion-competent cells. Effective cell-cell fusion occurred only between pairs of cell membranes in which EFF-1 localized. Downregulation of dynamin or RAB-5 caused EFF-1 mislocalization to all apical membrane domains and excessive fusion. Thus, internalization of EFF-1 is a safety mechanism preventing excessive cell fusion. PMID:26854231

  7. Tetraspanins CD9 and CD81 modulate HIV-1-induced membrane fusion.

    PubMed

    Gordón-Alonso, Mónica; Yañez-Mó, María; Barreiro, Olga; Alvarez, Susana; Muñoz-Fernández, M Angeles; Valenzuela-Fernández, Agustín; Sánchez-Madrid, Francisco

    2006-10-15

    Protein organization on the membrane of target cells may modulate HIV-1 transmission. Since the tetraspanin CD81 is associated to CD4, the receptor of HIV-1 envelope protein (Env; gp120/gp41), we have explored the possibility that this molecule may modulate the initial steps of HIV-1 infection. On the other hand, CD81 belongs to the tetraspanin family, which has been described as organizers of protein microdomains on the plasma membrane. Therefore, the role of CD81 and other related tetraspanin, CD9, on the cell-to-cell fusion process mediated by HIV-1 was studied. We found that anti-tetraspanin Abs enhanced the syncytia formation induced by HIV-1 envelope proteins and viral entry in human T lymphoblasts. In addition, anti-CD81 Abs triggered its clustering in patches, where CD4 and CXCR4 were included. Moreover, the knocking down of CD81 and CD9 expression resulted in an increase in syncytia formation and viral entry. Accordingly, overexpression of CD81 and CD9 rendered cells less susceptible to Env-mediated syncytia formation. These data indicate that CD9 and CD81 have an important role in membrane fusion induced by HIV-1 envelope.

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

    PubMed Central

    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. Impaired tethering and fusion of GLUT4 vesicles in insulin-resistant human adipose cells.

    PubMed

    Lizunov, Vladimir A; Lee, Jo-Ping; Skarulis, Monica C; Zimmerberg, Joshua; Cushman, Samuel W; Stenkula, Karin G

    2013-09-01

    Systemic glucose homeostasis is profoundly influenced by adipose cell function. Here we investigated GLUT4 dynamics in living adipose cells from human subjects with varying BMI and insulin sensitivity index (Si) values. Cells were transfected with hemagglutinin (HA)-GLUT4-green fluorescent protein (GFP)/mCherry (red fluorescence), and were imaged live using total internal reflection fluorescence and confocal microscopy. HA-GLUT4-GFP redistribution to the plasma membrane (PM) was quantified by surface-exposed HA epitope. In the basal state, GLUT4 storage vesicle (GSV) trafficking to and fusion with the PM were invariant with donor subject Si, as was total cell-surface GLUT4. In cells from insulin-sensitive subjects, insulin augmented GSV tethering and fusion approximately threefold, resulting in a corresponding increase in total PM GLUT4. However, with decreasing Si, these effects diminished progressively. All insulin-induced effects on GLUT4 redistribution and trafficking correlated strongly with Si and only weakly with BMI. Thus, while basal GLUT4 dynamics and total cell-surface GLUT4 are intact in human adipose cells, independent of donor Si, cells from insulin-resistant donors show markedly impaired GSV tethering and fusion responses to insulin, even after overnight culture. This altered insulin responsiveness is consistent with the hypothesis that adipose cellular dysfunction is a primary contributor to systemic metabolic dysfunction.

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

    PubMed

    Maeshima, Kazuhiro; Funakoshi, Tomoko; Imamoto, Naoko

    2014-01-01

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

  11. Ovine Herpesvirus 2 Glycoproteins B, H, and L Are Sufficient for, and Viral Glycoprotein Ov8 Can Enhance, Cell-Cell Membrane Fusion.

    PubMed

    AlHajri, Salim M; Cunha, Cristina W; Nicola, Anthony V; Aguilar, Hector C; Li, Hong; Taus, Naomi S

    2017-03-15

    Ovine herpesvirus 2 (OvHV-2) is a gammaherpesvirus in the genus Macavirus that is carried asymptomatically by sheep. Infection of poorly adapted animals with OvHV-2 results in sheep-associated malignant catarrhal fever, a fatal disease characterized by lymphoproliferation and vasculitis. There is no treatment or vaccine for the disease and no cell culture system to propagate the virus. The lack of cell culture has hindered studies of OvHV-2 biology, including its entry mechanism. As an alternative method to study OvHV-2 glycoproteins responsible for membrane fusion as a part of the entry mechanism, we developed a virus-free cell-to-cell membrane fusion assay to identify the minimum required OvHV-2 glycoproteins to induce membrane fusion. OvHV-2 glycoproteins B, H, and L (gB, gH, and gL) were able to induce membrane fusion together but not when expressed individually. Additionally, open reading frame Ov8, unique to OvHV-2, was found to encode a transmembrane glycoprotein that can significantly enhance membrane fusion. Thus, OvHV-2 gB, gH, and gL are sufficient to induce membrane fusion, while glycoprotein Ov8 plays an enhancing role by an unknown mechanism.IMPORTANCE Herpesviruses enter cells via attachment of the virion to the cellular surface and fusion of the viral envelope with cellular membranes. Virus-cell membrane fusion is an important step for a successful viral infection. Elucidating the roles of viral glycoproteins responsible for membrane fusion is critical toward understanding viral entry. Entry of ovine herpesvirus 2 (OvHV-2), the causative agent of sheep associated-malignant catarrhal fever, which is one of the leading causes of death in bison and other ungulates, has not been well studied due to the lack of a cell culture system to propagate the virus. The identification of OvHV-2 glycoproteins that mediate membrane fusion may help identify viral and/or cellular factors involved in OvHV-2 cell tropism and will advance investigation of cellular

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

    SciTech Connect

    Choi, Soyoung; Park, Sangeun; Kim, Suhyun; Lim, Chaeseung; Kim, Jungho; Cha, Dae Ryong; Oh, Junseo

    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

  13. Proteasome inhibitor MG-132 enhances histone deacetylase inhibitor SAHA-induced cell death of chronic myeloid leukemia cells by an ROS-mediated mechanism and downregulation of the Bcr-Abl fusion protein

    PubMed Central

    ZHOU, WENJING; ZHU, WEIWEI; MA, LIYA; XIAO, FENG; QIAN, WENBIN

    2015-01-01

    Recently, there has been progress in the treatment of chronic myeloid leukemia (CML). However, novel therapeutic strategies are required in order to address the emerging problem of imatinib resistance. Histone deacetylase inhibitors (HDACi) and proteasome inhibitors are promising alternatives, and may be amenable to integration with current therapeutic approaches. However, the mechanisms underlying the interaction between these two agents remain unclear. The present study assessed the cytotoxic effect of the HDACi, suberoylanilide hydroxamic acid (SAHA), in combination with the proteasome inhibitor, MG-132, in imatinib-sensitive K562 and imatinib-resistant K562G cells, and investigated the mechanism underlying this effect. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method and protein expression levels were determined by western blotting. Reactive oxygen species (ROS) generation levels were observed under a fluorescence microscope The results indicated that SAHA and MG-132 act in a synergistic manner to induce cell death in K562 and K562G cells. This effect was associated with Bcr-Abl downregulation and the production of ROS. Notably, the ROS scavenger, N-acetyl-L-cysteine, almost fully reversed the cell death and Bcr-Abl downregulation that was induced by the combination of SAHA and MG-132. By contrast, the pan-caspase inhibitor, z-VAD-fmk, only partially reversed the cell death induced by these two drugs in CML cells. These results indicated that increased intracellular ROS levels are important in the induction of cell death and the downregulation of Bcr-Abl. In conclusion, the present results suggested that combined SAHA and MG-132 may be a promising treatment for CML. PMID:26722260

  14. Role and organization of the actin cytoskeleton during cell-cell fusion.

    PubMed

    Martin, Sophie G

    2016-12-01

    Cell-cell fusion is a ubiquitous process that underlies fertilization and development of eukaryotes. This process requires fusogenic machineries to promote plasma membrane merging, and also relies on the organization of dedicated sub-cortical cytoskeletal assemblies. This review describes the role of actin structures, so called actin fusion foci, essential for the fusion of two distinct cell types: Drosophila myoblast cells, which fuse to form myotubes, and sexually differentiated cells of the fission yeast Schizosaccharomyces pombe, which fuse to form a zygote. I describe the respective composition and organization of the two structures, discuss their proposed role in promoting plasma membrane apposition, and consider the universality of similar structures for cell-cell fusion.

  15. Temperature dependence of calcium-induced fusion of sonicated phosphatidylserine vesicles.

    PubMed Central

    Sun, S T; Day, E P; Ho, J T

    1978-01-01

    We have measured the temperature dependence calcium-induced fusion of sonicated phosphatidylserine vesicles. The vesicles were incubated in the presence of calcium at a specified temperature until the resulting aggregation or fusion process had gone to completion. EDTA was then added and the resulting final size of the vesicle population was measured by using dynamic light scattering. This final size was plotted against incubation temperature to show the temperature dependence of calcium-induced fusion. This curve has a peak near 11 degrees C which may be associated with the phase transition of the sonicated phosphatidylserine vesicles in the presence of calcium prior to the aggregation or fusion process. PMID:279918

  16. Cell Therapy to Obtain Spinal Fusion

    DTIC Science & Technology

    2008-07-01

    hydrogel materials. We are also currently developing a near infrared dye IR800 that will enter cells and bind to a peptide moiety known as halo tag...Promega Corp). We believe the near infrared will provide the greatest sensitivity. However, we are including preliminary data comparing three...injection. We propose that introduction of the near infrared dye will allow us to detect as few as 100 cells. We propose to complete these

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

    PubMed Central

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

    2016-01-01

    Membrane fusion and fission are vital to eukaryotes’ life1–5. For three decades, it has been proposed that fusion is mediated by fusion between proximal leaflets of two bilayers (hemi-fusion) that produces a hemi-fused structure, followed by fusion between distal leaflets, whereas fission is via hemi-fission, which also produces a hemi-fused structure, followed by full fission1, 4, 6–10. This hypothesis remained unsupported owing to the lack of observation of hemi-fusion/hemi-fission in live cells. A competing fusion hypothesis involving protein-lined pore formation has also been proposed2, 11–15. Using confocal and super-resolution STED microscopy, we observed the hemi-fused Ω-shaped structure for the first time in live cells, neuroendocrine chromaffin cells and pancreatic β-cells. This structure was generated from fusion pore opening or closure (fission) at the plasma membrane. Unexpectedly, its transition to full fusion or fission was determined by competition between fusion and calcium/dynamin-dependent fission mechanisms, and was surprisingly slow (seconds to tens of seconds) in a significant fraction of the events. These results provide key missing evidence over the past three decades proving the hemi-fusion and hemi-fission hypothesis in live cells, and reveal the hemi-fused intermediate as a key structure controlling fusion/fission, as fusion and fission mechanisms compete to determine its transition to fusion or fission. PMID:27309816

  18. Active inclusion bodies of acid phosphatase PhoC: aggregation induced by GFP fusion and activities modulated by linker flexibility

    PubMed Central

    2013-01-01

    Background Biologically active inclusion bodies (IBs) have gained much attention in recent years. Fusion with IB-inducing partner has been shown to be an efficient strategy for generating active IBs. To make full use of the advantages of active IBs, one of the key issues will be to improve the activity yield of IBs when expressed in cells, which would need more choices on IB-inducing fusion partners and approaches for engineering IBs. Green fluorescent protein (GFP) has been reported to aggregate when overexpressed, but GFP fusion has not been considered as an IB-inducing approach for these fusion proteins so far. In addition, the role of linker in fusion proteins has been shown to be important for protein characteristics, yet impact of linker on active IBs has never been reported. Results Here we report that by fusing GFP and acid phosphatase PhoC via a linker region, the resultant PhoC-GFPs were expressed largely as IBs. These IBs show high levels of specific fluorescence and specific PhoC activities (phosphatase and phosphotransferase), and can account for up to over 80% of the total PhoC activities in the cells. We further demonstrated that the aggregation of GFP moiety in the fusion protein plays an essential role in the formation of PhoC-GFP IBs. In addition, PhoC-GFP IBs with linkers of different flexibility were found to exhibit different levels of activities and ratios in the cells, suggesting that the linker region can be utilized to manipulate the characteristics of active IBs. Conclusions Our results show that active IBs of PhoC can be generated by GFP fusion, demonstrating for the first time the potential of GFP fusion to induce active IB formation of another soluble protein. We also show that the linker sequence in PhoC-GFP fusion proteins plays an important role on the regulation of IB characteristics, providing an alternative and important approach for engineering of active IBs with the goal of obtaining high activity yield of IBs. PMID:23497261

  19. Glioma-associated Oncogene 2 Is Essential for Trophoblastic Fusion by Forming a Transcriptional Complex with Glial Cell Missing-a*

    PubMed Central

    Tang, Chao; Tang, Lanfang; Wu, Xiaokai; Xiong, Wenyi; Ruan, Hongfeng; Hussain, Musaddique; Wu, Junsong; Zou, Chaochun; Wu, Ximei

    2016-01-01

    Cell-cell fusion of human villous trophoblasts, referred to as a process of syncytialization, acts as a prerequisite for the proper development and functional maintenance of the human placenta. Given the fact that the main components of the Hedgehog signaling pathway are expressed predominantly in the syncytial layer of human placental villi, in this study, we investigated the potential roles and underlying mechanisms of Hedgehog signaling in trophoblastic fusion. Activation of Hedgehog signaling by a variety of approaches robustly induced cell fusion and the expression of syncytial markers, whereas suppression of Hedgehog signaling significantly attenuated cell fusion and the expression of syncytial markers in both human primary cytotrophoblasts and trophoblast-like BeWo cells. Moreover, among glioma-associated oncogene (GLI) family transcriptional factors in Hedgehog signaling, knockdown of GLI2 but not GLI1 and GLI3 significantly attenuated Hedgehog-induced cell fusion, whereas overexpression of the GLI2 activator alone was sufficient to induce cell fusion. Finally, GLI2 not only stabilized glial cell missing-a, a pivotal transcriptional factor for trophoblastic syncytialization, but also formed a transcriptional heterodimer with glial cell missing-a to transactivate syncytin-1, a trophoblastic fusogen, and promote trophoblastic syncytialization. Taken together, this study uncovered a so far uncharacterized role of Hedgehog/GLI2 signaling in trophoblastic fusion, implicating that Hedgehog signaling, through GLI2, could be required for human placental development and pregnancy maintenance. PMID:26769961

  20. Glioma-associated Oncogene 2 Is Essential for Trophoblastic Fusion by Forming a Transcriptional Complex with Glial Cell Missing-a.

    PubMed

    Tang, Chao; Tang, Lanfang; Wu, Xiaokai; Xiong, Wenyi; Ruan, Hongfeng; Hussain, Musaddique; Wu, Junsong; Zou, Chaochun; Wu, Ximei

    2016-03-11

    Cell-cell fusion of human villous trophoblasts, referred to as a process of syncytialization, acts as a prerequisite for the proper development and functional maintenance of the human placenta. Given the fact that the main components of the Hedgehog signaling pathway are expressed predominantly in the syncytial layer of human placental villi, in this study, we investigated the potential roles and underlying mechanisms of Hedgehog signaling in trophoblastic fusion. Activation of Hedgehog signaling by a variety of approaches robustly induced cell fusion and the expression of syncytial markers, whereas suppression of Hedgehog signaling significantly attenuated cell fusion and the expression of syncytial markers in both human primary cytotrophoblasts and trophoblast-like BeWo cells. Moreover, among glioma-associated oncogene (GLI) family transcriptional factors in Hedgehog signaling, knockdown of GLI2 but not GLI1 and GLI3 significantly attenuated Hedgehog-induced cell fusion, whereas overexpression of the GLI2 activator alone was sufficient to induce cell fusion. Finally, GLI2 not only stabilized glial cell missing-a, a pivotal transcriptional factor for trophoblastic syncytialization, but also formed a transcriptional heterodimer with glial cell missing-a to transactivate syncytin-1, a trophoblastic fusogen, and promote trophoblastic syncytialization. Taken together, this study uncovered a so far uncharacterized role of Hedgehog/GLI2 signaling in trophoblastic fusion, implicating that Hedgehog signaling, through GLI2, could be required for human placental development and pregnancy maintenance.

  1. Enhanced MyoD-induced transdifferentiation to a myogenic lineage by fusion to a potent transactivation domain.

    PubMed

    Kabadi, Ami M; Thakore, Pratiksha I; Vockley, Christopher M; Ousterout, David G; Gibson, Tyler M; Guilak, Farshid; Reddy, Timothy E; Gersbach, Charles A

    2015-06-19

    Genetic reprogramming holds great potential for disease modeling, drug screening, and regenerative medicine. Genetic reprogramming of mammalian cells is typically achieved by forced expression of natural transcription factors that control master gene networks and cell lineage specification. However, in many instances, the natural transcription factors do not induce a sufficiently robust response to completely reprogram cell phenotype. In this study, we demonstrate that protein engineering of the master transcription factor MyoD can enhance the conversion of human dermal fibroblasts and adult stem cells to a skeletal myocyte phenotype. Fusion of potent transcriptional activation domains to MyoD led to increased myogenic gene expression, myofiber formation, cell fusion, and global reprogramming of the myogenic gene network. This work supports a general strategy for synthetically enhancing the direct conversion between cell types that can be applied in both synthetic biology and regenerative medicine.

  2. Cell Therapy to Obtain Spinal Fusion

    DTIC Science & Technology

    2007-03-01

    shows the results of the BMP2 quantification. As can bee seen in figure 2, escalation of virus dose did not appear... Carpenter , T.C., Davie, N.J., and Stenmark, K.R. Circulating mononuclear cells with a dual, macrophage-fibroblast phenotype contribute robustly to hypoxia

  3. Fusion of CCL21 Non-Migratory Active Breast Epithelial and Breast Cancer Cells Give Rise to CCL21 Migratory Active Tumor Hybrid Cell Lines

    PubMed Central

    Reith, Georg; Keil, Silvia; Niggemann, Bernd; Zänker, Kurt S.; Dittmar, Thomas

    2013-01-01

    The biological phenomenon of cell fusion has been linked to tumor progression because several data provided evidence that fusion of tumor cells and normal cells gave rise to hybrid cell lines exhibiting novel properties, such as increased metastatogenic capacity and an enhanced drug resistance. Here we investigated M13HS hybrid cell lines, derived from spontaneous fusion events between M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics and HS578T-Hyg breast cancer cells, concerning CCL21/CCR7 signaling. Western Blot analysis showed that all cell lines varied in their CCR7 expression levels as well as differed in the induction and kinetics of CCR7 specific signal transduction cascades. Flow cytometry-based calcium measurements revealed that a CCL21 induced calcium influx was solely detected in M13HS hybrid cell lines. Cell migration demonstrated that only M13HS hybrid cell lines, but not parental derivatives, responded to CCL21 stimulation with an increased migratory activity. Knockdown of CCR7 expression by siRNA completely abrogated the CCL21 induced migration of hybrid cell lines indicating the necessity of CCL21/CCR7 signaling. Because the CCL21/CCR7 axis has been linked to metastatic spreading of breast cancer to lymph nodes we conclude from our data that cell fusion could be a mechanism explaining the origin of metastatic cancer (hybrid) cells. PMID:23667660

  4. Characterization of functionally active interleukin-18/eGFP fusion protein expression during cell cycle phases in recombinant chicken DF1 Cells.

    PubMed

    Wu, Hsing Chieh; Chen, Yu San; Shien, Jui Hung; Shen, Pin Chun; Lee, Long Huw

    2016-05-01

    The dependence of foreign gene expression on cell cycle phases in mammalian cells has been described. In this study, a DF1/chIL-18a cell line that stably expresses the fusion protein chIL-18 was constructed and the enhanced green fluorescence protein connected through a (G4 S)3 linker sequence investigated the relationship between cell cycle phases and fusion protein production. DF1/chIL-18a cells (1 × 10(5) ) were inoculated in 60-mm culture dishes containing 5 mL of media to achieve 50%-60% confluence and were cultured in the presence of the cycle-specific inhibitors 10058-F4, aphidicolin, and colchicine for 24 and 48 h. The percentage of cell density and mean fluorescence intensity in each cell cycle phase were assessed using flow cytometry. The inhibitors effectively arrested cell growth. The fusion protein production rate was higher in the S phase than in the G0/G1 and G2/M phases. When cell cycle progression was blocked in the G0/G1, S, and G2/M phases by the addition of 10058-F4, aphidicolin, and colchicine, respectively, the aphidicolin-induced single cells showed higher fusion protein levels than did the 10058-F4- or colchicine-induced phase cells and the uninduced control cells. Although the cells did not proliferate after the drug additions, the amount of total fusion protein accumulated in aphidicolin-treated cells was similar to that in the untreated cultures. Fusion protein is biologically active because it induces IFN-γ production in splenocyte cultures of chicken. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:581-591, 2016.

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

    PubMed

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

    2016-06-23

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

  6. Cell Fusion as a Cofactor in Prostate Cancer Metastasis

    DTIC Science & Technology

    2010-01-01

    fusion. However, our findings indicated a different and unexpected to us mechanism. We found that PC-3 released one or more infectious viruses that...and MLV are, we could not identify the virus secreted by PC-3 cells unambiguously. While were we conducting our studies , several reports questioned...vesicular stomatitis virus (VSV), to be reversibly activated by a brief incubation in mildly acidic medium. The advantages of this method are that it is not

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

    DOEpatents

    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.

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

    DOEpatents

    Gambhir, Sanjiv; Pritha, Ray

    2011-06-07

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

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

    SciTech Connect

    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.

  10. Fusion protein of CDR mimetic peptide with Fc inhibit TNF-alpha induced cytotoxicity.

    PubMed

    Qin, Weisong; Feng, Jiannan; Li, Yan; Lin, Zhou; Shen, Beifen

    2006-02-01

    The variable regions of antibodies play central roles in the binding with antigens. Based on the model of a tumour necrosis factor-alpha (TNF-alpha) neutralizing monoclonal antibody (named as Z12) with TNF-alpha, heavy chain CDR2 (HCDR2) and light chain CDR3 (LCDR3) of Z12 were found to be the most responsible to bind with TNF-alpha. A mimetic peptide (PT) was designed based on the sequence derived from HCDR2 and LCDR3. Fusion protein PT-Fc was constructed by linking PT with Fc of human IgG1 through a flexible linker (GGGGGS). The primary structural characteristics of Fc and PT-Fc were analyzed, including the flexibility, hydrophilicity and epitopes. It was demonstrated that PT and Fc in the fusion protein possessed bio-function properly and non-interfering with each other. Furthermore, PT-Fc was expressed in Escherichia coli by fusion with thioredoxin (Trx). After trx-PT-Fc was cleaved with recombinant enterokinase, PT-Fc was obtained. The results of in vitro cytotoxic assays showed that both PT and PT-Fc could efficiently inhibit TNF-alpha induced apoptosis on L929 cells. At the same micromole concentration, the inhibition activity of PT-Fc was significantly higher than PT.

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

    SciTech Connect

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

    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.

  12. [Experimental study on the immune response of fusion tumor vaccine of HepG2 and dendritic cells in vitro].

    PubMed

    Pang, Y B; Cui, B Y; He, J; Huang, X P; Liang, W; Li, L Q; Luo, X L

    2017-02-21

    Objective: To estimate the immune response of HepG2/dendritic cell (DC) fusion cells vaccines against HepG2 cells in vitro. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by Ficoll-Hypaque density-gradient centrifugation.Then DC were obtain from PBMCs by culturing in medium containing granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 5 days.DC and HepG2 fusion cells were induced by polythyleneglycol (PEG). The fusion cells were examined under fluorescence microscope by labeling DCs and HepG2 with green and red fluorescein, respectively, and then the fusion rates were analyzed by flow cytometry.The capacity of fusion cells to secrete interleukin (IL)-12 and stimulate the proliferation of T lymphocyte was assessed by ELISA and Flow cytometry, respectively.ELISPOT was used to assess the interferon gamma (IFN-γ) produced by cytotoxicity T lymphocyte (CTL), and the specific killing ability of fusion cells induce-CTL targeting HepG2 was estimated. Results: The fusion rate of HepG2/DC was 54.5%, and the fusion cells expressed a higher levels of DC mature marker CD80 and costimulatory molecules CD83, CD86 and MHC-Ⅰ, MHC-Ⅱ molecules HLA-ABC and HLA-DR than those in immature DCs (P<0.01). HepG2/DC showed a greater capacity to secrete high level of IL-12 (P<0.05) and activate proliferation of lymphocytes in vitro, as compared with DCs alone and DCs mix HepG2 (P<0.01). The HepG2/DC -activated CTL generated higher IFN-γ level and had a specific killing ability against HepG2 cells at the effecter/target ratio 30∶1 (31.4%±2.4%) and 100∶1 (57.6%±7.3%) (P<0.01). Conclusions: HepG2/DC fusion cells could efficiently stimulate T lymphocytes to generate specific CTL targeting HepG2 cells.It might be a promising strategy of immunotherapy for HCC.

  13. The Effect of Acute Microgravity on Mechanically-Induced Membrane Damage and Membrane-Membrane Fusion Events

    NASA Technical Reports Server (NTRS)

    Clarke, Mark, S. F.; Vanderburg, Charles R.; Feedback, Daniel L.

    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". This response is characterized by both membrane rupture and membrane resealing events mediated by membrane-membrane fusion. 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.

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

    NASA Technical Reports Server (NTRS)

    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.

  15. Search for systematic behavior of incomplete-fusion probability and complete-fusion suppression induced by {sup 9}Be on different targets

    SciTech Connect

    Gomes, P. R. S.; Linares, R.; Lubian, J.; Lopes, C. C.; Cardozo, E. N.; Pereira, B. H. F.

    2011-07-15

    We present a trial to obtain a systematic behavior of the results available in the literature on the complete and incomplete fusion induced by the weakly bound projectile {sup 9}Be on targets with different masses and/or charges. We stress that although the incomplete-fusion probability and the complete-fusion suppression are very closely related quantities, the first is an experimental value whereas the later is model dependent. A trend of systematic behavior for the incomplete-fusion probability as a function of the target charge is achieved, but not for the complete-fusion suppression.

  16. Fisetin antagonizes cell fusion, cytoskeletal organization and bone resorption in RANKL-differentiated murine macrophages.

    PubMed

    Kim, Yun-Ho; Kim, Jung-Lye; Lee, Eun-Jung; Park, Sin-Hye; Han, Seon-Young; Kang, Soon Ah; Kang, Young-Hee

    2014-03-01

    Osteoclastogenesis is comprised of several stage s including progenitor survival, differentiation to mononuclear preosteoclasts, cell fusion to multinuclear mature osteoclasts, and activation to osteoclasts with bone resorbing activity. Botanical antioxidants are now being increasingly investigated for their health-promoting effects on bone. This study investigated that fisetin, a flavonol found naturally in many fruits and vegetables, suppressed osteoclastogenesis by disturbing receptor activator of nuclear factor (NF)-κB ligand (RANKL)-mediated signaling pathway and demoting osteoclastogenic protein induction. Nontoxic fisetin at ≤10 μM inhibited the induction of RANK, tumor necrosis factor receptor associated factor 6 (TRAF6) and the activation of NF-κB in RANKL-stimulated RAW 264.7 macrophages. In RANKL-differentiated osteoclasts cell fusion protein of E-cadherin was induced, which was dampened by fisetin. The formation of tartrate-resistance acid phosphatase-positive multinucleated osteoclasts was suppressed by adding fisetin to RANKL-exposed macrophages. It was also found that fisetin reduced actin ring formation and gelsolin induction of osteclasts enhanced by RANKL through disturbing c-Src-proline-rich tyrosine kinase 2 signaling. Fisetin deterred preosteoclasts from the cell-cell fusion and the organization of the cytoskeleton to seal the resorbing area and to secret protons for bone resorption. Consistently, the 5 day-treatment of fisetin diminished RANKL-induced cellular expression of carbonic anhydrase II and integrin β3 concurrently with a reduction of osteoclast bone-resorbing activity. Therefore, fisetin was a natural therapeutic agent retarding osteoclast fusion and cytoskeletal organization such as actin rings and ruffled boarder, which is a property of mature osteoclasts and is required for osteoclasts to resorb bone.

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

    PubMed

    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

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

    PubMed

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

    2009-05-01

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

  19. Dual Split Protein (DSP) Assay to Monitor Cell-Cell Membrane Fusion.

    PubMed

    Nakane, Shuhei; Matsuda, Zene

    2015-01-01

    Fusion between viral and cellular membranes is the essential first step in infection of enveloped viruses. This step is mediated by viral envelope glycoproteins (Env) that recognize cellular receptors. The membrane fusion between the effector cells expressing viral Env and the target cells expressing its receptors can be monitored by several methods. We have recently developed a pair of chimeric reporter protein composed of split Renilla luciferase (RL) and split GFP. We named this reporter dual split protein (DSP), since it recovers both RL and GFP activities upon self reassociation. By using DSP, pore formation and content mixing between the effector and target cells can be monitored upon the recovery of RL and GFP activities after the membrane fusion. This quick assay provides quantitative as well as spatial information about membrane fusion mediated by viral Env.

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

    SciTech Connect

    Karentz, D.; Cleaver, J.E.

    1986-10-01

    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. 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.

  1. Laser-assisted cell fusion and cytoplast transfer in early mammalian embryos

    NASA Astrophysics Data System (ADS)

    Clement-Sengewald, Annette; Schutze, Karin; Heinze, A.; Palma, G. A.; Poesl, H.; Brem, G.

    1993-07-01

    A UV-laser microbeam was successfully used to induce fusion of early embryonic cells. The developmental capacity of the laser-fused cells was examined using in vitro culture methods. Blastomeres within mouse two-cell embryos were fused with 3 - 10 subsequent laser pulses in order to produce tetraploid embryos. Thirty-one percent of the laser treated embryos fused and 10% of those developed to the morula or blastocyst stage. With 1 - 10 successive laser pulses cattle oocytes were fused with cytoplasts. Thirty-six percent of the laser treated cells fused and 10% of those cleaved to the 6- and 8-cell stage. These preliminary results indicate that a UV- laser microbeam combined with an optical tweezers may facilitate the manipulation of embryonic cells and can be a helpful tool in polyploidy studies and in cytoplasmic transfer experiments.

  2. Two modes of lytic granule fusion during degranulation by natural killer cells.

    PubMed

    Liu, Dongfang; Martina, Jose A; Wu, Xufeng S; Hammer, John A; Long, Eric O

    2011-08-01

    Lytic granules in cytotoxic lymphocytes, which include T cells and natural killer (NK) cells, are secretory lysosomes that release their content upon fusion with the plasma membrane (PM), a process known as degranulation. Although vesicle exocytosis has been extensively studied in endocrine and neuronal cells, much less is known about the fusion of lytic granules in cytotoxic lymphocytes. Here, we used total internal reflection fluorescence microscopy to examine lytic granules labeled with fluorescently tagged Fas ligand (FasL) in the NK cell line NKL stimulated with phorbol ester and ionomycin and in primary NK cells activated by physiological receptor-ligand interactions. Two fusion modes were observed: complete fusion, characterized by loss of granule content and rapid diffusion of FasL at the PM; and incomplete fusion, characterized by transient fusion pore opening and retention of FasL at the fusion site. The pH-sensitive green fluorescence protein (pHluorin) fused to the lumenal domain of FasL was used to visualize fusion pore opening with a time resolution of 30 ms. Upon incomplete fusion, pHluorin emission lasted several seconds in the absence of noticeable diffusion. Thus, we conclude that lytic granules in NK cells undergo both complete and incomplete fusion with the PM, and propose that incomplete fusion may promote efficient recycling of lytic granule membrane after the release of cytotoxic effector molecules.

  3. MiR-106b-mediated Mfn2 suppression is critical for PKM2 induced mitochondrial fusion

    PubMed Central

    Wu, Haili; Li, Zhuoyu; Wang, Yingying; Yang, Peng; Li, Zongrui; Li, Hanqing; Wu, Changxin

    2016-01-01

    Cancer cells preferentially metabolize glucose through aerobic glycolysis. This phenomenon, known as the Warburg effect, is a characteristic of glucose metabolism in cancer cells. PKM2 is reported to imply an important role in glycolysis. However, whether and how PKM2 can cause mitochondrial dysfunction, then subsequently forcing cancer cells using glycolysis instead of oxidation phosphorylation is poorly understood. Here we reported that overexpression of PKM2 disrupted mitochondrial dynamics by enhancing fusion. And PKM2 overexpression increased the expression of fusion protein Mfn2. Simultaneously, PKM2 overexpression induced mitochondrial dysfunctions shown by the decreased ATP level and increased mitochondrial DNA (mtDNA) copy number. Reduction of Mfn2 expression by siRNA attenuated the PKM2-enhanced mitochondrial fusion and restored the functions. Quantitative and morphological analyses showed that the expression of microRNA-106b (miR-106b) was decreased in the PKM2 overexpressed cells, and the reduction of Mfn2 expression and the recovery of mitochondrial functions were induced by the treatment of miR-106b mimics, demonstrating that miR-106b played important roles in the down-regulation of Mfn2 expression and the PKM2 mediation of mitochondrial fusion. Clinical investigation was performed and results showed that the higher expression levels of PKM2 corresponded with the higher expression levels of Mfn2 in breast cancer tissues by comparison of their expression in adjacent normal tissues. Taken together, our data demonstrate that the overexpression of PKM2 and Mfn2 causes mitochondrial dysfunction via enhancing mitochondrial fusion and miR-106b play crucial roles in PKM2 mediated mitochondrial function through its regulation of Mfn2 expression, which provides new insights into the molecular mechanisms underlying glycolysis and oxidative phosphorylation. PMID:27822413

  4. High perfluorooctanoic acid exposure induces autophagy blockage and disturbs intracellular vesicle fusion in the liver.

    PubMed

    Yan, Shengmin; Zhang, Hongxia; Guo, Xuejiang; Wang, Jianshe; Dai, Jiayin

    2017-01-01

    Perfluorooctanoic acid (PFOA) has been shown to cause hepatotoxicity and other toxicological effects. Though PPARα activation by PFOA in the liver has been well accepted as an important mechanism of PFOA-induced hepatotoxicity, several pieces of evidence have shown that the hepatotoxic effects of PFOA may not be fully explained by PPARα activation. In this study, we observed autophagosome accumulation in mouse livers as well as HepG2 cells after PFOA exposure. Further in vitro study revealed that the accumulation of autophagosomes was not caused by autophagic flux stimulation. In addition, we observed that PFOA exposure affected the proteolytic activity of HepG2 cells while significant dysfunction of lysosomes was not detected. Quantitative proteomic analysis of crude lysosomal fractions from HepG2 cells treated with PFOA revealed that 54 differentially expressed proteins were related to autophagy or vesicular trafficking and fusion. The proteomic results were further validated in the cells in vitro and livers in vivo after PFOA exposure, which implied potential dysfunction at the late stage of autophagy. However, in HepG2 cells, it seemed that further inhibition of autophagy did not significantly alter the effects of PFOA on cell viability. Although these findings demonstrate that PFOA blocked autophagy and disturbed intracellular vesicle fusion in the liver, the changes in autophagy were observed only at high cytotoxic concentrations of PFOA, suggesting that autophagy may not be a primary target or mode of toxicity. Furthermore, since altered liver autophagy was not observed at concentrations of PFOA associated with human exposures, the relevance of these findings must be questioned.

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

    PubMed

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

    2015-08-07

    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.

  6. The dissimilar effect of diacylglycerols on Ca(2+)-induced phosphatidylserine vesicle fusion.

    PubMed Central

    Sánchez-Migallón, M P; Aranda, F J; Gómez-Fernández, J C

    1995-01-01

    We have studied the effect of physiological concentrations of different diacylglycerols on Ca(2+)-induced fusion between phosphatidylserine vesicles. We monitored vesicle fusion as mixing of membrane lipids under conditions where the limiting factor was the aggregation and also in conditions where this aggregation was not the limiting factor. We found that diacylglycerols have a different modulating effect on the Ca(2+)-induced fusion: i) depending on their interfacial conformation, so that 1,2-isomers of diacylglycerols containing unsaturated or short saturated acyl chains stimulated fusion and their 1,3-isomers did not, and ii) depending on their specific type of bilayer interior perturbation, so that diacylglycerols containing unsaturated or short chain saturated acyl chains stimulated fusion but those containing long-chain saturated acyl chains did not. These requirements resembled those required for the diacylglycerol activation of protein kinase C, suggesting that diacylglycerol acts in both the specific activation of this enzyme and the induction of membrane fusion through the same perturbation of lipid structure. We found that polylysine affected the stimulatory role of 1,2-dioleoylglycerol differently, depending on whether aggregation was the limiting factor of fusion. When we studied the effect of very low concentrations of diacylglycerols on the bulk structural properties of phosphatidylserine, we found that they neither significantly perturbed the thermotropic transitions of phosphatidylserine nor affected the interaction of Ca2+ with the phosphate group of phosphatidylserine. The underlying mechanism of fusion between phosphatidylserine vesicles is discussed. PMID:7696508

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

    PubMed

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

    2013-05-28

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

  8. Direct transfer of learned behaviour via cell fusion in non-neural organisms.

    PubMed

    Vogel, David; Dussutour, Audrey

    2016-12-28

    Cell fusion is a fundamental phenomenon observed in all eukaryotes. Cells can exchange resources such as molecules or organelles during fusion. In this paper, we ask whether a cell can also transfer an adaptive response to a fusion partner. We addressed this question in the unicellular slime mould Physarum polycephalum, in which cell-cell fusion is extremely common. Slime moulds are capable of habituation, a simple form of learning, when repeatedly exposed to an innocuous repellent, despite lacking neurons and comprising only a single cell. In this paper, we present a set of experiments demonstrating that slime moulds habituated to a repellent can transfer this adaptive response by cell fusion to individuals that have never encountered the repellent. In addition, we show that a slime mould resulting from the fusion of a minority of habituated slime moulds and a majority of unhabituated ones still shows an adaptive response to the repellent. Finally, we further reveal that fusion must last a certain time to ensure an effective transfer of the behavioural adaptation between slime moulds. Our results provide strong experimental evidence that slime moulds exhibit transfer of learned behaviour during cell fusion and raise the possibility that similar phenomena may occur in other cell-cell fusion systems.

  9. The Glycoprotein B Cytoplasmic Domain Lysine Cluster Is Critical for Varicella-Zoster Virus Cell-Cell Fusion Regulation and Infection.

    PubMed

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

    2017-01-01

    The conserved glycoproteins gB and gH-gL are essential for herpesvirus entry and cell-cell fusion induced syncytium formation, a characteristic of varicella-zoster virus (VZV) pathology in skin and sensory ganglia. VZV syncytium formation, which has been implicated in the painful condition of postherpetic neuralgia, is regulated by the cytoplasmic domains of gB (gBcyt) via an immunoreceptor tyrosine-based inhibition motif (ITIM) and gH (gHcyt). A lysine cluster (K894, K897, K898, and K900) in the VZV gBcyt was identified by sequence alignment to be conserved among alphaherpesviruses, suggesting a functional role. Alanine and arginine substitutions were used to determine if the positive charge and susceptibility to posttranslational modifications of these lysines contributed to gB/gH-gL cell-cell fusion. Critically, the positive charge of the lysine residues was necessary for fusion regulation, as alanine substitutions induced a 440% increase in fusion compared to that of the wild-type gBcyt while arginine substitutions had wild-type-like fusion levels in an in vitro gB/gH-gL cell fusion assay. Consistent with these results, the alanine substitutions in the viral genome caused exaggerated syncytium formation, reduced VZV titers (-1.5 log10), and smaller plaques than with the parental Oka (pOka) strain. In contrast, arginine substitutions resulted in syncytia with only 2-fold more nuclei, a -0.5-log10 reduction in titers, and pOka-like plaques. VZV mutants with both an ITIM mutation and either alanine or arginine substitutions had reduced titers and small plaques but differed in syncytium morphology. Thus, effective VZV propagation is dependent on cell-cell fusion regulation by the conserved gBcyt lysine cluster, in addition to the gBcyt ITIM and the gHcyt.

  10. Investigation of contribution of incomplete fusion in the total fusion process induced by 9Be on 181Ta target at near barrier energies

    NASA Astrophysics Data System (ADS)

    Kharab, Rajesh; Chahal, Rajiv; Kumar, Rajiv

    2016-02-01

    We have studied the relative contribution of incomplete fusion (ICF) and complete fusion (CF) in total fusion (TF) induced by 9Be on 181Ta target at energies in the vicinity of Coulomb barrier using classical dynamical model and Wong's formula in conjugation with energy dependent Woods-Saxon formula. It is found that at above barrier energies ICF contributes almost 30% in TF while at energies below the barrier qualitatively its contribution is much more than thirty percent.

  11. Rapid fusion between mesenchymal stem cells and cardiomyocytes yields electrically active, non-contractile hybrid cells.

    PubMed

    Shadrin, Ilya Y; Yoon, Woohyun; Li, Liqing; Shepherd, Neal; Bursac, Nenad

    2015-07-10

    Cardiac cell therapies involving bone marrow-derived human mesenchymal stem cells (hMSCs) have shown promising results, although their mechanisms of action are still poorly understood. Here, we investigated direct interactions between hMSCs and cardiomyocytes in vitro. Using a genetic Ca(2+) indicator gCaMP3 to efficiently label hMSCs in co-cultures with neonatal rat ventricular myocytes (NRVMs), we determined that 25-40% of hMSCs (from 4 independent donors) acquired periodic Ca(2+) transients and cardiac markers through spontaneous fusion with NRVMs. Sharp electrode and voltage-clamp recordings in fused cells showed action potential properties and Ca(2+) current amplitudes in between those of non-fused hMSCs and NRVMs. Time-lapse video-microscopy revealed the first direct evidence of active fusion between hMSCs and NRVMs within several hours of co-culture. Application of blebbistatin, nifedipine or verapamil caused complete and reversible inhibition of fusion, suggesting potential roles for actomyosin bridging and Ca(2+) channels in the fusion process. Immunostaining for Cx43, Ki67, and sarcomeric α-actinin showed that fused cells remain strongly coupled to surrounding NRVMs, but downregulate sarcomeric structures over time, acquiring a non-proliferative and non-contractile phenotype. Overall, these results describe the phenotype and mechanisms of hybrid cell formation via fusion of hMSCs and cardiomyocytes with potential implications for cardiac cell therapy.

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

    PubMed

    Yao, Fei; Kausalya, Jaya P; Sia, Yee Yen; Teo, Audrey S M; Lee, Wah Heng; Ong, Alicia G M; Zhang, Zhenshui; Tan, Joanna H J; Li, Guoliang; Bertrand, Denis; Liu, Xingliang; Poh, Huay Mei; Guan, Peiyong; Zhu, Feng; Pathiraja, Thushangi Nadeera; Ariyaratne, Pramila N; Rao, Jaideepraj; Woo, Xing Yi; Cai, Shaojiang; Mulawadi, Fabianus H; Poh, Wan Ting; Veeravalli, Lavanya; Chan, Chee Seng; Lim, Seong Soo; Leong, See Ting; Neo, Say Chuan; Choi, Poh Sum D; Chew, Elaine G Y; Nagarajan, Niranjan; Jacques, Pierre-Étienne; So, Jimmy B Y; Ruan, Xiaoan; Yeoh, Khay Guan; Tan, Patrick; Sung, Wing-Kin; Hunziker, Walter; Ruan, Yijun; Hillmer, Axel M

    2015-07-14

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

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

    SciTech Connect

    Wu Liguo; Hutt-Fletcher, Lindsey M. . E-mail: lhuttf@lsuhsc.edu

    2007-06-20

    Cell fusion mediated by Epstein-Barr virus requires three conserved glycoproteins, gB and gHgL, but activation is cell type specific. B cell fusion requires interaction between MHC class II and a fourth virus glycoprotein, gp42, which complexes non-covalently with gHgL. Epithelial cell fusion requires interaction between gHgL and a novel epithelial cell coreceptor and is blocked by excess gp42. We show here that gp42 interacts directly with gH and that point mutations in the region of gH recognized by an antibody that differentially inhibits epithelial and B cell fusion significantly impact both the core fusion machinery and cell-specific events. Substitution of alanine for glycine at residue 594 completely abrogates fusion with either B cells or epithelial cells. Substitution of alanine for glutamic acid at residue 595 reduces fusion with epithelial cells, greatly enhances fusion with B cells and allows low levels of B cell fusion even in the absence of gL.

  14. Role of Ca++ in virus-induced membrane fusion. Ca++ accumulation and ultrastructural changes induced by Sendai virus in chicken erythrocytes

    PubMed Central

    1978-01-01

    Some of the ultrastructural (freeze-etching technique), morphological, and biochemical effects of Sendai virus interaction with chicken erythrocytes have been studied under fusogenic (in the presence of CaCl2) and nonfusogenic (in the presence of ethyleneglycol-bis-N,N'- tetraacetic acid, [EGTA]) conditions. The following phenomena occur, irrespective of the presence of CaCl2 or EGTA: (a) binding of iodinated virus particles to chicken erythrocytes at 4 degrees C and their partial release from the cells at 37 degrees C; (b) gradual incorporation of the viral envelope and viral M-protein into plasma membrane, as visualized in the protoplasmic and exoplasmic fracture (P and E, respectively) faces of the membrane; and (c) virus-dependent transient clustering of intramembrane particles at 4 degrees C, which is reversible after transferring the cells back to 37 degrees C. The following virus-induced phenomena occur only in the presence of CaCl2: (a) rounding of cells followed by their fusion; (b) transient decrease in the density of intramembrane particles; and (c) the virus induces uptake of 45CaCl2 by chicken erythrocytes. The uptake is specific as it is inhibited by LaCl3, and no accumulation of [14C]glucose-1-phosphate ([14C]G-1-P) could be observed under the 45 CaCl2 uptake conditions. The data show that fusion of virus with plasma membrane is a Ca++- independent process and, as such, it should be distinguished from the virus-induced membrane-membrane and cell fusion processes. The latter is absolutely dependent on the rise of intracellular Ca++, as reflected by the fact that Ca++-induced rounding of chicken erythrocytes always precedes fusion (Volsky, D. and A. Loyter. 1977.Biochim. Biophys. Acta 471:253--259). PMID:211140

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

    NASA Astrophysics Data System (ADS)

    Wang, Ning; Ou, Li; Zhang, Yingxun; Li, Zhuxia

    2014-06-01

    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 O16 + Ge76, O16 + Sm154, Ca40 + Zr96, and Sn132 + Ca40 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 ≈78 MeV and the surface energy coefficient is gsur=18±1.5 MeV fm2. 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 neck side result in the lowering of the fusion barrier.

  16. Increasing the physical size and nucleation status of human pluripotent stem cell-derived ventricular cardiomyocytes by cell fusion.

    PubMed

    Kong, Chi-Wing; Chen, Shuxun; Geng, Lin; Shum, Angie Man-Yee; Sun, Dong; Li, Ronald A

    2017-03-01

    Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) provide an unlimited source of donor cells for potential cardiac regenerative therapies. However, hPSC-CMs are immature. For instance, hPSC-CMs are only 1/10 of the physical size of their adult counterparts; the majority are mono- rather than bi- or multi-nucleated, which is an evolutionary adaptive feature in metabolically active cells such as adult CMs. Here, we attempted to increase the physical size and nucleation status of hPSC-derived ventricular (V) cardiomyocytes (hPSC-VCMs) using chemically-induced cell fusion, and examined the subsequent functional effects. Polyethylene glycol (PEG) was employed to fuse a 1:1 mixture of lentiviral vectors LV-MLC2v-GFP- or -tdTomato-labeled hPSC-VCMs, such that hPSC-VCMs fused syncytia (FS) were identified as doubly GFP(+)/tdTomato(+) multi-nucleated cells. These microscopically-identified FS were doubled in size as gauged by their capacitance when compared to the control mononucleated hPSC-VCMs using patch-clamp analysis. Reduced automaticity or action potential (AP) firing rate and moderately prolonged AP duration were observed in FS from day 6 post-fusion induction. However, Ca(2+) handling, mitochondrial biogenesis and the extent of apoptosis were not significantly altered. We conclude that larger, multi-nucleated hPSC-VCMs FS can be created by chemically-induced cell fusion but global maturation requires additional triggering cues.

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

    PubMed

    Oleinick, Alexander; Svir, Irina; Amatore, Christian

    2017-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Oleinick, Alexander; Svir, Irina; Amatore, Christian

    2017-01-01

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

  19. Identification of CCDC6-RET fusion in the human lung adenocarcinoma cell line, LC-2/ad.

    PubMed

    Matsubara, Daisuke; Kanai, Yoshihiko; Ishikawa, Shumpei; Ohara, Shiori; Yoshimoto, Taichiro; Sakatani, Takashi; Oguni, Sachiko; Tamura, Tomoko; Kataoka, Hiroaki; Endo, Shunsuke; Murakami, Yoshinori; Aburatani, Hiroyuki; Fukayama, Masashi; Niki, Toshiro

    2012-12-01

    Rearranged during transfection (RET) fusions have been newly identified in approximately 1% of patients with primary lung tumors. However, patient-derived lung cancer cell lines harboring RET fusions have not yet been established or identified, and therefore, the effectiveness of an RET inhibitor on lung tumors with endogenous RET fusion has not yet been studied. In this study, we report identification of CCDC6-RET fusion in the human lung adenocarcinoma cell line LC-2/ad. LC-2/ad showed distinctive sensitivity to the RET inhibitor, vandetanib, among 39 non-small lung cancer cell lines. The xenograft tumor of LC-2/ad showed cribriform acinar structures, a morphologic feature of primary RET fusion-positive lung adenocarcinomas. LC-2/ad cells could provide useful resources to analyze molecular functions of RET-fusion protein and its response to RET inhibitors.

  20. Laser-induced tissue fluorescence in radiofrequency tissue-fusion characterization

    NASA Astrophysics Data System (ADS)

    Su, Lei; Fonseca, Martina B.; Arya, Shobhit; Kudo, Hiromi; Goldin, Robert; Hanna, George B.; Elson, Daniel S.

    2014-01-01

    Heat-induced tissue fusion is an important procedure in modern surgery and can greatly reduce trauma, complications, and mortality during minimally invasive surgical blood vessel anastomosis, but it may also have further benefits if applied to other tissue types such as small and large intestine anastomoses. We present a tissue-fusion characterization technology using laser-induced fluorescence spectroscopy, which provides further insight into tissue constituent variations at the molecular level. In particular, an increase of fluorescence intensity in 450- to 550-nm range for 375- and 405-nm excitation suggests that the collagen cross-linking in fused tissues increased. Our experimental and statistical analyses showed that, by using fluorescence spectral data, good fusion could be differentiated from other cases with an accuracy of more than 95%. This suggests that the fluorescence spectroscopy could be potentially used as a feedback control method in online tissue-fusion monitoring.

  1. Microtubule-dependent balanced cell contraction and luminal-matrix modification accelerate epithelial tube fusion

    PubMed Central

    Kato, Kagayaki; Dong, Bo; Wada, Housei; Tanaka-Matakatsu, Miho; Yagi, Yoshimasa; Hayashi, Shigeo

    2016-01-01

    Connection of tubules into larger networks is the key process for the development of circulatory systems. In Drosophila development, tip cells of the tracheal system lead the migration of each branch and connect tubules by adhering to each other and simultaneously changing into a torus-shape. We show that as adhesion sites form between fusion cells, myosin and microtubules form polarized bundles that connect the new adhesion site to the cells' microtubule-organizing centres, and that E-cadherin and retrograde recycling endosomes are preferentially deposited at the new adhesion site. We demonstrate that microtubules help balancing tip cell contraction, which is driven by myosin, and is required for adhesion and tube fusion. We also show that retrograde recycling and directed secretion of a specific matrix protein into the fusion-cell interface promote fusion. We propose that microtubule bundles connecting these cell–cell interfaces coordinate cell contractility and apical secretion to facilitate tube fusion. PMID:27067650

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

    SciTech Connect

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

    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.

  3. Characterization of the plasma membrane localization and orientation of HPV16 E5 for cell-cell fusion

    SciTech Connect

    Hu Lulin; Ceresa, Brian P.

    2009-10-10

    Human papillomavirus (HPV) is a non-enveloped DNA virus with an approx 8000 base pair genome. Infection with certain types of HPV is associated with cervical cancer, although the molecular mechanism by which HPV induces carcinogenesis is poorly understood. Three genes encoded by HPV16 are regarded as oncogenic - E5, E6, and E7. The role of E5 has been controversial. Expression of HPV16 E5 causes cell-cell fusion, an event that can lead to increased chromosomal instability, particularly in the presence of cell cycle checkpoint inhibitors like HPV16 E6 and E7. Using biochemical and cell biological assays to better understand HPV16 E5, we find that HPV16 E5 localizes to the plasma membrane with an intracellular amino terminus and an extracellular carboxyl-terminus. Further, HPV16 E5 must be expressed on both cells for cell fusion to occur. When the extracellular epitope of HPV16 E5 is targeted with an antibody, the number of bi-nucleated cells decreases.

  4. The p10 FAST protein fusion peptide functions as a cystine noose to induce cholesterol-dependent liposome fusion without liposome tubulation.

    PubMed

    Key, Tim; Sarker, Muzaddid; de Antueno, Roberto; Rainey, Jan K; Duncan, Roy

    2015-02-01

    The reovirus p10 fusion-associated small transmembrane (FAST) proteins are the smallest known membrane fusion proteins, and evolved specifically to mediate cell-cell, rather than virus-cell, membrane fusion. The 36-40-residue ectodomains of avian reovirus (ARV) and Nelson Bay reovirus (NBV) p10 contain an essential intramolecular disulfide bond required for both cell-cell fusion and lipid mixing between liposomes. To more clearly define the functional, biochemical and biophysical features of this novel fusion peptide, synthetic peptides representing the p10 ectodomains of ARV and NBV were analyzed by solution-state NMR spectroscopy, circular dichroism spectroscopy, fluorescence spectroscopy-based hydrophobicity analysis, and liposome binding and fusion assays. Results indicate that disulfide bond formation promotes exposure of hydrophobic residues, as indicated by bis-ANS binding and time-dependent peptide aggregation under aqueous conditions, implying the disulfide bond creates a small, geometrically constrained, cystine noose. Noose formation is required for peptide partitioning into liposome membranes and liposome lipid mixing, and electron microscopy revealed that liposome-liposome fusion occurs in the absence of liposome tubulation. In addition, p10 fusion peptide activity, but not membrane partitioning, is dependent on membrane cholesterol.

  5. TMPRSS2-ERG gene fusion in small cell carcinoma of the prostate.

    PubMed

    Guo, Charles C; Dancer, Jane Y; Wang, Yan; Aparicio, Ana; Navone, Nora M; Troncoso, Patricia; Czerniak, Bogdan A

    2011-01-01

    Recent studies have shown that most prostate cancers carry the TMPRSS2-ERG gene fusion. Here we evaluated the TMPRSS2-ERG gene fusion in small cell carcinoma of the prostate (n = 12) in comparison with small cell carcinoma of the urinary bladder (n = 12) and lung (n = 11). Fluorescence in situ hybridization demonstrated rearrangement of the ERG gene in 8 cases of prostatic small cell carcinoma (67%), and the rearrangement was associated with deletion of the 5' ERG gene in 7 cases, but rearrangement of the ERG gene was not present in any small cell carcinoma of the urinary bladder or lung. Next we evaluated the TMPRSS2-ERG gene fusion in nude mouse xenografts that were derived from 2 prostatic small cell carcinomas carrying the TMPRSS2-ERG gene fusion. Two transcripts encoded by the TMPRSS2-ERG gene fusion were detected by reverse transcriptase polymerase chain reaction, and DNA sequencing demonstrated that the 2 transcripts were composed of fusions of exon 1 of the TMPRSS2 gene to exon 4 or 5 of the ERG gene. Our study demonstrates the specific presence of TMPRSS2-ERG gene fusion in prostatic small cell carcinoma, which may be helpful in distinguishing small cell carcinoma of prostatic origin from nonprostatic origins. The high prevalence of the TMPRSS2-ERG gene fusion in prostatic small cell carcinoma as well as adenocarcinoma implies that small cell carcinoma may share a common pathogenic pathway with adenocarcinoma in the prostate.

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

    SciTech Connect

    Kim, Kyoungmi; Kim, Hwain; Lee, Daekee

    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.

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

    PubMed

    Zhang, Wenbin; Chen, Yaomin; Yang, Qun; Che, Honglei; Chen, Xiangjun; Yao, Ting; Zhao, Fang; Liu, Mingchao; Ke, Tao; Chen, Jingyuan; Luo, Wenjing

    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 degrees C) for 2, 4, 8, or 12h. Western blot analyses showed that these cells expressed decreased fission-related protein Drp1 and increased fusion-related protein Mfn2 at 4h; 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.

  8. Laser fusion of mouse embryonic cells and intra-embryonic fusion of blastomeres without affecting the embryo integrity.

    PubMed

    Krivokharchenko, Alexander; Karmenyan, Artashes; Sarkisov, Oleg; Bader, Michael; Chiou, Arthur; Shakhbazyan, Avetik

    2012-01-01

    Manipulation with early mammalian embryos is the one of the most important approach to study preimplantation development. Artificial cell fusion is a research tool for various biotechnological experiments. However, the existing methods have various disadvantages, first of them impossibility to fuse selected cells within multicellular structures like mammalian preimplantation embryos. In our experiments we have successfully used high repetition rate picosecond near infrared laser beam for fusion of pairs of oocytes and oocytes with blastomeres. Fused cells looked morphologically normal and keep their ability for further divisions in vitro. We also fused two or three blastomeres inside four-cell mouse embryos. The presence of one, two or three nuclei in different blastomeres of the same early preimplantation mouse embryo was confirmed under UV-light after staining of DNA with the vital dye Hoechst-33342. The most of established embryos demonstrated high viability and developed in vitro to the blastocyst stage. We demonstrated for the first time the use of laser beam for the fusion of various embryonic cells of different size and of two or three blastomeres inside of four-cell mouse embryos without affecting the embryo's integrity and viability. These embryos with blastomeres of various ploidy maybe unique model for numerous purposes. Thus, we propose laser optical manipulation as a new tool for investigation of fundamental mechanisms of mammalian development.

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

    SciTech Connect

    Filone, Claire Marie; Heise, Mark; Doms, Robert W. . E-mail: doms@mail.med.upenn.edu; Bertolotti-Ciarlet, Andrea . E-mail: aciarlet@mail.med.upenn.edu

    2006-12-20

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

  10. Design of Recombinant Stem Cell Factor macrophage Colony Stimulating Factor Fusion Proteins and their Biological Activity In Vitro

    NASA Astrophysics Data System (ADS)

    Chen, Tao; Yang, Jie; Wang, Yuelang; Zhan, Chenyang; Zang, Yuhui; Qin, Junchuan

    2005-05-01

    Stem cell factor (SCF) and macrophage colony stimulating factor (M-CSF) can act in synergistic way to promote the growth of mononuclear phagocytes. SCF-M-CSF fusion proteins were designed on the computer using the Homology and Biopolymer modules of the software packages InsightII. Several existing crystal structures were used as templates to generate models of the complexes of receptor with fusion protein. The structure rationality of the fusion protein incorporated a series of flexible linker peptide was analyzed on InsightII system. Then, a suitable peptide GGGGSGGGGSGG was chosen for the fusion protein. Two recombinant SCF-M-CSF fusion proteins were generated by construction of a plasmid in which the coding regions of human SCF (1-165aa) and M-CSF (1-149aa) cDNA were connected by this linker peptide coding sequence followed by subsequent expression in insect cell. The results of Western blot and activity analysis showed that these two recombinant fusion proteins existed as a dimer with a molecular weight of 84 KD under non-reducing conditions and a monomer of 42 KD at reducing condition. The results of cell proliferation assays showed that each fusion protein induced a dose-dependent proliferative response. At equimolar concentration, SCF/M-CSF was about 20 times more potent than the standard monomeric SCF in stimulating TF-1 cell line growth, while M-CSF/SCF was 10 times of monomeric SCF. No activity difference of M-CSF/SCF or SCF/M-CSF to M-CSF (at same molar) was found in stimulating the HL-60 cell linear growth. The synergistic effect of SCF and M-CSF moieties in the fusion proteins was demonstrated by the result of clonogenic assay performed with human bone mononuclear, in which both SCF/M-CSF and M-CSF/SCF induced much higher number of CFU-M than equimolar amount of SCF or M-CSF or that of two cytokines mixture.

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

    PubMed

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

    2016-10-18

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

  12. Expression and activity analysis of a new fusion protein targeting ovarian cancer cells.

    PubMed

    Su, Manman; Chang, Weiqin; Wang, Dingding; Cui, Manhua; Lin, Yang; Wu, Shuying; Xu, Tianmin

    2015-09-01

    The aim of the present study was to develop a new therapeutic drug to improve the prognosis of ovarian cancer patients. Human urokinase-type plasminogen activator (uPA)17-34-kunitz-type protease inhibitor (KPI) eukaryotic expression vector was constructed and recombinant human uPA17-34-KPI (rhuPA17-34-KPI) in P. pastoris was expressed. In the present study, the DNA sequences that encode uPA 17-34 amino acids were created according to the native amino acids sequence and inserted into the KPI-pPICZαC vector, which was constructed. Then, uPA17‑34-KPI-pPICZαC was transformed into P. pastoris X-33, and rhuPA17-34-KPI was expressed by induction of methanol. The bioactivities of a recombinant fusion protein were detected with trypsin inhibition analysis, and the inhibitory effects on the growth of ovarian cancer cells were identified using the TUNEL assay, in vitro wound‑healing assay and Matrigel model analysis. The results of the DNA sequence analysis of the recombinant vector uPA17-34-KPI‑pPICZα demonstrated that the DNA‑encoding human uPA 17-34 amino acids, 285-288 amino acids of amyloid precursor protein (APP) and 1-57 amino acids of KPI were correctly inserted into the pPICZαC vector. Following induction by methonal, the fusion protein with a molecular weight of 8.8 kDa was observed using SDS-PAGE and western blot analysis. RhuPA17-34-KPI was expressed in P. pastoris with a yield of 50 mg/l in a 50-ml tube. The recombinant fusion protein was able to inhibit the activity of trypsin, inhibit growth and induce apoptosis of SKOV3 cells, and inhibit the invasion and metastasis of ovarian cancer cells. By considering uPA17-34 amino acid specific binding uPAR as the targeted part of fusion protein and utilizing the serine protease inhibitor activity of KPI, it was found that the recombinant fusion protein uPA17-34-KPI inhibited the invasion and metastasis of ovarian tumors, and may therefore be regarded as effective in targeted treatment.

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

    DTIC Science & Technology

    2015-06-01

    stem cells , which are one of the main fusion partners of cancer cells , invade vertically into the assay we are developing when under the influence of...successfully deleted Histone H3.1 coupling from one half of the BiFC probe set. With this approach proliferation and cell viability long term were... viability and proliferative capacity of all cell types of interest for this proposal (previous report)   4   • Indication of spontaneous fusion between

  14. Analysis of polyethylene glycol (PEG) fusion in cultured neuroblastoma cells via flow cytometry: Techniques & optimization.

    PubMed

    Hoffman, Ashley N; Bamba, Ravinder; Pollins, Alonda C; Thayer, Wesley P

    2017-02-01

    Polyethylene glycol (PEG) has long been used as a membrane fusogen, but recently it has been adopted as a technique for peripheral nerve repair. Vertebrate models using PEG fusion have shown improved outcomes when PEG is applied during repair of severed peripheral nerves. The cellular mechanism of PEG fusion in the peripheral nerve repair model has not previously been assessed via flow cytometry. PEG fusion was assessed in this experiment by dying B35 rat neuroblastoma cells with different color fluorescent labels. The different color cells were combined and PEG was applied in concentrations of 50%, 75% and 100%. The amount of cell fusion was assessed via flow cytometry as the percentage of double positive cells. Results showed increasing fusion and decreasing viability with increasing concentrations of PEG.

  15. Development of a Lentivirus Vector-Based Assay for Non-Destructive Monitoring of Cell Fusion Activity

    PubMed Central

    Neshati, Zeinab; Liu, Jia; Zhou, Guangqian; Schalij, Martin J.; de Vries, Antoine A. F.

    2014-01-01

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

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

    PubMed

    Neshati, Zeinab; Liu, Jia; Zhou, Guangqian; Schalij, Martin J; de Vries, Antoine A F

    2014-01-01

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

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

    PubMed

    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

  18. Flagellar membrane fusion and protein exchange in trypanosomes; a new form of cell-cell communication?

    PubMed Central

    Imhof, Simon; Fragoso, Cristina; Hemphill, Andrew; von Schubert, Conrad; Li, Dong; Legant, Wesley; Betzig, Eric; Roditi, Isabel

    2016-01-01

    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. PMID:27239276

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

    SciTech Connect

    Rowe, Cynthia L.; Connolly, Sarah A.; Chen, Jia; Jardetzky, Theodore S.; Longnecker, Richard

    2013-02-05

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

  20. Type II integral membrane protein, TM of J paramyxovirus promotes cell-to-cell fusion.

    PubMed

    Li, Zhuo; Hung, Cher; Paterson, Reay G; Michel, Frank; Fuentes, Sandra; Place, Ryan; Lin, Yuan; Hogan, Robert J; Lamb, Robert A; He, Biao

    2015-10-06

    Paramyxoviruses include many important animal and human pathogens. Most paramyxoviruses have two integral membrane proteins: fusion protein (F) and attachment proteins hemagglutinin, hemagglutinin-neuraminidase, or glycoprotein (G), which are critical for viral entry into cells. J paramyxovirus (JPV) encodes four integral membrane proteins: F, G, SH, and transmembrane (TM). The function of TM is not known. In this work, we have generated a viable JPV lacking TM (JPV∆TM). JPV∆TM formed opaque plaques compared with JPV. Quantitative syncytia assays showed that JPV∆TM was defective in promoting cell-to-cell fusion (i.e., syncytia formation) compared with JPV. Furthermore, cells separately expressing F, G, TM, or F plus G did not form syncytia whereas cells expressing F plus TM formed some syncytia. However, syncytia formation was much greater with coexpression of F, G, and TM. Biochemical analysis indicates that F, G, and TM interact with each other. A small hydrophobic region in the TM ectodomain from amino acid residues 118 to 132, the hydrophobic loop (HL), was important for syncytial promotion, suggesting that the TM HL region plays a critical role in cell-to-cell fusion.

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

    PubMed

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

    2012-08-01

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

  2. The Influenza Hemagglutinin Fusion Domain Is an Amphipathic Helical Hairpin That Functions by Inducing Membrane Curvature*

    PubMed Central

    Smrt, Sean T.; Draney, Adrian W.; Lorieau, Justin L.

    2015-01-01

    The highly conserved N-terminal 23 residues of the hemagglutinin glycoprotein, known as the fusion peptide domain (HAfp23), is vital to the membrane fusion and infection mechanism of the influenza virus. HAfp23 has a helical hairpin structure consisting of two tightly packed amphiphilic helices that rest on the membrane surface. We demonstrate that HAfp23 is a new class of amphipathic helix that functions by leveraging the negative curvature induced by two tightly packed helices on membranes. The helical hairpin structure has an inverted wedge shape characteristic of negative curvature lipids, with a bulky hydrophobic region and a relatively small hydrophilic head region. The F3G mutation reduces this inverted wedge shape by reducing the volume of its hydrophobic base. We show that despite maintaining identical backbone structures and dynamics as the wild type HAfp23, the F3G mutant has an attenuated fusion activity that is correlated to its reduced ability to induce negative membrane curvature. The inverted wedge shape of HAfp23 is likely to play a crucial role in the initial stages of membrane fusion by stabilizing negative curvature in the fusion stalk. PMID:25398882

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

    SciTech Connect

    Robinson, Claire; Kolb, Andreas F.

    2009-02-01

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

  4. Discriminating in vitro cell fusion from cell aggregation by flow cytometry combined with fluorescence resonance energy transfer.

    PubMed

    Huerta, Leonor; López-Balderas, Nayali; Larralde, Carlos; Lamoyi, Edmundo

    2006-12-01

    Expression of fusion proteins in the plasma membrane enables cells to bind and fuse with surrounding cells to form syncytia. Cell fusion can have important functional outcomes for the interacting cells, as syncytia formation does in AIDS pathogenesis. Studies on cell fusion would be facilitated by a quantitative method able to discriminate between cellular aggregates and bona fide fused cells in a cell population. Flow cytometry with fluorescence resonance energy transfer is applied here for analyzing fusion of HIV-1 envelope-expressing cells with CD4+ Jurkat cells. Fusion partners were labeled with the vital lipophilic fluorescent probes DiO (green) and DiI (red) and FRET is manifested by an enhancement of the DiI red fluorescence intensity in double fluorescent cells, thus allowing discrimination between fused and aggregated cells. The inhibitory effect of anti-CD4 monoclonal antibodies and the inhibitory peptide T-20 upon cell fusion were readily quantified by this technique. This method allows the distinction of fused and aggregated cells even when they are at low frequencies.

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

    PubMed

    Liao, Jen-Chung

    2016-07-05

    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.

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

    PubMed

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

    2011-02-15

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

  7. Spine fusion using cell matrix composites enriched in bone marrow-derived cells.

    PubMed

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

    2003-02-01

    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 fusion model and cancellous bone matrix was used to compare an enriched cellular composite bone graft alone, bone matrix plus bone marrow clot, and an enriched bone matrix composite graft plus bone marrow clot. Union score, quantitative computed tomography, and mechanical testing were used to define outcome. The union score for the enriched bone matrix plus bone marrow clot composite was superior to the enriched bone matrix alone and the bone matrix plus bone marrow clot. The enriched bone matrix plus bone marrow clot composite also was superior to the enriched bone matrix alone in fusion volume and in fusion area. These data confirm that the addition of a bone marrow clot to an enriched cell-matrix composite graft results in significant improvement in graft performance. Enriched composite grafts prepared using this strategy provide a rapid, simple, safe, and inexpensive method for intraoperative concentration and delivery of bone marrow-derived cells and connective tissue progenitors that may improve the outcome of bone grafting.

  8. Fusion reactions in collisions induced by Li isotopes on Sn targets

    SciTech Connect

    Fisichella, M.; Shotter, A. C.; Di Pietro, A.; Figuera, P.; Lattuada, M.; Marchetta, C.; Musumarra, A.; Pellegriti, M. G.; Ruiz, C.; Scuderi, V.; Strano, E.; Torresi, D.; Zadro, M.

    2012-10-20

    Fusion cross sections for the {sup 6}Li+{sup 120}Sn and {sup 7}Li+{sup 119}Sn systems have been measured. We aim to search for possible effects due to the different neutron transfer Q-values, by comparing the fusion cross sections for the two systems below the barrier. This experiment is the first step of a wider systematic aiming to study the above problems in collisions induced by stable and unstable Li isotopes on tin all forming the same compound nucleus.

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

    SciTech Connect

    Petit, Chad M.; Chouljenko, Vladimir N.; Iyer, Arun; Colgrove, Robin; Farzan, Michael; Knipe, David M.; Kousoulas, K.G. . E-mail: vtgusk@lsu.edu

    2007-04-10

    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 {sup 3}H-palmitic acid in wild-type S molecules. S glycoprotein palmitoylation was significantly reduced for mutant glycoproteins having cluster I and II cysteine changes, but was largely unaffected for cysteine cluster III and IV mutants. These results show that the S cytoplasmic domain is palmitoylated and that palmitoylation of the membrane proximal cysteine clusters I and II may be important for S-mediated cell fusion.

  10. The temperature arrested intermediate of virus-cell fusion is a functional step in HIV infection.

    PubMed

    Henderson, Hamani I; Hope, Thomas J

    2006-05-25

    HIV entry occurs via membrane-mediated fusion of virus and target cells. Interactions between gp120 and cellular co-receptors lead to both the formation of fusion pores and release of the HIV genome into target cells. Studies using cell-cell fusion assays have demonstrated that a temperature-arrested state (TAS) can generate a stable intermediate in fusion related events. Other studies with MLV pseudotyped with HIV envelope also found that a temperature sensitive intermediate could be generated as revealed by the loss of a fluorescently labeled membrane. However, such an intermediate has never been analyzed in the context of virus infection. Therefore, we used virus-cell infection with replication competent HIV to gain insights into virus-cell fusion. We find that the TAS is an intermediate in the process culminating in the HIV infection of a target cell. In the virion-cell TAS, CD4 has been engaged, the heptad repeats of gp41 are exposed and the complex is kinetically predisposed to interact with coreceptor to complete the fusion event leading to infection.

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

    PubMed Central

    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

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

    PubMed Central

    2014-01-01

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

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

    SciTech Connect

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

    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

  14. HIV cell-to-cell transmission requires the production of infectious virus particles and does not proceed through env-mediated fusion pores.

    PubMed

    Monel, Blandine; Beaumont, Elodie; Vendrame, Daniela; Schwartz, Olivier; Brand, Denys; Mammano, Fabrizio

    2012-04-01

    Direct cell-to-cell transmission of human immunodeficiency virus (HIV) is a more potent and efficient means of virus propagation than infection by cell-free virus particles. The aim of this study was to determine whether cell-to-cell transmission requires the assembly of enveloped virus particles or whether nucleic acids with replication potential could translocate directly from donor to target cells through envelope glycoprotein (Env)-induced fusion pores. To this end, we characterized the transmission properties of viruses carrying mutations in the matrix protein (MA) that affect the incorporation of Env into virus particles but do not interfere with Env-mediated cell-cell fusion. By use of cell-free virus, the infectivity of MA mutant viruses was below the detection threshold both in single-cycle and in multiple-cycle assays. Truncation of the cytoplasmic tail (CT) of Env restored the incorporation of Env into MA mutant viruses and rescued their cell-free infectivity to different extents. In cell-to-cell transmission assays, MA mutations prevented HIV transmission from donor to target cells, despite efficient Env-dependent membrane fusion. HIV transmission was blocked at the level of virus core translocation into the cytosol of target cells. As in cell-free assays, rescue of Env incorporation by truncation of the Env CT restored the virus core translocation and cell-to-cell infectivity of MA mutant viruses. These data show that HIV cell-to-cell transmission requires the assembly of enveloped virus particles. The increased efficiency of this infection route may thus be attributed to the high local concentrations of virus particles at sites of cellular contacts rather than to a qualitatively different transmission process.

  15. Tumor necrosis factor-{alpha} enhanced fusions between oral squamous cell carcinoma cells and endothelial cells via VCAM-1/VLA-4 pathway

    SciTech Connect

    Song, Kai; Zhu, Fei; Zhang, Han-zhong; Shang, Zheng-jun

    2012-08-15

    Fusion between cancer cells and host cells, including endothelial cells, may strongly modulate the biological behavior of tumors. However, no one is sure about the driving factors and underlying mechanism involved in such fusion. We hypothesized in this study that inflammation, one of the main characteristics in tumor microenvironment, serves as a prominent catalyst for fusion events. Our results showed that oral cancer cells can fuse spontaneously with endothelial cells in co-culture and inflammatory cytokine tumor necrosis factor-{alpha} (TNF-{alpha}) increased fusion of human umbilical vein endothelium cells and oral cancer cells by up to 3-fold in vitro. Additionally, human oral squamous cell carcinoma cell lines and 35 out of 50 (70%) oral squamous carcinoma specimens express VLA-4, an integrin, previously implicated in fusions between human peripheral blood CD34-positive cells and murine cardiomyocytes. Expression of VCAM-1, a ligand for VLA-4, was evident on vascular endothelium of oral squamous cell carcinoma. Moreover, immunocytochemistry and flow cytometry analysis revealed that expression of VCAM-1 increased obviously in TNF-{alpha}-stimulated endothelial cells. Anti-VLA-4 or anti-VCAM-1 treatment can decrease significantly cancer-endothelial adhesion and block such fusion. Collectively, our results suggested that TNF-{alpha} could enhance cancer-endothelial cell adhesion and fusion through VCAM-1/VLA-4 pathway. This study provides insights into regulatory mechanism of cancer-endothelial cell fusion, and has important implications for the development of novel therapeutic strategies for prevention of metastasis. -- Highlights: Black-Right-Pointing-Pointer Spontaneous oral cancer-endothelial cell fusion. Black-Right-Pointing-Pointer TNF-{alpha} enhanced cell fusions. Black-Right-Pointing-Pointer VCAM-1/VLA-4 expressed in oral cancer. Black-Right-Pointing-Pointer TNF-{alpha} increased expression of VCAM-1 on endothelial cells. Black

  16. United we stand: Adhesion and molecular mechanisms driving cell fusion across species.

    PubMed

    Zito, Francesca; Lampiasi, Nadia; Kireev, Igor; Russo, Roberta

    2016-12-01

    Cell-cell fusion is a physiological process playing an essential role for fertilization, shaping organs, tissue repair and immune defense in multicellular organisms. Recent research in the field aims to understand why two or more cells fuse each other and to decipher the general mechanisms regulating this process. Few basic and general steps can be identified, i.e. migration, adhesion and fusion, which are common to different types of cells. As pre-fused and fused cells undergo dramatic changes in their ultrastructure and behavior, the coordinated action of multiple factors is required, including adhesion molecules, cell surface receptors, intracellular kinases, transcription factors, and miRNAs. Although a number of reviews on cell-cell fusion have been published over the years, comprehensive reviews that broadly summarize this process including extracellular and intracellular cues are lacking. For example, a link between cell fusion and adhesive molecules and/or miRNAs has rarely been highlighted in the recent literature. In this review, we will summarize some molecular mechanisms controlling the process of somatic cell-cell fusion during embryonic development. We will specially focus on adhesive molecules, ECM components and miRNAs, providing a summary of important findings on their role in mediating this process in few model systems, in vertebrate and invertebrate organisms.

  17. The EFF-1A Cytoplasmic Domain Influences Hypodermal Cell Fusions in C. elegans But Is Not Dependent on 14-3-3 Proteins

    PubMed Central

    Shinn-Thomas, Jessica H.; del Campo, Jacob J.; Wang, Jianjun; Mohler, William A.

    2016-01-01

    Background Regulatory and biophysical mechanisms of cell-cell fusion are largely unknown despite the fundamental requirement for fused cells in eukaryotic development. Only two cellular fusogens that are not of clear recent viral origin have been identified to date, both in nematodes. One of these, EFF-1, is necessary for most cell fusions in Caenorhabditis elegans. Unregulated EFF-1 expression causes lethality due to ectopic fusion between cells not developmentally programmed to fuse, highlighting the necessity of tight fusogen regulation for proper development. Identifying factors that regulate EFF-1 and its paralog AFF-1 could lead to discovery of molecular mechanisms that control cell fusion upstream of the action of a membrane fusogen. Bioinformatic analysis of the EFF-1A isoform’s predicted cytoplasmic domain (endodomain) previously revealed two motifs that have high probabilities of interacting with 14-3-3 proteins when phosphorylated. Mutation of predicted phosphorylation sites within these motifs caused measurable loss of eff-1 gene function in cell fusion in vivo. Moreover, a human 14-3-3 isoform bound to EFF-1::GFP in vitro. We hypothesized that the two 14-3-3 proteins in C. elegans, PAR-5 and FTT-2, may regulate either localization or fusion-inducing activity of EFF-1. Methodology/Principal Findings Timing of fusion events was slightly but significantly delayed in animals unable to produce full-length EFF-1A. Yet, mutagenesis and live imaging showed that phosphoserines in putative 14-3-3 binding sites are not essential for EFF-1::GFP accumulation at the membrane contact between fusion partner cells. Moreover, although the EFF-1A endodomain was required for normal rates of eff-1-dependent epidermal cell fusions, reduced levels of FTT-2 and PAR-5 did not visibly affect the function of wild-type EFF-1 in the hypodermis. Conclusions/Significance Deletion of the EFF-1A endodomain noticeably affects the timing of hypodermal cell fusions in vivo. However

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

    SciTech Connect

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

    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.

  19. Modulation of non steroidal anti-inflammatory drug induced membrane fusion by copper coordination of these drugs: anchoring effect.

    PubMed

    Majumdar, Anupa; Chakraborty, Sreeja; Sarkar, Munna

    2014-12-04

    Membrane fusion, an integral event in several biological processes, is characterized by several intermediate steps guided by specific energy barriers. Hence, it requires the aid of fusogens to complete the process. Common fusogens, such as proteins/peptides, have the ability to overcome theses barriers by their conformational reorganization, an advantage not shared by small drug molecules. Hence, drug induced fusion at physiologically relevant drug concentrations is rare and occurs only in the case of the oxicam group of non steroidal anti-inflammatory drugs (NSAIDs). To use drugs to induce and control membrane fusion in various biochemical processes requires the understanding of how different parameters modulate fusion. Also, fusion efficacy needs to be enhanced. Here we have synthesized and used Cu(II) complexes of fusogenic oxicam NSAIDs, Meloxicam and Piroxicam, to induce fusion in model membranes monitored by using DSC, TEM, steady-state, and time-resolved spectroscopy. The ability of the complexes to anchor apposing model membranes to initiate/facilitate fusion has been demonstrated. This results in better fusion efficacy compared to the bare drugs. These complexes can take the fusion to its final step. Unlike other designed membrane anchors, the role of molecular recognition and strength of interaction between molecular partners is obliterated for these preformed Cu(II)-NSAIDs.

  20. Use of RecA fusion proteins to induce genomic modifications in zebrafish

    PubMed Central

    Liao, Hsin-Kai; Essner, Jeffrey J.

    2011-01-01

    The bacterial recombinase RecA forms a nucleic acid-protein filament on single-stranded (ss) DNA during the repair of double-strand breaks (DSBs) that efficiently undergoes a homology search and engages in pairing with the complementary DNA sequence. We utilized the pairing activity of RecA–DNA filaments to tether biochemical activities to specific chromosomal sites. Different filaments with chimeric RecA proteins were tested for the ability to induce loss of heterozygosity at the golden locus in zebrafish after injection at the one-cell stage. A fusion protein between RecA containing a nuclear localization signal (NLS) and the DNA-binding domain of Gal4 (NLS-RecA-Gal4) displayed the most activity. Our results demonstrate that complementary ssDNA filaments as short as 60 nucleotides coated with NLS-RecA-Gal4 protein are able to cause loss of heterozygosity in ∼3% of the injected embryos. We demonstrate that lesions in ∼9% of the F0 zebrafish are transmitted to subsequent generations as large chromosomal deletions. Co-injection of linear DNA with the NLS-RecA-Gal4 DNA filaments promotes the insertion of the DNA into targeted genomic locations. Our data support a model whereby NLS-RecA-Gal4 DNA filaments bind to complementary target sites on chromatin and stall DNA replication forks, resulting in a DNA DSB. PMID:21266475

  1. Use of RecA fusion proteins to induce genomic modifications in zebrafish.

    PubMed

    Liao, Hsin-Kai; Essner, Jeffrey J

    2011-05-01

    The bacterial recombinase RecA forms a nucleic acid-protein filament on single-stranded (ss) DNA during the repair of double-strand breaks (DSBs) that efficiently undergoes a homology search and engages in pairing with the complementary DNA sequence. We utilized the pairing activity of RecA-DNA filaments to tether biochemical activities to specific chromosomal sites. Different filaments with chimeric RecA proteins were tested for the ability to induce loss of heterozygosity at the golden locus in zebrafish after injection at the one-cell stage. A fusion protein between RecA containing a nuclear localization signal (NLS) and the DNA-binding domain of Gal4 (NLS-RecA-Gal4) displayed the most activity. Our results demonstrate that complementary ssDNA filaments as short as 60 nucleotides coated with NLS-RecA-Gal4 protein are able to cause loss of heterozygosity in ∼3% of the injected embryos. We demonstrate that lesions in ∼9% of the F0 zebrafish are transmitted to subsequent generations as large chromosomal deletions. Co-injection of linear DNA with the NLS-RecA-Gal4 DNA filaments promotes the insertion of the DNA into targeted genomic locations. Our data support a model whereby NLS-RecA-Gal4 DNA filaments bind to complementary target sites on chromatin and stall DNA replication forks, resulting in a DNA DSB.

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

    NASA Astrophysics Data System (ADS)

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

    2000-06-01

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

  3. Increased proliferation and chemosensitivity of human mesenchymal stromal cells expressing fusion yeast cytosine deaminase.

    PubMed

    Kucerova, Lucia; Poturnajova, Martina; Tyciakova, Silvia; Matuskova, Miroslava

    2012-03-01

    Mesenchymal stromal cells (MSCs) are considered to be suitable vehicles for cellular therapy in various conditions. The expression of reporter and/or effector protein(s) enabled both the identification of MSCs within the organism and the exploitation in targeted tumor therapies. The aim of this study was to evaluate cellular changes induced by retrovirus-mediated transgene expression in MSCs in vitro. Human Adipose Tissue-derived MSCs (AT-MSCs) were transduced to express (i) the enhanced green fluorescent protein (EGFP) reporter transgene, (ii) the fusion yeast cytosine deaminase::uracil phosphoribosyltransferase (CDy::UPRT) enzyme along with the expression of dominant positive selection gene NeoR or (iii) the selection marker NeoR alone (MOCK). CDy::UPRT expression resulted in increased proliferation of CDy::UPRT-MSCs versus naïve AT-MSCs, MOCK-MSCs or EGFP-MSCs. Furthermore, CDy::UPRT-MSCs were significantly more sensitive to 5-fluorouracil (5FU), cisplatin, cyclophosphamide and cytosine arabinoside as determined by increased Caspase 3/7 activation and/or decreased relative proliferation. CDy::UPRT-MSCs in direct cocultures with breast cancer cells MDA-MB-231 increased tumor cell killing induced by low concentrations of 5FU. Our data demonstrated the changes in proliferation and chemoresistance in engineered MSCs expressing transgene with enzymatic function and suggested the possibilities for further augmentation of targeted MSC-mediated antitumor therapy.

  4. Different host cell proteases activate the SARS-coronavirus spike-protein for cell-cell and virus-cell fusion

    SciTech Connect

    Simmons, Graham; Bertram, Stephanie; Glowacka, Ilona; Steffen, Imke; Chaipan, Chawaree; Agudelo, Juliet; Lu Kai; Rennekamp, Andrew J.; Hofmann, Heike; Bates, Paul; Poehlmann, Stefan

    2011-05-10

    Severe acute respiratory syndrome coronavirus (SARS-CoV) poses a considerable threat to human health. Activation of the viral spike (S)-protein by host cell proteases is essential for viral infectivity. However, the cleavage sites in SARS-S and the protease(s) activating SARS-S are incompletely defined. We found that R667 was dispensable for SARS-S-driven virus-cell fusion and for SARS-S-activation by trypsin and cathepsin L in a virus-virus fusion assay. Mutation T760R, which optimizes the minimal furin consensus motif 758-RXXR-762, and furin overexpression augmented SARS-S activity, but did not result in detectable SARS-S cleavage. Finally, SARS-S-driven cell-cell fusion was independent of cathepsin L, a protease essential for virus-cell fusion. Instead, a so far unknown leupeptin-sensitive host cell protease activated cellular SARS-S for fusion with target cells expressing high levels of ACE2. Thus, different host cell proteases activate SARS-S for virus-cell and cell-cell fusion and SARS-S cleavage at R667 and 758-RXXR-762 can be dispensable for SARS-S activation.

  5. Selective retention of bone marrow-derived cells to enhance spinal fusion.

    PubMed

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

    2005-03-01

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

  6. Transformation by myc prevents fusion but not biochemical differentiation of C2C12 myoblasts: mechanisms of phenotypic correction in mixed culture with normal cells

    PubMed Central

    1994-01-01

    To study the effects of myc oncogene on muscle differentiation, we infected the murine skeletal muscle cell line C2C12 with retroviral vectors encoding various forms of avian c- or v-myc oncogene. myc expression induced cell transformation but, unlike many other oncogenes, prevented neither biochemical differentiation, nor commitment (irreversible withdrawal from the cell cycle). Yet, myotube formation by fusion of differentiated cells was strongly inhibited. Comparison of uninfected C2C12 myotubes with differentiated myc- expressing C2C12 did not reveal consistent differences in the expression of several muscle regulatory or structural genes. The present results lead us to conclude that transformation by myc is compatible with differentiation in C2C12 cells. myc expression induced cell death under growth restricting conditions. Differentiated cells escaped cell death despite continuing expression of myc, suggesting that the muscle differentiation programme interferes with the mechanism of myc-induced cell death. Cocultivation of v-myc-transformed C2C12 cells with normal fibroblasts or myoblasts restored fusion competence and revealed two distinguishable mechanisms that lead to correction of the fusion defect. PMID:8195295

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

    DTIC Science & Technology

    2012-07-01

    optimized electroporation conditions for T47D and human mesenchymal stem cell populations. As a result we have been able to conduct our first co...using T47D cells and human mesenchymal stem cells (hMSCs). Electroporation conditions for hMSCs had been previously optimized in the lab. Each cell ...can allow it to acquire the ability to proliferate inappropriately. Fusion of a tumor cell with a mesenchymal stem cell can allow it to degrade

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

    SciTech Connect

    McBride, Corrin E.; Machamer, Carolyn E.

    2010-09-15

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

  9. Mechanism of reduction of virus release and cell-cell fusion in persistent canine distemper virus infection.

    PubMed

    Meertens, Nadine; Stoffel, Michael H; Cherpillod, Pascal; Wittek, Riccardo; Vandevelde, Marc; Zurbriggen, Andreas

    2003-10-01

    Canine distemper virus (CDV), a mobillivirus related to measles virus causes a chronic progressive demyelinating disease, associated with persistence of the virus in the central nervous system (CNS). CNS persistence of morbilliviruses has been associated with cell-to-cell spread, thereby limiting immune detection. The mechanism of cell-to-cell spread remains uncertain. In the present study we studied viral spread comparing a cytolytic (non-persistent) and a persistent CDV strain in cell cultures. Cytolytic CDV spread in a compact concentric manner with extensive cell fusion and destruction of the monolayer. Persistent CDV exhibited a heterogeneous cell-to-cell pattern of spread without cell fusion and 100-fold reduction of infectious viral titers in supernatants as compared to the cytolytic strain. Ultrastructurally, low infectious titers correlated with limited budding of persistent CDV as compared to the cytolytic strain, which shed large numbers of viral particles. The pattern of heterogeneous cell-to-cell viral spread can be explained by low production of infectious viral particles in only few areas of the cell membrane. In this way persistent CDV only spreads to a small proportion of the cells surrounding an infected one. Our studies suggest that both cell-to-cell spread and limited production of infectious virus are related to reduced expression of fusogenic complexes in the cell membrane. Such complexes consist of a synergistic configuration of the attachment (H) and fusion (F) proteins on the cell surface. F und H proteins exhibited a marked degree of colocalization in cytolytic CDV infection but not in persistent CDV as seen by confocal laser microscopy. In addition, analysis of CDV F protein expression using vaccinia constructs of both strains revealed an additional large fraction of uncleaved fusion protein in the persistent strain. This suggests that the paucity of active fusion complexes is due to restricted intracellular processing of the viral fusion

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

    SciTech Connect

    Beck, C.; Keeley, N.

    2008-05-12

    The influence on the fusion process of coupling transfer/breakup channels is investigated for the medium weight {sup 6,7}Li+{sup 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 {sup 6}He+{sup 59}Co induced by the borromean halo nucleus {sup 6}He are also proposed.

  11. Near-barrier Fusion Induced by Stable Weakly Bound and Exotic Halo Light Nuclei

    SciTech Connect

    Beck, C.; Zafra, A. Sanchez I.; Diaz-Torres, A.; Thompson, I. J.; Keeley, N.

    2006-08-14

    The effect of breakup is investigated for the medium weight 6Li+59Co system in the vicinity of the Coulomb barrier. The strong coupling of breakup/transfer channels to fusion is discussed within a comparison of predictions of the Continuum Discretized Coupled-Channels model which is also applied to 6He+59Co a reaction induced by the borromean halo nucleus 6He.

  12. Mitochondrial fusion and fission in cell life and death.

    PubMed

    Westermann, Benedikt

    2010-12-01

    Mitochondria are dynamic organelles that constantly fuse and divide. These processes (collectively termed mitochondrial dynamics) are important for mitochondrial inheritance and for the maintenance of mitochondrial functions. The core components of the evolutionarily conserved fusion and fission machineries have now been identified, and mechanistic studies have revealed the first secrets of the complex processes that govern fusion and fission of a double membrane-bound organelle. Mitochondrial dynamics was recently recognized as an important constituent of cellular quality control. Defects have detrimental consequences on bioenergetic supply and contribute to the pathogenesis of neurodegenerative diseases. These findings open exciting new directions to explore mitochondrial biology.

  13. Homotypic Fusion of Immature Secretory Granules during Maturation in a Cell-free Assay

    PubMed Central

    Urbé, Sylvie; Page, Lesley J.; Tooze, Sharon A.

    1998-01-01

    The biogenesis of secretory granules embodies several morphological and biochemical changes. In particular, in neuroendocrine cells maturation of secretory granules is characterized by an increase in size which has been proposed to reflect homotypic fusion of immature secretory granules (ISGs). Here we describe an assay that provides the first biochemical evidence for such a fusion event and allows us to analyze its regulation. The assay reconstitutes homotypic fusion between one population of ISGs containing a [35S]sulfate-labeled substrate, secretogranin II (SgII), and a second population containing the prohormone convertase PC2. Both substrate and enzyme are targeted exclusively to ISGs. Fusion is measured by quantification of a cleavage product of SgII produced by PC2. With this assay we show that fusion only occurs between ISGs and not between ISGs and MSGs, is temperature dependent, and requires ATP and GTP and cytosolic proteins. NSF (N-ethylmaleimide–sensitive fusion protein) is amongst the cytosolic proteins required, whereas we could not detect a requirement for p97. The ability to reconstitute ISG fusion in a cell-free assay is an important advance towards the identification of molecules involved in the maturation of secretory granules and will increase our understanding of this process. PMID:9864358

  14. Cold fusion, Alchemist's dream

    SciTech Connect

    Clayton, E.D.

    1989-09-01

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

  15. Atomic force microscopy: Unraveling the fundamental principles governing secretion and membrane fusion in cells.

    PubMed

    Jena, Bhanu P

    2009-07-01

    The story of cell secretion and membrane fusion is as old as life itself. Without these fundamental cellular processes known to occur in yeast to humans, life would cease to exist. In the last 15 years, primarily using the atomic force microscope, a detailed understanding of the molecular process and of the molecular machinery and mechanism of secretion and membrane fusion in cells has come to light. This has led to a paradigm shift in our understanding of the underlying mechanism of cell secretion. The journey leading to the discovery of a new cellular structure the 'porosome',-the universal secretory machinery in cells, and the contributions of the AFM in our understanding of the general molecular machinery and mechanism of cell secretion and membrane fusion, is briefly discussed in this article.

  16. Isolation and characterization of Escherichia coli strains containing new gene fusions (soi::lacZ) inducible by superoxide radicals.

    PubMed Central

    Mito, S; Zhang, Q M; Yonei, S

    1993-01-01

    Gene fusions in Escherichia coli that showed increased beta-galactosidase expression in response to treatment with a superoxide radical (O2-) generator, methyl viologen (MV), were obtained. These fusions were constructed by using a Mud(Ap lac) phage to insert the lactose structural genes randomly into the E. coli chromosome. Ampicillin-resistant colonies were screened for increased expression of beta-galactosidase on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) plates containing MV at 1.25 micrograms/ml. Other O2- generators, menadione and plumbagin, also induced beta-galactosidase activity in these fusion strains. The induction by these drugs occurred only under aerobic conditions. Hyperoxygenation also elicited an induction of the fusions. On the other hand, no significant induction was observed with hydrogen peroxide and cumene hydroperoxide. The induction of these fusions by MV was not dependent on the peroxide stress control mediated by the oxyR gene or on the recA-dependent SOS system. These fusions were named soi (superoxide inducible)::lacZ. The induction of beta-galactosidase was significantly reduced by introducing a soxS::Tn10 locus into the fusion strains, indicating that the soi genes are members of the soxRS regulon. Five of the fusions were located in 6 to 26 min of the E. coli genetic map, while three fusions were located in 26 to 36 min, indicating that these fusions are not related to genes already known to be inducible by O2- under the control of soxRS. At least five mutants containing the soi::lacZ fusion were more sensitive to MV and menadione than the wild-type strain, suggesting that the products of these soi genes play an important role in protection against oxidative stress. PMID:8386722

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

    PubMed

    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.

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

    PubMed

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

    2016-07-08

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

  19. Ankle arthrodesis fusion rates for mesenchymal stem cell bone allograft versus proximal tibia autograft.

    PubMed

    Anderson, John J; Boone, Joshua J; Hansen, Myron; Brady, Chad; Gough, Adam; Swayzee, Zflan

    2014-01-01

    Ankle arthrodesis is commonly used in the treatment of ankle arthritis. The present study compared mesenchymal stem cell (MSC) bone allografts and proximal tibia autografts as adjuncts in performing ankle arthrodesis. A total of 109 consecutive ankle fusions performed from 2002 to 2008 were evaluated retrospectively. Of the 109 fusions, 24 were excluded from the present study, leaving 85 patients who had undergone ankle arthrodesis. Of the 85 patients, 41 had received a proximal tibia autograft and 44, an MSC bone allograft. These 2 groups were reviewed and compared retrospectively at least 2 years postoperatively for the overall fusion rate, interval to radiographic fusion, and interval to clinical fusion. A modified and adjusted American College of Foot and Ankle Surgeons ankle scale was used to measure patient satisfaction. The overall fusion rate was 84.1% in the MSC bone allograft group and 95.1% in the proximal tibia autograft group (p = .158). The corresponding mean intervals to radiographic fusion were 13.0 ± 2.5 weeks and 11.3 ± 2.8 weeks (p ≤ .001). The interval to clinical fusion was 13.1 ± 2.1 weeks and 11.0 ± 1.5 weeks (p ≤ .001) in the MSC bone allograft and proximal tibia autograft group, respectively. No statistically significant difference was found in the fusion rates between the MSC bone allograft and proximal tibia autograft groups. Also, no statistically significant difference was found between the preoperative and postoperative scores using a modified and adjusted American College of Foot and Ankle Surgeons ankle scale between the 2 groups (p = .41 and p = .44, respectively). A statistically significant delay to radiographic and clinical fusion was present in the MSC bone allograft group compared with the proximal tibia autograft group; however, no difference was found in patient satisfaction.

  20. Broad target cell selectivity of Kaposi's sarcoma-associated herpesvirus glycoprotein-mediated cell fusion and virion entry

    SciTech Connect

    Kaleeba, Johnan A.R.; Berger, Edward A. . E-mail: edward_berger@nih.gov

    2006-10-10

    The molecular mechanism of Kaposi's sarcoma-associated herpesvirus (KSHV, human herpesvirus 8) entry is poorly understood. We tested a broad variety of cell types of diverse species and tissue origin for their ability to function as targets in a quantitative reporter gene assay for KSHV-glycoprotein-mediated cell fusion. Several human, non-human primate, and rabbit cell lines were efficient targets, whereas rodent and all human lymphoblastoid cell lines were weak targets. Parallel findings were obtained with a virion entry assay using a recombinant KSHV encoding a reporter gene. No correlation was observed between target cell activity and surface expression of {alpha}3{beta}1 integrin, a proposed KSHV receptor. We hypothesize that target cell permissiveness in both the cell fusion and virion entry assays reflects the presence of a putative KSHV fusion-entry receptor.

  1. Cytolethal distending toxin B as a cell-killing component of tumor-targeted anthrax toxin fusion proteins.

    PubMed

    Bachran, C; Hasikova, R; Leysath, C E; Sastalla, I; Zhang, Y; Fattah, R J; Liu, S; Leppla, S H

    2014-01-16

    Cytolethal distending toxin (Cdt) is produced by Gram-negative bacteria of several species. It is composed of three subunits, CdtA, CdtB, and CdtC, with CdtB being the catalytic subunit. We fused CdtB from Haemophilus ducreyi to the N-terminal 255 amino acids of Bacillus anthracis toxin lethal factor (LFn) to design a novel, potentially potent antitumor drug. As a result of this fusion, CdtB was transported into the cytosol of targeted cells via the efficient delivery mechanism of anthrax toxin. The fusion protein efficiently killed various human tumor cell lines by first inducing a complete cell cycle arrest in the G2/M phase, followed by induction of apoptosis. The fusion protein showed very low toxicity in mouse experiments and impressive antitumor effects in a Lewis Lung carcinoma model, with a 90% cure rate. This study demonstrates that efficient drug delivery by a modified anthrax toxin system combined with the enzymatic activity of CdtB has great potential as anticancer treatment and should be considered for the development of novel anticancer drugs.

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

    PubMed Central

    Dhanyasi, Nagaraju; Segal, Dagan; Shimoni, Eyal; Shinder, Vera

    2015-01-01

    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. PMID:26459604

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

    PubMed Central

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

    2016-01-01

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

  4. HES6 reverses nuclear reprogramming of insulin-producing cells following cell fusion

    SciTech Connect

    Ball, Andrew J.; Abrahamsson, Annelie E.; Tyrberg, Bjoern; Itkin-Ansari, Pamela; Levine, Fred; E-mail: flevine@ucsd.edu

    2007-04-06

    To examine the mechanism by which growth-stimulated pancreatic {beta}-cells dedifferentiate, somatic cell fusions were performed between MIN6, a highly differentiated mouse insulinoma, and {beta}lox5, a cell line derived from human {beta}-cells which progressively dedifferentiated in culture. MIN6/{beta}lox5 somatic cells hybrids underwent silencing of insulin expression and a marked decline in PDX1, NeuroD, and MafA, indicating that {beta}lox5 expresses a dominant transacting factor(s) that represses {beta}-cell differentiation. Expression of Hes1, which inhibits endocrine differentiation was higher in hybrid cells than in parental MIN6 cells. Hes6, a repressor of Hes1, was highly expressed in primary {beta}-cells as well as MIN6, but was repressed in hybrids. Hes6 overexpression using a retroviral vector led to a decrease in Hes1 levels, an increase in {beta}-cell transcription factors and partial restoration of insulin expression. We conclude that the balance of Notch activators and inhibitors may play an important role in maintaining the {beta}-cell differentiated state.

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

    PubMed

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

    2015-06-01

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

  6. Regulation of Fusion Pore Closure and Compound Exocytosis in Neuroendocrine PC12 Cells by SCAMP1

    PubMed Central

    Zhang, Jie; Castle, David

    2011-01-01

    During exocytosis, neuroendocrine cells can achieve partial release of stored secretory products from dense core vesicles (DCVs) by coupling endocytosis directly at fusion sites and without full discharge. The physiological role of partial secretion is of substantial interest. Much is known about SNARE-mediated initiation of exocytosis and dynamin-mediated completion of endocytosis, but little is known about coupling events. We have used real-time microscopy to examine the role of secretory carrier membrane protein SCAMP1 in exo-endocytic coupling in PC12 cells. While reduced SCAMP1 expression is known to impede dilation of newly opened fusion pores during onset of DCV exocytosis, we now show that SCAMP1 deficiency also inhibits closure of fusion pores after they have opened. Inhibition causes accumulation of fusion figures at the plasma membrane. Closure is recovered by restoring expression and accelerated slightly by overexpression. Interestingly, inhibited pore closure resulting from loss of SCAMP1 appears to increase secondary fusion of DCVs to already-fused DCVs (compound exocytosis). Unexpectedly, reinternalization of expanded DCV membranes following compound exocytosis appears to proceed normally in SCAMP1-deficient cells. SCAMP1’s apparent dual role in facilitating dilation and closure of fusion pores implicates its function in exo-endocytic coupling and in the regulation of partial secretion. Secondarily, SCAMP1 may serve to limit the extent of compound exocytosis. PMID:21272170

  7. Fusion of Aequorea victoria GFP and aequorin provides their Ca(2+)-induced interaction that results in red shift of GFP absorption and efficient bioluminescence energy transfer.

    PubMed

    Gorokhovatsky, Andrey Yu; Marchenkov, Victor V; Rudenko, Natalia V; Ivashina, Tanya V; Ksenzenko, Vladimir N; Burkhardt, Nils; Semisotnov, Gennady V; Vinokurov, Leonid M; Alakhov, Yuli B

    2004-07-30

    The bioluminescence emitted by Aequorea victoria jellyfish is greenish while its single bioluminescent photoprotein aequorin emits blue light. This phenomenon may be explained by a bioluminescence resonance energy transfer (BRET) from aequorin chromophore to green fluorescent protein (GFP) co-localized with it. However, a slight overlapping of the aequorin bioluminescence spectrum with the GFP absorption spectrum and the absence of marked interaction between these proteins in vitro pose a question on the mechanism providing the efficient BRET in A. victoria. Here we report the in vitro study of BRET between homologous Ca(2+)-activated photoproteins, aequorin or obelin (Obelia longissima), as bioluminescence energy donors, and GFP, as an acceptor. The fusions containing donor and acceptor proteins linked by a 19 aa peptide were purified after expressing their genes in Escherichia coli cells. It was shown that the GFP-aequorin fusion has a significantly greater BRET efficiency, compared to the GFP-obelin fusion. Two main factors responsible for the difference in BRET efficiency of these fusions were revealed. First, it is the presence of Ca(2+)-induced interaction between the donor and acceptor in the aequorin-containing fusion and the absence of the interaction in the obelin-containing fusion. Second, it is a red shift of GFP absorption toward better overlapping with aequorin bioluminescence induced by the interaction of aequorin with GFP. Since the connection of the two proteins in vitro mimics their proximity in vivo, Ca(2+)-induced interaction between aequorin and GFP may occur in A. victoria jellyfish providing efficient BRET in this organism.

  8. Myelin-reactive antibodies mediate the pathology of MBP-PLP fusion protein MP4-induced EAE.

    PubMed

    Kuerten, Stefanie; Pauly, Robert; Rottlaender, Andrea; Rodi, Michael; Gruppe, Traugott L; Addicks, Klaus; Tary-Lehmann, Magdalena; Lehmann, Paul V

    2011-07-01

    Experimental autoimmune encephalomyelitis (EAE) is frequently used for studies of multiple sclerosis (MS). Because in most EAE models T cells mediate the pathology in the absence of B cells/autoantibodies, the notion has evolved that also MS may be a primarily T cell-mediated disease. We have previously introduced MBP-PLP fusion protein (MP4)-induced EAE in C57BL/6 mice. Here we show that the disease in this model is antibody-dependent. Immunization of B cell-deficient mice did not induce EAE. When such B cell-deficient mice were, however, injected with MBP/PLP-specific antibodies in addition to the immunization with MP4, they developed disease of a severity and course that was similar to the wild-type mice. The deposition of antibodies in demyelinated lesions provided further evidence for the contribution of MBP/PLP-specific antibodies to CNS lesion formation. Based upon these data we suggest a two-stage model for the involvement of MBP/PLP-specific antibodies in autoimmune CNS pathology.

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

    PubMed

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

    2016-06-02

    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.

  10. Nanoscale organization of beta2-adrenergic receptor-Venus fusion protein domains on the surface of mammalian cells.

    PubMed

    Vobornik, Dusan; Rouleau, Yanouchka; Haley, Jennifer; Bani-Yaghoub, Mahmud; Taylor, Rod; Johnston, Linda J; Pezacki, John Paul

    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(2)-adrenergic receptors (beta(2)AR) fused to the GFP analogue Venus at the nanoscale on HEK293 cells. The expression of the beta(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(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(2)AR are observed in the membrane of the HEK293 cells that stably overexpress beta(2)AR-GFP and beta(2)AR-eYFP. We conclude that precise chemical control of gene expression is highly advantageous for the use beta(2)AR-Venus fusion proteins as models for beta(2)AR function. These observations are critical for designing future cell models and assays based on beta(2)AR, since the receptor biology is consistent with a relatively low density of nanoscale receptor domains.

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

    SciTech Connect

    Vobornik, Dusan; Rouleau, Yanouchka; Haley, Jennifer; Bani-Yaghoub, Mahmud; Taylor, Rod; Johnston, Linda J.; Pezacki, John Paul

    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.

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

    SciTech Connect

    Holmlid, Leif

    2015-08-15

    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{sup −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 H{sub N}(0) of ultra-dense hydrogen (size of a few pm) escape with a substantial fraction of the energy. Heat loss to the D{sub 2} 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.

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

    DOE PAGES

    Lattuada, D.; Barbarino, M.; Bonasera, A.; ...

    2016-04-19

    In this paper, 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 2H(d,n)3He. The experiment was performed with the Texas Petawatt Laser, which delivered 150–270 fs pulses of energy ranging from 90 to 180 J to D2 or CD4 molecular clusters (where D denotes 2H). 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,more » 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 2H(d,n)3He and 3He(d,p)4He cases discussed here, it is very important to apply the background subtraction for the energetic ions and to measure the fusion yields with high precision. While the available data on both ion distribution and fusion yields allow us to determine with good precision the S factor in the d+d case (lower Gamow energies), for the d+3He case the data are not precise enough to obtain the S factor using this method. Our results agree with other experiments within the experimental error, even though smaller values of the S factor were obtained. This might be due to the plasma environment differing from the beam target conditions in a conventional accelerator experiment.« less

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

    PubMed Central

    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

  15. Henipavirus membrane fusion and viral entry.

    PubMed

    Aguilar, Hector C; Iorio, Ronald M

    2012-01-01

    Nipah (NiV) and Hendra (HeV) viruses cause cell-cell fusion (syncytia) in brain, lung, heart, and kidney tissues, leading to encephalitis, pneumonia, and often death. Membrane fusion is essential to both viral entry and virus-induced cell-cell fusion, a hallmark of henipavirus infections. Elucidiation of the mechanism(s) of membrane fusion is critical to understanding henipavirus pathobiology and has the potential to identify novel strategies for the development of antiviral therapeutic agents. Henipavirus membrane fusion requires the coordinated actions of the viral attachment (G) and fusion (F) glycoproteins. Current henipavirus fusion models posit that attachment of NiV or HeV G to its cell surface receptors releases F from its metastable pre-fusion conformation to mediate membrane fusion. The identification of ephrinB2 and ephrinB3 as henipavirus receptors has paved the way for recent advances in our understanding of henipavirus membrane fusion. These advances highlight mechanistic similarities and differences between membrane fusion for the henipavirus and other genera within the Paramyxoviridae family. Here, we review these mechanisms and the current gaps in our knowledge in the field.

  16. Cell and organ printing 2: fusion of cell aggregates in three-dimensional gels.

    PubMed

    Boland, Thomas; Mironov, Vladimir; Gutowska, Anna; Roth, Elisabeth A; Markwald, Roger R

    2003-06-01

    We recently developed a cell printer (Wilson and Boland, 2003) that enables us to place cells in positions that mimic their respective positions in organs. However, this technology was limited to the printing of two-dimensional (2D) tissue constructs. Here we describe the use of thermosensitive gels to generate sequential layers for cell printing. The ability to drop cells on previously printed successive layers provides a real opportunity for the realization of three-dimensional (3D) organ printing. Organ printing will allow us to print complex 3D organs with computer-controlled, exact placing of different cell types, by a process that can be completed in several minutes. To demonstrate the feasibility of this novel technology, we showed that cell aggregates can be placed in the sequential layers of 3D gels close enough for fusion to occur. We estimated the optimum minimal thickness of the gel that can be reproducibly generated by dropping the liquid at room temperature onto a heated substrate. Then we generated cell aggregates with the corresponding (to the minimal thickness of the gel) size to ensure a direct contact between printed cell aggregates during sequential printing cycles. Finally, we demonstrated that these closely-placed cell aggregates could fuse in two types of thermosensitive 3D gels. Taken together, these data strongly support the feasibility of the proposed novel organ-printing technology.

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

    PubMed

    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.

  18. Characterisation of the role of Vrp1 in cell fusion during the development of visceral muscle of Drosophila melanogaster

    PubMed Central

    2010-01-01

    Background In Drosophila muscle cell fusion takes place both during the formation of the somatic mesoderm and the visceral mesoderm, giving rise to the skeletal muscles and the gut musculature respectively. The core process of myoblast fusion is believed to be similar for both organs. The actin cytoskeleton regulator Verprolin acts by binding to WASP, which in turn binds to the Arp2/3 complex and thus activates actin polymerization. While Verprolin has been shown to be important for somatic muscle cell fusion, the function of this protein in visceral muscle fusion has not been determined. Results Verprolin is specifically expressed in the fusion competent myoblasts of the visceral mesoderm, suggesting a role in visceral mesoderm fusion. We here describe a novel Verprolin mutant allele which displays subtle visceral mesoderm fusion defects in the form of mislocalization of the immunoglobulin superfamily molecule Duf/Kirre, which is required on the myoblast cell surface to facilitate attachment between cells that are about to fuse, indicating a function for Verprolin in visceral mesoderm fusion. We further show that Verprolin mutant cells are capable of both migrating and fusing and that the WASP-binding domain of Verprolin is required for rescue of the Verprolin mutant phenotype. Conclusions Verprolin is expressed in the visceral mesoderm and plays a role in visceral muscle fusion as shown by mislocalization of Duf/Kirre in the Verprolin mutant, however it is not absolutely required for myoblast fusion in either the visceral or the somatic mesoderm. PMID:20701765

  19. Requirement of N-terminal amino acid residues of gp41 for human immunodeficiency virus type 1-mediated cell fusion.

    PubMed Central

    Schaal, H; Klein, M; Gehrmann, P; Adams, O; Scheid, A

    1995-01-01

    An expression vector was designed to test the structural requirements of the gp41 N terminus for human immunodeficiency virus type 1-induced membrane fusion. Mutations in the region coding for the N terminus of gp41 were found to disrupt glycoprotein expression because of deleterious effects on the Rev-responsive element (RRE). Insertion of an additional RRE in the 3'-noncoding sequence of env made possible efficient glycoprotein expression, irrespective of the mutations introduced into the RRE in the natural location. This permitted the insertion of the unique restriction site SpeI within the N-terminal sequences of gp41, allowing convenient and efficient mutation of the gp41 N terminus by using double-stranded synthetic oligonucleotides. Mutants with deletions of 1 to 7 amino acids of the N terminus were constructed. Expression and cleavage of all mutants were confirmed by Western immunoblot analysis with anti-gp41 antibodies. The capability of mutants to induce membrane fusion was monitored following transfection of HeLa-T4+ cell lines with wild-type and mutant expression vectors by electroporation and microinjection. The efficiency of cell-fusing activity decreased drastically with deletion of 3 and 4 amino acids and was completely lost with deletion of 5 amino acids. Cotransfection of the parent and mutant expression vectors resulted in reduced cell-fusing activity. The extent of this dominant interference by mutant glycoprotein paralleled the decrease in cell-fusing activity of the mutants alone. This suggests the existence of a specific N-terminal structure required for fusing activity. However, there does not appear to be a stringent requirement for the precise length of the N terminus. This finding is supported by the length variation of this region among natural human immunodeficiency virus type 1 isolates and is in contrast to the apparent stringency in the length of analogous N-terminal structures of influenza A virus and paramyxovirus fusion

  20. Cell fusion as the formation mechanism of unreduced gametes in the gynogenetic diploid hybrid fish

    PubMed Central

    Wang, Jing; Liu, Qingfeng; Luo, Kaikun; Chen, Xuan; Xiao, Jun; Zhang, Chun; Tao, Min; Zhao, Rurong; Liu, Shaojun

    2016-01-01

    The gynogenetic diploid hybrid clone line (GDH) derived from red crucian carp (♀ RCC) × common carp (♂ CC) possesses the unusual reproductive trait of producing unreduced diploid eggs. To identify the mechanism underlying this phenomenon, we examined the structure, in vivo developmental process and in vitro dynamic development of the GDH gonad. In summary, compared with RCC and CC, GDH showed certain special straits. First, a high frequency (84.7%) of germ cell fusion occurred in gonadal tissue culture in vitro as observed by time-lapse microscopy. Second, microstructural and ultrastructural observation showed numerous binucleated and multinucleated germ cells in the gonad, providing evidence of germ cell fusion in vivo. By contrast, in the diploid RCC and CC ovaries, neither cell fusion nor multinucleated cells were observed during the development of gonads. Third, the ovary of GDH remained at stage I for 10 months, whereas those of RCC and CC remained at that stage for 2 months, indicating that the GDH germ cells underwent abnormal development before meiosis. This report is the first to demonstrate that cell fusion facilitates the formation of unreduced gametes in vertebrates, which is a valuable finding for both evolutionary biology and reproductive biology. PMID:27530321

  1. Fusion of mitochondria in tobacco suspension cultured cells is dependent on the cellular ATP level but not on actin polymerization.

    PubMed

    Wakamatsu, Kairo; Fujimoto, Masaru; Nakazono, Mikio; Arimura, Shin-ichi; Tsutsumi, Nobuhiro

    2010-10-01

    Mitochondria in plant cells undergo fusion and fission frequently. Although the mechanisms and proteins of mitochondrial fusion are well known in yeast and mammalian cells, they remain poorly understood in plant cells. To clarify the physiological requirements for plant mitochondrial fusion, we investigated the fusion frequency of mitochondria in tobacco cultured cells using the photoconvertible fluorescent protein Kaede and some physiological inhibitors. The latter included two uncouplers, 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an inhibitor of mitochondrial ATP synthase, oligomycin, and an actin polymerization inhibitor, latrunculin B (Lat B). The frequency of mitochondrial fusion was clearly reduced by DNP, CCCP and oligomycin, but not by Lat B, although Lat B severely inhibited mitochondrial movement. Moreover, DNP, CCCP and oligomycin evidently lowered the cellular ATP levels. These results indicate that plant mitochondrial fusion depends on the cellular ATP level, but not on actin polymerization.

  2. Electro-acoustic fusion of erythrocytes and of myeloma cells.

    PubMed

    Vienken, J; Zimmermann, U; Zenner, H P; Coakley, W T; Gould, R K

    1985-11-07

    Mammalian cells can be concentrated in a sound field. A method is introduced, which combines the reversible aggregation of cells in a sound field with the electrical breakdown of cell membranes to fuse cells, which are in contact. Human red blood cells and mouse myeloma cells are fused by means of that procedure.

  3. Evaluation of EML4-ALK Fusion Proteins in Non-Small Cell Lung Cancer Using Small Molecule Inhibitors12

    PubMed Central

    Li, Yongjun; Ye, Xiaofen; Liu, Jinfeng; Zha, Jiping; Pei, Lin

    2011-01-01

    The echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion gene resulting from an inversion within chromosome 2p occurs in approximately 5% of non-small cell lung cancer and is mutually exclusive with Ras and EGFR mutations. In this study, we have used a potent and selective ALK small molecule inhibitor, NPV-TAE684, to assess the oncogenic role of EML4-ALK in non-small cell lung cancer (NSCLC). We show here that TAE684 inhibits proliferation and induces cell cycle arrest, apoptosis, and tumor regression in two NSCLC models that harbor EML4-ALK fusions. TAE684 inhibits EML4-ALK activation and its downstream signaling including ERK, AKT, and STAT3. We used microarray analysis to carry out targeted pathway studies of gene expression changes in H2228 NSCLC xenograft model after TAE684 treatment and identified a gene signature of EML4-ALK inhibition. The gene signature represents 1210 known human genes, and the top biologic processes represented by these genes are cell cycle, DNA synthesis, cell proliferation, and cell death. We also compared the effect of TAE684 with PF2341066, a c-Met and ALK small molecule inhibitor currently in clinical trial in cancers harboring ALK fusions, and demonstrated that TAE684 is a much more potent inhibitor of EML4-ALK. Our data demonstrate that EML4-ALK plays an important role in the pathogenesis of a subset of NSCLC and provides insight into the mechanism of EML4-ALK inhibition by a small molecule inhibitor. PMID:21245935

  4. Generation and preclinical characterization of an NKp80-Fc fusion protein for redirected cytolysis of natural killer (NK) cells against leukemia.

    PubMed

    Deng, Gang; Zheng, Xiaodong; Zhou, Jing; Wei, Haiming; Tian, Zhigang; Sun, Rui

    2015-09-11

    The capacity of natural killer (NK) cells to mediate Fc receptor-dependent effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), largely contributes to their clinical application. Given that activation-induced C-type lectin (AICL), an identified ligand for the NK-activating receptor NKp80, is frequently highly expressed on leukemia cells, the lack of therapeutic AICL-specific antibodies limits clinical application. Here we explore a strategy to reinforce NK anti-leukemia reactivity by combining targeting AICL-expressing leukemia cells with the induction of NK cell ADCC using NKp80-Fc fusion proteins. The NKp80-Fc fusion protein we generated bound specifically to leukemia cells in an AICL-specific manner. Cell binding assays between NK and leukemia cells showed that NKp80-Fc significantly increased NK target cell conjugation. In functional analyses, treatment with NKp80-Fc clearly induced the ADCC effect of NK cells. NKp80-Fc not only promoted NK-mediated leukemia cell apoptosis in the early stage of cell conjugation but also enhanced NK cell degranulation and cytotoxicity activity in the late stage. The bifunctional NKp80-Fc could redirect NK cells toward leukemia cells and triggered NK cell killing in vitro. Moreover, NKp80-Fc enhanced the lysis of NK cells against tumors in leukemia xenograft non-obese diabetic/severe combined immunodeficiency mice. Taken together, our results demonstrate that NKp80-Fc potently amplifies NK cell anti-leukemia effects in vitro and in vivo through induction of the NK cell ADCC effect. This method could potentially be useful for molecular targeted therapy, and the fusion proteins may be a promising drug for immunotherapy of leukemia.

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

    NASA Astrophysics Data System (ADS)

    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.

  6. Fusion-derived epithelial cancer cells express hematopoietic markers and contribute to stem cell and migratory phenotype in ovarian carcinoma.

    PubMed

    Ramakrishnan, Mallika; Mathur, Sandeep R; Mukhopadhyay, Asok

    2013-09-01

    For a long time, the external milieu of cancer cells was considered to be of secondary importance when compared with its intrinsic properties. That has changed now as the microenvironment is considered to be a major contributing factor toward the progression of tumor. In this study, we show that in human and mouse epithelial ovarian carcinoma and mouse lung carcinoma, the interaction between tumor-infiltrating hematopoietic cells and epithelial cancer cells results in their fusion. Intriguingly, even after the fusion event, cancer cells retain the expression of the pan-hematopoietic marker (CD45) and various markers of hematopoietic lineage, including those of hematopoietic stem cells, indicating that the hematopoietic genome is not completely reprogrammed. This observation may have implications on the bone marrow contribution to the cancer stem cell population. Interestingly, it was seen that in both cancer models, the expression of chemokine receptor CXCR4 was largely contributed to by the fused compartment of cancer cells. We hypothesize that the superior migratory potential gained by the cancer cells due to the fusion helps in its dissemination to various secondary organs upon activation of the CXCR4/CXCL12 axis. We are the first to report the presence of a hemato-epithelial cancer compartment, which contributes to stem cell markers and CXCR4 in epithelial carcinoma. This finding has repercussions on CXCR4-based therapeutics and opens new avenues in discovering novel molecular targets against fusion and metastasis.

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

    SciTech Connect

    Maric, Martina; Haugo, Alison C.; Dauer, William; Johnson, David; Roller, Richard J.

    2014-07-15

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

  8. The Problem of Colliding Networks and its Relation to Cell Fusion and Cancer

    NASA Astrophysics Data System (ADS)

    Koulakov, Alexei A.; Lazebnik, Yuri

    2012-11-01

    Complex systems, ranging from living cells to human societies, can be represented as attractor networks, whose basic property is to exist in one of allowed states, or attractors. We noted that merging two systems that are in distinct attractors creates uncertainty, as the hybrid system cannot assume two attractors at once. As a prototype of this problem, we explore cell fusion, whose ability to combine distinct cells into hybrids was proposed to cause cancer. By simulating cell types as attractors, we find that hybrids are prone to assume spurious attractors, which are emergent and sporadic states of networks, and propose that cell fusion can make a cell cancerous by placing it into normally inaccessible spurious states. We define basic features of hybrid networks and suggest that the problem of colliding networks has general significance in processes represented by attractor networks, including biological, social, and political phenomena.

  9. Synesthetes show normal sound-induced flash fission and fusion illusions.

    PubMed

    Whittingham, Karen M; McDonald, J Scott; Clifford, Colin W G

    2014-12-01

    Idiopathic synesthesia, a neurological condition in which a stimulus in one sense generates a concurrent experience in a different sense, is often considered an example of multisensory integration. Consequently it has been suggested that synesthetes should experience multisensory illusions more consistently and compellingly than typical participants. To test this we measured the sound induced flash fission and fusion illusions in 22 coloured hearing synesthetes and 31 control participants. Analysis of the data using signal detection analysis, however, indicated no difference between the groups, either in perception or response bias, but a secondary analysis of the data did show evidence of a decline in the illusions for synesthetes with increasing age.

  10. A FINE-STRUCTURAL ANALYSIS OF THE FUSION OF MYOGENIC CELLS

    PubMed Central

    Lipton, Bruce H.; Konigsberg, Irwin R.

    1972-01-01

    The fusion of myogenic cells has been examined on the fine-structural level in muscle cell cultures of embryonic Japanese Coturnix quail. Cells, selected by light microscopy, were serially sectioned normal to their long axis. In this plane, oblique sections of cell membranes are rare and plasmalemmal profiles are more easily traced between adjacent cells. In seven cases, pairs of cells, apparently fixed in the process of fusion, are joined by a single cytoplasmic bridge. Since obliquely sectioned membranes often suggest cytoplasmic confluence, tilting stage analysis was employed to resolve cell membranes in suspect cases. In contrast to such artifacts of superposition, however, the observed intercommunicating pores are contained within a pair of culs-de-sac formed by the fused membranes of both cells. These blind pouches can be traced back between the cells to the external space. The confluent regions are clearly demarcated and they are not simply areas between vesicular profiles. The results of this analysis suggest that (a) at no time is there any loss of integrity of the cellular envelope, and (b) fusion is most probably initiated at single sites between pairs of cells, the pore enlarging, leaving first vestiges and eventually no trace of the original intervening membranes. PMID:4554365

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

    PubMed

    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.

  12. Using GFP--ligand fusions to measure receptor-mediated endocytosis in living cells.

    PubMed

    Medina-Kauwe, Lali K; Chen, Xinhua

    2002-01-01

    Recombinant DNA technology has enabled the production of many types of chimeric proteins containing heterologous functional domains that have served a variety of useful capacities for cell biology research. Among proteins gaining wide use as a fusion partner is Aequorea victoria green fluorescent protein (GFP). GFP has been employed by numerous groups as a reporter gene for cell transfection and as an autofluorescent tag by recombinant fusion to foreign sequences. Here we describe the use of GFP as a tag for ligands, and provide examples of how purified recombinant GFP-ligand fusion proteins may be used to detect ligand-receptor interactions, including receptor-mediated endocytosis. Both its utility and limitations are discussed.

  13. Cell Fusion as a Cause of Prostate Cancer Metastasis

    DTIC Science & Technology

    2009-03-01

    understand how clonogenic survival of fused cells or cells that become tetraploid by other means is regulated. Considering that tetraploidy is...Thus, we compared the fate of cells that were treated identically in the same dish, but became either binuclear ( tetraploid ) or mononuclear (diploid...only one of the mechanisms that prevent clonogenic expansion of tetraploid cells. Some tetraploid cells remained clonogenic if the parental cells

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

    DTIC Science & Technology

    2013-07-01

    populations. Last cycle we optimized electroporation conditions for T47D and human mesenchymal stem cell populations and this cycle we have improved our... stem cells at a frequency 0.003% + 0.003%. Similarly, highly metastatic breast tumor line, MDA-MB-231 can also fuse spontaneously with human...mesenchymal stem cells at a frequency somewhat higher than that of normal epithelium (0.03% + 0.01%, Figure 1). Spontaneous fusion occurred under physiologic

  15. Cell-fusion-mediated reprogramming: pluripotency or transdifferentiation? Implications for regenerative medicine.

    PubMed

    Sanges, Daniela; Lluis, Frederic; Cosma, Maria Pia

    2011-01-01

    Cell-cell fusion is a natural process that occurs not only during development, but as has emerged over the last few years, also with an important role in tissue regeneration. Interestingly, in-vitro studies have revealed that after fusion of two different cell types, the developmental potential of these cells can change. This suggests that the mechanisms by which cells differentiate during development to acquire their identities is not irreversible, as was considered until a few years ago. To date, it is well established that the fate of a cell can be changed by a process known as reprogramming. This mainly occurs in two different ways: the differentiated state of a cell can be reversed back into a pluripotent state (pluripotent reprogramming), or it can be switched directly to a different differentiated state (lineage reprogramming). In both cases, these possibilities of obtaining sources of autologous somatic cells to maintain, replace or rescue different tissues has provided new and fundamental insights in the stem-cell-therapy field. Most interestingly, the concept that cell reprogramming can also occur in vivo by spontaneous cell fusion events is also emerging, which suggests that this mechanism can be implicated not only in cellular plasticity, but also in tissue regeneration. In this chapter, we will summarize the present knowledge of the molecular mechanisms that mediate the restoration of pluripotency in vitro through cell fusion, as well as the studies carried out over the last 3 decades on lineage reprogramming, both in vitro and in vivo. How the outcome of these studies relate to regenerative medicine applications will also be discussed.

  16. Lipids as modulators of membrane fusion mediated by viral fusion proteins.

    PubMed

    Teissier, Elodie; Pécheur, Eve-Isabelle

    2007-11-01

    Enveloped viruses infect host cells by fusion of viral and target membranes. This fusion event is triggered by specific glycoproteins in the viral envelope. Fusion glycoproteins belong to either class I, class II or the newly described third class, depending upon their arrangement at the surface of the virion, their tri-dimensional structure and the location within the protein of a short stretch of hydrophobic amino acids called the fusion peptide, which is able to induce the initial lipid destabilization at the onset of fusion. Viral fusion occurs either with the plasma membrane for pH-independent viruses, or with the endosomal membranes for pH-dependent viruses. Although, viral fusion proteins are parted in three classes and the subcellular localization of fusion might vary, these proteins have to act, in common, on lipid assemblies. Lipids contribute to fusion through their physical, mechanical and/or chemical properties. Lipids can thus play a role as chemically defined entities, or through their preferential partitioning into membrane microdomains called "rafts", or by modulating the curvature of the membranes involved in the fusion process. The purpose of this review is to make a state of the art on recent findings on the contribution of cholesterol, sphingolipids and glycolipids in cell entry and membrane fusion of a number of viral families, whose members bear either class I or class II fusion proteins, or fusion proteins of the recently discovered third class.

  17. A novel bioinformatics pipeline for identification and characterization of fusion transcripts in breast cancer and normal cell lines.

    PubMed

    Asmann, Yan W; Hossain, Asif; Necela, Brian M; Middha, Sumit; Kalari, Krishna R; Sun, Zhifu; Chai, High-Seng; Williamson, David W; Radisky, Derek; Schroth, Gary P; Kocher, Jean-Pierre A; Perez, Edith A; Thompson, E Aubrey

    2011-08-01

    SnowShoes-FTD, developed for fusion transcript detection in paired-end mRNA-Seq data, employs multiple steps of false positive filtering to nominate fusion transcripts with near 100% confidence. Unique features include: (i) identification of multiple fusion isoforms from two gene partners; (ii) prediction of genomic rearrangements; (iii) identification of exon fusion boundaries; (iv) generation of a 5'-3' fusion spanning sequence for PCR validation; and (v) prediction of the protein sequences, including frame shift and amino acid insertions. We applied SnowShoes-FTD to identify 50 fusion candidates in 22 breast cancer and 9 non-transformed cell lines. Five additional fusion candidates with two isoforms were confirmed. In all, 30 of 55 fusion candidates had in-frame protein products. No fusion transcripts were detected in non-transformed cells. Consideration of the possible functions of a subset of predicted fusion proteins suggests several potentially important functions in transformation, including a possible new mechanism for overexpression of ERBB2 in a HER-positive cell line. The source code of SnowShoes-FTD is provided in two formats: one configured to run on the Sun Grid Engine for parallelization, and the other formatted to run on a single LINUX node. Executables in PERL are available for download from our web site: http://mayoresearch.mayo.edu/mayo/research/biostat/stand-alone-packages.cfm.

  18. Pre-stem cell formation by non-platelet RNA-containing particle fusion

    PubMed Central

    Kong, Wuyi; Nuo, Mu; Zhu, Xiao Ping; Han, Xiu Juan; Luo, Lihua; Wang, Xian

    2013-01-01

    1. We found a group of non-platelet RNA-containing particles (NPRCP) in human umbilical cord blood. To understand the origin, characterization and differentiation of NPRCP, we examined cord blood-isolated NPRCP in vitro. 2. The NPRCP range in size from < 1 to 5 μm, have a thin bilayer membrane and various morphological features, contain short RNA and microRNA and express octamer-binding transcription factor 4 (OCT4), sex-determining region Y 2 (SOX2) and DEAD box polypeptide 4 (DDX4). On coculture with nucleated cells from umbilical cord blood, NPRCP fuse to small, active, non-nucleated cells called ‘particle fusion-derived non-nucleated cells’ (PFDNC). The PFDNC are approximately 8 μm in diameter and are characterized by their twisting movement in culture plates. They can easily move into and out of nucleated cells and finally differentiate into mesenchymal-like cells. In addition, the larger non-nucleated cellular structures that are derived from the aggregation and fusion of multiple NPRCP can further differentiate into large stem cells that also release OCT4- and SOX2-positive non-nucleated small cells. 3. Our data provide strong evidence that NPRCP can fuse into PFDNC, which further differentiate into mesenchymal-like cells. Multiple NPRCP also fuse into other types of large stem cells. We believe that stem cells are derived from NPRCP fusion. There is considerable potential for the use of NPRCP in clinical therapy. PMID:23611023

  19. TGFβ and BMP Dependent Cell Fate Changes Due to Loss of Filamin B Produces Disc Degeneration and Progressive Vertebral Fusions

    PubMed Central

    Zieba, Jennifer; Forlenza, Kimberly Nicole; Khatra, Jagteshwar Singh; Sarukhanov, Anna; Duran, Ivan; Rigueur, Diana; Lyons, Karen M.; Cohn, Daniel H.; Merrill, Amy E.; Krakow, Deborah

    2016-01-01

    Spondylocarpotarsal synostosis (SCT) is an autosomal recessive disorder characterized by progressive vertebral fusions and caused by loss of function mutations in Filamin B (FLNB). FLNB acts as a signaling scaffold by linking the actin cytoskleteon to signal transduction systems, yet the disease mechanisms for SCT remain unclear. Employing a Flnb knockout mouse, we found morphologic and molecular evidence that the intervertebral discs (IVDs) of Flnb–/–mice undergo rapid and progressive degeneration during postnatal development as a result of abnormal cell fate changes in the IVD, particularly the annulus fibrosus (AF). In Flnb–/–mice, the AF cells lose their typical fibroblast-like characteristics and acquire the molecular and phenotypic signature of hypertrophic chondrocytes. This change is characterized by hallmarks of endochondral-like ossification including alterations in collagen matrix, expression of Collagen X, increased apoptosis, and inappropriate ossification of the disc tissue. We show that conversion of the AF cells into chondrocytes is coincident with upregulated TGFβ signaling via Smad2/3 and BMP induced p38 signaling as well as sustained activation of canonical and noncanonical target genes p21 and Ctgf. These findings indicate that FLNB is involved in attenuation of TGFβ/BMP signaling and influences AF cell fate. Furthermore, we demonstrate that the IVD disruptions in Flnb–/–mice resemble aging degenerative discs and reveal new insights into the molecular causes of vertebral fusions and disc degeneration. PMID:27019229

  20. Introduction of hypoxia-targeting p53 fusion protein for the selective therapy of non-small cell lung cancer.

    PubMed

    Zhao, Yu; Wu, Shaoping; Wu, Junhua; Jia, Peiyuan; Gao, Shan; Yan, Xiangdong; Wang, Yuxia

    2011-01-01

    Non-small cell lung cancer (NSCLC), which accounts for ~85% of lung cancer, is the major cause of malignancy mortality around the world. TP53 dysfunction and hypoxia are the typical biological features of the diverse solid tumors, including NSCLC. To develop an effective and low cytotoxic biological agent for targeted therapy, a p53 fusion protein, which was conjugated with the minimum motif of oxygen-dependent degradation domain (ODD) and the basic domain of TAT of HIV-1 named as TAT-ODD-p53, was evaluated for the treatment of NSCLC established by grafting H1299 cell line in which TP53 is homozygously deleted. We provide the evidence that this p53 fusion protein could significantly induce the cell-cycle arrest and/or apoptosis to inhibit H1299 cells' growth via p53-dependent pathways, including up-regulation of p21 expression and activation of pro-caspase-3, especially under hypoxia in vitro. The results in vivo indicated that this protein could selectively accumulate in the low oxygen tension areas of solid tumor tissues, inhibiting tumor growth via a similar mechanism to that in vitro. No obvious side effects were observed. Therefore, this recombinant p53 protein is likely to become a good candidate for targeted therapy of NSCLC.

  1. Mild cerebellar neurodegeneration of aged heterozygous PCD mice increases cell fusion of Purkinje and bone marrow-derived cells.

    PubMed

    Díaz, David; Recio, Javier S; Weruaga, Eduardo; Alonso, José R

    2012-01-01

    Bone marrow-derived cells have different plastic properties, especially regarding cell fusion, which increases with time and is prompted by tissue injury. Several recessive mutations, including Purkinje Cell Degeneration, affect the number of Purkinje cells in homozygosis; heterozygous young animals have an apparently normal phenotype but they undergo Purkinje cell loss as they age. Our findings demonstrate that heterozygous pcd mice undergo Purkinje cell loss at postnatal day 300, this slow but steadily progressing cell death starting sooner than has been reported previously and without massive reactive gliosis or inflammation. Here, transplantation of bone marrow stem cells was performed to assess the arrival of bone marrow-derived cells in the cerebellum in these heterozygous mice. Our results reveal that a higher number of cell fusion events occurs in heterozygous animals than in the controls, on days 150 and 300 postnatally. In sum, this study indicates that mild cell death promotes the fusion of bone marrow-derived cells with surviving Purkinje neurons. This phenomenon suggests new therapies for long-lasting neurodegenerative disorders.

  2. Myosin 2 Maintains an Open Exocytic Fusion Pore in Secretory Epithelial Cells

    PubMed Central

    Bhat, Purnima

    2009-01-01

    Many studies have implicated F-actin and myosin 2 in the control of regulated secretion. Most recently, evidence suggests a role for the microfilament network in regulating the postfusion events of vesicle dynamics. This is of potential importance as postfusion behavior can influence the loss of vesicle content and may provide a new target for drug therapy. We have investigated the role of myosin 2 in regulating exocytosis in secretory epithelial cells by using novel assays to determine the behavior of the fusion pore in individual granules. We immunolocalize myosin 2A to the apical region of pancreatic acinar cells, suggesting it is this isoform that plays a role in granule exocytosis. We further show myosin 2 phosphorylation increased on cell stimulation, consistent with a regulatory role in secretion. Importantly, in a single-cell, single-granule secretion assay, neither the myosin 2 inhibitor (−)-blebbistatin nor the myosin light chain kinase inhibitor ML-9 had any effect on the numbers of granules stimulated to fuse after cell stimulation. These data indicate that myosin 2, if it has any action on secretion, must be targeting postfusion granule behavior. This interpretation is supported by direct study of fusion pore opening in which we show that (−)-blebbistatin and ML-9 promote fusion pore closure and decrease fusion pore lifetimes. Our work now adds to a growing body of evidence showing that myosin 2 is an essential regulator of postfusion granule behavior. In particular, in the case of the secretory epithelial cells, myosin 2 activity is necessary to maintain fusion pore opening. PMID:19158378

  3. Ewing Sarcoma, an enigmatic malignancy of likely progenitor cell origin, driven by transcription factor oncogenic fusions

    PubMed Central

    Jedlicka, Paul

    2010-01-01

    Since its first description by James Ewing in 1921, Ewing Sarcoma has been a cryptic malignancy. A poorly differentiated tumor of uncertain histogenesis and aggressive biologic behavior, it is the second most common malignancy of bone and soft tissue affecting adolescents and young adults. Some two decades ago, the understanding of Ewing Sarcoma biology took a leap forward with the identification of recurrent EWS/Ets fusions, which drive onco-genesis in this disease. A further leap forward occurred over the last half decade with the application of gene silencing, global expression profiling and primary cell culture technologies to the study of this disease. Resulting work has revealed EWS/Ets fusions to be surprisingly versatile regulators of gene expression, and has narrowed the search for the elusive cell of origin. Improved understanding of EWS/Ets biology and relevant oncogenic pathways has in turn led to the development of targeted therapies, including, recently, small molecules targeting key complexes involving the oncogenic fusion itself. In many respects still remaining an enigma, Ewing Sarcoma is an important model for cancers originating in progenitor-type cells or manifesting progenitor-type cell features, and cancers containing recurrent oncogenic fusions, the latter a surprisingly expanding number. PMID:20490326

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

    PubMed

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

    2003-06-24

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

  5. Data fusion-based assessment of raw materials in mammalian cell culture.

    PubMed

    Lee, Hae Woo; Christie, Andrew; Xu, Jin; Yoon, Seongkyu

    2012-11-01

    In mammalian cell culture producing therapeutic proteins, one of the important challenges is the use of several complex raw materials whose compositional variability is relatively high and their influences on cell culture is poorly understood. Under these circumstances, application of spectroscopic techniques combined with chemometrics can provide fast, simple, and non-destructive ways to evaluate raw material quality, leading to more consistent cell culture performance. In this study, a comprehensive data fusion strategy of combining multiple spectroscopic techniques is investigated for the prediction of raw material quality in mammalian cell culture. To achieve this purpose, four different spectroscopic techniques of near-infrared, Raman, 2D fluorescence, and X-ray fluorescence spectra were employed for comprehensive characterization of soy hydrolysates which are commonly used as supplements in culture media. First, the different spectra were compared separately in terms of their prediction capability. Then, ensemble partial least squares (EPLS) was further employed by combining all of these spectral datasets in order to produce a more accurate estimation of raw material properties, and compared with other data fusion techniques. The results showed that data fusion models based on EPLS always exhibit best prediction accuracy among all the models including individual spectroscopic methods, demonstrating the synergetic effects of data fusion in characterizing the raw material quality.

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

    NASA Astrophysics Data System (ADS)

    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.

  7. An induced junction photovoltaic cell

    NASA Technical Reports Server (NTRS)

    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.

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

    PubMed Central

    Wong, Carmen M.; Poulin, Kathy L.; Tong, Grace; Christou, Carin; Kennedy, Michael A.; Falls, Theresa; Bell, John C.; Parks, Robin J.

    2016-01-01

    Adenoviruses (Ads) are used in numerous preclinical and clinical studies for delivery of anti-cancer therapeutic genes. Unfortunately, Ad has a poor ability to distribute throughout a tumor mass after intratumoral injection, and infects cells primarily within the immediate area of the injection tract. Thus, Ad-encoded transgene expression is typically limited to only a small percentage of cells within the tumor. One method to increase the proportion of the tumor impacted by Ad is through expression of fusogenic proteins. Infection of a single cell with an Ad vector encoding a fusogenic protein should lead to syncytium formation with adjacent cells, effectively spreading the effect of Ad and Ad-encoded therapeutic transgenes to a greater percentage of the tumor mass. Moreover, syncytium formation can be cytotoxic, suggesting that such proteins may be effective sole therapeutics. We show that an early region 1 (E1)-deleted Ad expressing reptilian reovirus p14 fusion-associated small transmembrane (FAST) protein caused extensive cell fusion in the replication-permissive 293 cell line and at high multiplicity of infection in non-permissive human lung adenocarcinoma A549 cells in vitro. FAST protein expression in the A549 cancer cell line led to a loss of cellular metabolic activity and membrane integrity, which correlated with induction of apoptosis. However, in an A549 xenograft CD-1 nude mouse cancer model, Ad-mediated FAST gene delivery did not induce detectable cell fusion, reduce tumor burden nor enhance mouse survival compared to controls. Taken together, our results show that, although AdFAST can enhance cancer cell killing in vitro, it is not effective as a sole therapeutic in the A549 tumor model in vivo. PMID:26986751

  9. Targeting of Cytolytic T-Cells for Breast Cancer Therapy Using Novel-Fusion Proteins

    DTIC Science & Technology

    1999-07-01

    1 construct was subsequently subcloned into the Pichia pastoris expression plasmid pPICZcxB (Invitrogen) which contains the alcohol oxidase promoter...breast carcinomas, and the extracellular domain of B7.2 (CD86). This fusion protein was expressed and purified from Pichia pastoris, shown to retain...year’s report, the hB7.2/B1 chimeric fusion protein produced in Pichia pastoris, was shown to bind to both recombinant and cell surface tumor marker erbB

  10. Photoinduced Fusion of Lipid Bilayer Membranes.

    PubMed

    Suzuki, Yui; Nagai, Ken H; Zinchenko, Anatoly; Hamada, Tsutomu

    2017-03-14

    We have developed a novel system for photocontrol of the fusion of lipid vesicles through the use of a photosensitive surfactant containing an azobenzene moiety (AzoTAB). Real-time microscopic observations clarified a change in both the surface area and internal volume of vesicles during fusion. We also determined the optimal cholesterol concentrations and temperature for inducing fusion. The mechanism of fusion can be attributed to a change in membrane tension, which is caused by the solubilization of lipids through the isomerization of AzoTAB. We used a micropipet technique to estimate membrane tension and discuss the mechanism of fusion in terms of membrane elastic energy. The obtained results regarding this novel photoinduced fusion could lead to a better understanding of the mechanism of membrane fusion in living cells and may also see wider applications, such as in drug delivery and biomimetic material design.

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

    NASA Technical Reports Server (NTRS)

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

    1992-01-01

    The influence of microgravity on lymphocyte activation is central to the understanding of immunological function in space. Moreover, the adaptation of ground-based 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, mitogen-driven B lymphocyte stimulation and culture were adapted that allow for the in vitro generation of large numbers of antibody forming cells suitable for cell fusion over a period of 1-2 weeks. It is believed 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 bioseparation.

  12. Mechanistic Insight into Bunyavirus-Induced Membrane Fusion from Structure-Function Analyses of the Hantavirus Envelope Glycoprotein Gc

    PubMed Central

    Stettner, Eva; Jeffers, Scott Allen; Pérez-Vargas, Jimena; Pehau-Arnaudet, Gerard; Tortorici, M. Alejandra; Jestin, Jean-Luc; England, Patrick; Tischler, Nicole D.; Rey, Félix A.

    2016-01-01

    Hantaviruses are zoonotic viruses transmitted to humans by persistently infected rodents, giving rise to serious outbreaks of hemorrhagic fever with renal syndrome (HFRS) or of hantavirus pulmonary syndrome (HPS), depending on the virus, which are associated with high case fatality rates. There is only limited knowledge about the organization of the viral particles and in particular, about the hantavirus membrane fusion glycoprotein Gc, the function of which is essential for virus entry. We describe here the X-ray structures of Gc from Hantaan virus, the type species hantavirus and responsible for HFRS, both in its neutral pH, monomeric pre-fusion conformation, and in its acidic pH, trimeric post-fusion form. The structures confirm the prediction that Gc is a class II fusion protein, containing the characteristic β-sheet rich domains termed I, II and III as initially identified in the fusion proteins of arboviruses such as alpha- and flaviviruses. The structures also show a number of features of Gc that are distinct from arbovirus class II proteins. In particular, hantavirus Gc inserts residues from three different loops into the target membrane to drive fusion, as confirmed functionally by structure-guided mutagenesis on the HPS-inducing Andes virus, instead of having a single “fusion loop”. We further show that the membrane interacting region of Gc becomes structured only at acidic pH via a set of polar and electrostatic interactions. Furthermore, the structure reveals that hantavirus Gc has an additional N-terminal “tail” that is crucial in stabilizing the post-fusion trimer, accompanying the swapping of domain III in the quaternary arrangement of the trimer as compared to the standard class II fusion proteins. The mechanistic understandings derived from these data are likely to provide a unique handle for devising treatments against these human pathogens. PMID:27783711

  13. Mechanistic Insight into Bunyavirus-Induced Membrane Fusion from Structure-Function Analyses of the Hantavirus Envelope Glycoprotein Gc.

    PubMed

    Guardado-Calvo, Pablo; Bignon, Eduardo A; Stettner, Eva; Jeffers, Scott Allen; Pérez-Vargas, Jimena; Pehau-Arnaudet, Gerard; Tortorici, M Alejandra; Jestin, Jean-Luc; England, Patrick; Tischler, Nicole D; Rey, Félix A

    2016-10-01

    Hantaviruses are zoonotic viruses transmitted to humans by persistently infected rodents, giving rise to serious outbreaks of hemorrhagic fever with renal syndrome (HFRS) or of hantavirus pulmonary syndrome (HPS), depending on the virus, which are associated with high case fatality rates. There is only limited knowledge about the organization of the viral particles and in particular, about the hantavirus membrane fusion glycoprotein Gc, the function of which is essential for virus entry. We describe here the X-ray structures of Gc from Hantaan virus, the type species hantavirus and responsible for HFRS, both in its neutral pH, monomeric pre-fusion conformation, and in its acidic pH, trimeric post-fusion form. The structures confirm the prediction that Gc is a class II fusion protein, containing the characteristic β-sheet rich domains termed I, II and III as initially identified in the fusion proteins of arboviruses such as alpha- and flaviviruses. The structures also show a number of features of Gc that are distinct from arbovirus class II proteins. In particular, hantavirus Gc inserts residues from three different loops into the target membrane to drive fusion, as confirmed functionally by structure-guided mutagenesis on the HPS-inducing Andes virus, instead of having a single "fusion loop". We further show that the membrane interacting region of Gc becomes structured only at acidic pH via a set of polar and electrostatic interactions. Furthermore, the structure reveals that hantavirus Gc has an additional N-terminal "tail" that is crucial in stabilizing the post-fusion trimer, accompanying the swapping of domain III in the quaternary arrangement of the trimer as compared to the standard class II fusion proteins. The mechanistic understandings derived from these data are likely to provide a unique handle for devising treatments against these human pathogens.

  14. Characterisation of Weibel-Palade body fusion by amperometry in endothelial cells reveals fusion pore dynamics and the effect of cholesterol on exocytosis.

    PubMed

    Cookson, Emma A; Conte, Ianina L; Dempster, John; Hannah, Matthew J; Carter, Tom

    2013-12-01

    Regulated secretion from endothelial cells is mediated by Weibel-Palade body (WPB) exocytosis. Plasma membrane cholesterol is implicated in regulating secretory granule exocytosis and fusion pore dynamics; however, its role in modulating WPB exocytosis is not clear. To address this we combined high-resolution electrochemical analysis of WPB fusion pore dynamics, by amperometry, with high-speed optical imaging of WPB exocytosis following cholesterol depletion or supplementation in human umbilical vein endothelial cells. We identified serotonin (5-HT) immunoreactivity in WPBs, and VMAT1 expression allowing detection of secreted 5-HT as discrete current spikes during exocytosis. A high proportion of spikes (∼75%) had pre-spike foot signals, indicating that WPB fusion proceeds via an initial narrow pore. Cholesterol depletion significantly reduced pre-spike foot signal duration and increased the rate of fusion pore expansion, whereas cholesterol supplementation had broadly the reverse effect. Cholesterol depletion slowed the onset of hormone-evoked WPB exocytosis, whereas its supplementation increased the rate of WPB exocytosis and hormone-evoked proregion secretion. Our results provide the first analysis of WPB fusion pore dynamics and highlight an important role for cholesterol in the regulation of WPB exocytosis.

  15. Reduced susceptibility to the sound-induced flash fusion illusion in schizophrenia.

    PubMed

    Vanes, Lucy D; White, Thomas P; Wigton, Rebekah L; Joyce, Dan; Collier, Tracy; Shergill, Sukhi S

    2016-11-30

    Schizophrenia is characterised by the presence of abnormal complex sensory perceptual experiences. Such experiences could arise as a consequence of dysfunctional multisensory integration. We used the sound-induced flash illusion paradigm, which probes audiovisual integration using elementary visual and auditory cues, in a sample of individuals with schizophrenia (n=40) and matched controls (n=22). Signal detection theory analyses were performed to characterise patients' and controls' sensitivity in distinguishing 1 and 2 flashes under varying auditory conditions. Both groups experienced significant fission illusions (whereby one visual flash, accompanied by two auditory beeps, is misperceived as two flashes) and fusion illusions (whereby two flashes, accompanied by one beep, are perceived as one flash). Patients showed significantly lower fusion illusion rates compared to HC, while the fission illusion occurred similarly frequently in both groups. However, using an SDT approach, we compared illusion conditions with unimodal visual conditions, and found that illusory visual perception was overall more strongly influenced by auditory input in HC compared to patients for both illusions. This suggests that multisensory integration may be impaired on a low perceptual level in SZ.

  16. ELT-5 and ELT-6 are required continuously to regulate epidermal seam cell differentiation and cell fusion in C. elegans.

    PubMed

    Koh, K; Rothman, J H

    2001-08-01

    The C. elegans epidermis is a simple epithelium comprised of three major cell types, the seam, syncytial and P cells. While specification of all major epidermal cells is known to require the ELT-1 GATA transcription factor, little is known about how the individual epidermal cell types are specified. We report that elt-5 and -6, adjacent genes encoding GATA factors, are essential for the development of the lateral epidermal cells, the seam cells. Inhibition of elt-5 and -6 function by RNA-mediated interference results in penetrant late embryonic and early larval lethality. Seam cells in affected animals do not differentiate properly: the alae, seam-specific cuticular structures, are generally absent and expression of several seam-specific markers is blocked. In addition, elt-3, which encodes another GATA factor normally expressed in non-seam epidermis, is often ectopically expressed in the seam cells of affected animals, demonstrating that ELT-5 and -6 repress elt-3 expression in wild-type seam cells. Seam cells in affected animals often undergo inappropriate fusion with the epidermal syncytia. Interference of elt-5 and -6 function during larval development can cause fusion of all seam cells with the surrounding syncytia and pronounced defects in molting. elt-5 and -6 are both expressed in seam cells and many other cells, and are apparently functionally interchangeable. Their expression is controlled by separable tissue-specific regulatory elements and the apportionment of monocistronic versus dicistronic transcription of both genes appears to be subject to cell-type-specific regulation. Collectively, these findings indicate that elt-5 and -6 function continuously throughout C. elegans development to regulate seam cell differentiation and cell fusion.

  17. Identification of Targetable FGFR Gene Fusions in Diverse Cancers

    PubMed Central

    Wu, Yi-Mi; Su, Fengyun; Kalyana-Sundaram, Shanker; Khazanov, Nick; Ateeq, Bushra; Cao, Xuhong; Lonigro, Robert J.; Vats, Pankaj; Wang, Rui; Lin, Su-Fang; Cheng, Ann-Joy; Kunju, Lakshmi P.; Siddiqui, Javed; Tomlins, Scott A.; Wyngaard, Peter; Sadis, Seth; Roychowdhury, Sameek; Hussain, Maha H.; Feng, Felix Y.; Zalupski, Mark M.; Talpaz, Moshe; Pienta, Kenneth J.; Rhodes, Daniel R.; Robinson, Dan R.; Chinnaiyan, Arul M.

    2013-01-01

    Through a prospective clinical sequencing program for advanced cancers, four index cases were identified which harbor gene rearrangements of FGFR2 including patients with cholangiocarcinoma, breast cancer, and prostate cancer. After extending our assessment of FGFR rearrangements across multiple tumor cohorts, we identified additional FGFR gene fusions with intact kinase domains in lung squamous cell cancer, bladder cancer, thyroid cancer, oral cancer, glioblastoma, and head and neck squamous cell cancer. All FGFR fusion partners tested exhibit oligomerization capability, suggesting a shared mode of kinase activation. Overexpression of FGFR fusion proteins induced cell proliferation. Two bladder cancer cell lines that harbor FGFR3 fusion proteins exhibited enhanced susceptibility to pharmacologic inhibition in vitro and in vivo. Due to the combinatorial possibilities of FGFR family fusion to a variety of oligomerization partners, clinical sequencing efforts which incorporate transcriptome analysis for gene fusions are poised to identify rare, targetable FGFR fusions across diverse cancer types. PMID:23558953

  18. Identification of targetable FGFR gene fusions in diverse cancers.

    PubMed

    Wu, Yi-Mi; Su, Fengyun; Kalyana-Sundaram, Shanker; Khazanov, Nickolay; Ateeq, Bushra; Cao, Xuhong; Lonigro, Robert J; Vats, Pankaj; Wang, Rui; Lin, Su-Fang; Cheng, Ann-Joy; Kunju, Lakshmi P; Siddiqui, Javed; Tomlins, Scott A; Wyngaard, Peter; Sadis, Seth; Roychowdhury, Sameek; Hussain, Maha H; Feng, Felix Y; Zalupski, Mark M; Talpaz, Moshe; Pienta, Kenneth J; Rhodes, Daniel R; Robinson, Dan R; Chinnaiyan, Arul M

    2013-06-01

    Through a prospective clinical sequencing program for advanced cancers, four index cases were identified which harbor gene rearrangements of FGFR2, including patients with cholangiocarcinoma, breast cancer, and prostate cancer. After extending our assessment of FGFR rearrangements across multiple tumor cohorts, we identified additional FGFR fusions with intact kinase domains in lung squamous cell cancer, bladder cancer, thyroid cancer, oral cancer, glioblastoma, and head and neck squamous cell cancer. All FGFR fusion partners tested exhibit oligomerization capability, suggesting a shared mode of kinase activation. Overexpression of FGFR fusion proteins induced cell proliferation. Two bladder cancer cell lines that harbor FGFR3 fusion proteins exhibited enhanced susceptibility to pharmacologic inhibition in vitro and in vivo. Because of the combinatorial possibilities of FGFR family fusion to a variety of oligomerization partners, clinical sequencing efforts, which incorporate transcriptome analysis for gene fusions, are poised to identify rare, targetable FGFR fusions across diverse cancer types.

  19. Expression of a connexin 43/beta-galactosidase fusion protein inhibits gap junctional communication in NIH3T3 cells

    PubMed Central

    1995-01-01

    Gap junctions contain membrane channels that mediate the cell-to-cell movement of ions, metabolites and cell signaling molecules. As gap junctions are comprised of a hexameric array of connexin polypeptides, the expression of a mutant connexin polypeptide may exert a dominant negative effect on gap junctional communication. To examine this possibility, we constructed a connexin 43 (Cx43)/beta-galactosidase (beta-gal) expression vector in which the bacterial beta-gal protein is fused in frame to the carboxy terminus of Cx43. This vector was transfected into NIH3T3 cells, a cell line which is well coupled via gap junctions and expresses high levels of Cx43. Transfectant clones were shown to express the fusion protein by northern and western analysis. X-Gal staining further revealed that all of the fusion protein containing cells also expressed beta-gal enzymatic activity. Double immunostaining with a beta-gal and Cx43 antibody demonstrated that the fusion protein is immunolocalized to the perinuclear region of the cytoplasm and also as punctate spots at regions of cell-cell contact. This pattern is similar to that of Cx43 in the parental 3T3 cells, except that in the fusion protein expressing cells, Cx43 expression was reduced at regions of cell-cell contact. Examination of gap junctional communication (GJC) with dye injection studies further showed that dye coupling was inhibited in the fusion protein expressing cells, with the largest reduction in coupling found in a clone exhibiting little Cx43 localization at regions of cell-cell contact. When the fusion protein expression vector was transfected into the communication poor C6 cell line, abundant fusion protein expression was observed, but unlike the transfected NIH3T3 cells, no fusion protein was detected at the cell surface. Nevertheless, dye coupling was inhibited in these C6 cells. Based on these observations, we propose that the fusion protein may inhibit GJC by sequestering the Cx43 protein intracellularly

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

    DTIC Science & Technology

    2005-07-01

    regulate the development of anti-tumor immune responses . Importantly, our results show that, compared to unfused DC and tumor cells, the DC/ breast tumor...AD Award Number: DAMD17-03-1-0487 TITLE: Fusions of Breast Carcinoma and Dendritic Cells as a Vaccine for the Treatment of Metastatic Breast Cancer ...4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Fusions of Breast Carcinoma and Dendritic Cells as a Vaccine for the Treatment of Metastatic Breast Cancer

  1. The FIP1L1-PDGFRA fusion gene cooperates with IL-5 to induce murine hypereosinophilic syndrome (HES)/chronic eosinophilic leukemia (CEL)-like disease.

    PubMed

    Yamada, Yoshiyuki; Rothenberg, Marc E; Lee, Andrew W; Akei, Hiroko Saito; Brandt, Eric B; Williams, David A; Cancelas, Jose A

    2006-05-15

    Dysregulated tyrosine kinase activity by the Fip1-like1 (FIP1L1)-platelet-derived growth factor receptor alpha (PDGFRA) (F/P) fusion gene has been identified as a cause of clonal hypereosinophilic syndrome (HES), called F/P-positive chronic eosinophilic leukemia (CEL) in humans. However, transplantation of F/P-transduced hematopoietic stem cells/progenitors (F/P(+) HSCs/Ps) into mice results in a chronic myelogenous leukemia-like disease, which does not resemble HES. Because a subgroup of patients with HES show T-cell-dependent interleukin-5 (IL-5) overexpression, we determined if expression of the F/P fusion gene in the presence of transgenic T-cell IL-5 overexpression in mice induces HES-like disease. Mice that received a transplant of CD2-IL-5-transgenic F/P(+) HSC/Ps (IL-5Tg-F/P) developed intense leukocytosis, strikingly high eosinophilia, and eosinophilic infiltration of nonhematopoietic as well as hematopoietic tissues, a phenotype resembling human HES. The disease phenotype was transferable to secondary transplant recipients of a high cell dose, suggesting involvement of a short-term repopulating stem cell or an early myeloid progenitor. Induction of significant eosinophilia was specific for F/P since expression of another fusion oncogene, p210-BCR/ABL, in the presence of IL-5 overexpression was characterized by a significantly lower eosinophilia than IL-5Tg-F/P recipients. These results suggest that F/P is not sufficient to induce a HES/CEL-like disease but requires a second event associated with IL-5 overexpression.

  2. Melanoma-Derived BRAF(V600E) Mutation in Peritumoral Stromal Cells: Implications for in Vivo Cell Fusion.

    PubMed

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

    2016-06-21

    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 BRAF(V600E) melanoma, melanoma antigen recognized by T-cells (MART1)-negative peritumoral stromal cells express BRAF(V600E) protein. To confirm the presence of the oncogene at the genetic level, peritumoral stromal cells were microdissected and screened for the presence of BRAF(V600E) with a mutation-specific polymerase chain reaction. Interestingly, cells carrying the BRAF(V600E) 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 BRAF(V600E) mutation or protein in the peritumoral stroma of BRAF(WT) 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.

  3. Helicobacter pylori TlyA agglutinates liposomes and induces fusion and permeabilization of the liposome membranes.

    PubMed

    Lata, Kusum; Chattopadhyay, Kausik

    2014-06-10

    Helicobacter pylori TlyA is a pore-forming hemolysin with potent cytotoxic activity. To explore the potential membrane-damaging activity of H. pylori TlyA, we have studied its interaction with the synthetic liposome vesicles. In our study, H. pylori TlyA shows a prominent ability to associate with the liposome vesicles without displaying an obligatory requirement for any protein receptor on the liposome membranes. Interaction of TlyA triggers agglutination of the liposome vesicles. Such agglutinating activity of TlyA could also be observed with erythrocytes before the induction of its pore-forming hemolytic activity. In addition to its agglutinating activity against liposomes, TlyA also induces fusion and disruption of the liposome membranes. Altogether, our study highlights novel membrane-damaging properties of H. pylori TlyA that have not been documented previously with any other TlyA family protein.

  4. Transformation from Globular to Cylindrical Mixed Micelles through Molecular Exchange that Induces Micelle Fusion.

    PubMed

    Jensen, Grethe V; Lund, Reidar; Narayanan, Theyencheri; Pedersen, Jan Skov

    2016-06-02

    Transformations between different micellar morphologies in solution induced by changes in composition, salt, or temperature are well-known phenomena; however, the understanding of the associated kinetic pathways is still limited. Especially for mixed surfactant systems, the micelles can take a very wide range of structures, depending on the surfactant packing parameter and other thermodynamic conditions. Synchrotron-based small-angle X-ray scattering (SAXS) in combination with fast mixing using a stopped-flow apparatus can give direct access to the structural kinetics on a millisecond time scale. Here, this approach is used to study the formation of cylindrical micelles after mixing two solutions with globular micelles of the nonionic surfactant dodecyl maltoside (DDM) and the anionic surfactant sodium dodecyl sulfate (SDS), respectively. Two separate processes were identified: (i) a transition in micellar shell structure, interpreted as exchange of surfactant molecules resulting in mixed globular micelles, and subsequently, (ii) fusion into larger, cylindrical structures.

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

    PubMed

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

    2016-01-07

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

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

    PubMed Central

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

    2016-01-01

    The nucleoporin Nup98 is frequently rearranged to form leukemogenic Nup98-fusion proteins with various partners. However, their function remains largely elusive. Here, we show that Nup98-HoxA9, a fusion between Nup98 and the homeobox transcription factor HoxA9, forms nuclear aggregates that frequently associate with facultative heterochromatin. We demonstrate that stable expression of Nup98-HoxA9 in mouse embryonic stem cells selectively induces the expression of Hox cluster genes. Genome-wide binding site analysis revealed that Nup98-HoxA9 is preferentially targeted and accumulated at Hox cluster regions where the export factor Crm1 is originally prebound. In addition, leptomycin B, an inhibitor of Crm1, disassembled nuclear Nup98-HoxA9 dots, resulting in the loss of chromatin binding of Nup98-HoxA9 and Nup98-HoxA9-mediated activation of Hox genes. Collectively, our results indicate that highly selective targeting of Nup98-fusion proteins to Hox cluster regions via prebound Crm1 induces the formation of higher order chromatin structures that causes aberrant Hox gene regulation. DOI: http://dx.doi.org/10.7554/eLife.09540.001 PMID:26740045

  7. Magnesium-induced lipid bilayer microdomain reorganizations: implications for membrane fusion.

    PubMed

    Schultz, Zachary D; Pazos, Ileana M; McNeil-Watson, Fraser K; Lewis, E Neil; Levin, Ira W

    2009-07-23

    Interactions between dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylserine (DPPS), combined both as binary lipid bilayer assemblies and separately, under the influence of divalent Mg2+, a membrane bilayer fusogenic agent, are reported. Infrared vibrational spectroscopic analyses of the lipid acyl chain methylene symmetric stretching modes indicate that aggregates of the two phospholipid components exist as domains heterogeneously distributed throughout the binary bilayer system. In the presence of Mg2+, DPPS maintains an ordered orthorhombic subcell gel phase structure through the phase transition temperature, while the DPPC component is only minimally perturbed with respect to the gel to liquid crystalline phase change. The addition of Mg2+ induces a reorganization of the lipid domains in which the gel phase acyl chain planes rearrange from a hexagonal configuration toward a triclinic, parallel chain subcell. Examination of the acyl chain methylene deformation modes at low temperatures allows a determination of DPPS microdomain sizes, which decrease upon the addition of DPPC-d62 in the absence of Mg2+. On adding Mg2+, a uniform DPPS domain size is observed in the binary mixtures. In either the presence or absence of Mg2+, DPPC-d62 aggregates remain in a configuration for which microdomain sizes are not spectroscopically measurable. Analysis of the acyl chain methylene deformation modes for DPPC-d62 in the binary system suggests that clusters of the deuterated lipids are distributed throughout the DPPS matrix. Light scattering and fluorescence measurements indicate that Mg2+ induces both the aggregation and the fusion of the lipid assemblies as a function of the ratio of DPPS to DPPC. The structural reorganizations of the lipid microdomains within the DPPS-DPPC bilayer are interpreted in the context of current concepts regarding lipid bilayer fusion.

  8. Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions.

    PubMed

    Wolters, Manuel; Zobiak, Bernd; Nauth, Theresa; Aepfelbacher, Martin

    2015-10-13

    Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

  9. Nuclear export of the glucocorticoid receptor is accelerated by cell fusion-dependent release of calreticulin.

    PubMed

    Walther, Rhian F; Lamprecht, Claudia; Ridsdale, Andrew; Groulx, Isabelle; Lee, Stephen; Lefebvre, Yvonne A; Haché, Robert J G

    2003-09-26

    Nucleocytoplasmic exchange of nuclear hormone receptors is hypothesized to allow for rapid and direct interactions with cytoplasmic signaling factors. In addition to recycling between a naïve, chaperone-associated cytoplasmic complex and a liganded chaperone-free nuclear form, the glucocorticoid receptor (GR) has been observed to shuttle between nucleus and cytoplasm. Nuclear export of GR and other nuclear receptors has been proposed to depend on direct interactions with calreticulin, which is predominantly localized to the lumen of the endoplasmic reticulum. We show that rapid calreticulin-mediated nuclear export of GR is a specific response to transient disruption of the endoplasmic reticulum that occurs during polyethylene glycol-mediated cell fusion. Using live and digitonin-permeabilized cells we demonstrate that, in the absence of cell fusion, GR nuclear export occurs slowly over a period of many hours independent of direct interaction with calreticulin. Our findings temper expectations that nuclear receptors respond rapidly and directly to cytoplasmic signals in the absence of additional regulatory control. These results highlight the importance of verifying findings of nucleocytoplasmic trafficking using techniques in addition to heterokaryon cell fusion.

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

    PubMed

    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.

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

    PubMed Central

    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

  12. Targeting tumor cells via EGF receptors: selective toxicity of an HBEGF-toxin fusion protein.

    PubMed

    Chandler, L A; Sosnowski, B A; McDonald, J R; Price, J E; Aukerman, S L; Baird, A; Pierce, G F; Houston, L L

    1998-09-25

    Over-expression of the epidermal growth factor receptor (EGFR) is a hallmark of numerous solid tumors, thus providing a means of selectively targeting therapeutic agents. Heparin-binding epidermal growth factor (HBEGF) binds to EGFRs with high affinity and to heparan sulfate proteoglycans, resulting in increased mitogenic potential compared to other EGF family members. We have investigated the feasibility of using HBEGF to selectively deliver a cytotoxic protein into EGFR-expressing tumor cells. Recombinant fusion proteins consisting of mature human HBEGF fused to the plant ribosome-inactivating protein saporin (SAP) were expressed in Escherichia coli. Purified HBEGF-SAP chimeras inhibited protein synthesis in a cell-free assay and competed with EGF for binding to receptors on intact cells. A construct with a 22-amino-acid flexible linker (L22) between the HBEGF and SAP moieties exhibited an affinity for the EGFR that was comparable to that of HBEGF. The sensitivity to HBEGF-L22-SAP was determined for a variety of human tumor cell lines, including the 60 cell lines comprising the National Cancer Institute Anticancer Drug Screen. HBEGF-L22-SAP was cytotoxic in vitro to a variety of EGFR-bearing cell lines and inhibited growth of EGFR-over-expressing human breast carcinoma cells in vivo. In contrast, the fusion protein had no effect on small-cell lung carcinoma cells, which are EGFR-deficient. Our results demonstrate that fusion proteins composed of HBEGF and SAP exhibit targeting specificity and cytotoxicity that may be of therapeutic value in treating a variety of EGFR-bearing malignancies.

  13. Variations for susceptibilities to ultraviolet induced cellular inactivation and gene segregation among protoplast fusion hybrids of Candida albicans.

    PubMed

    Sarachek, A; Henderson, L A

    1988-01-01

    Hybrids of the naturally diploid, asexual and opportunistically pathogenic yeast, Candida albicans, can be obtained artificially by protoplast fusion. Evidence is presented that gene conversion and reciprocal recombination contribute to ultraviolet (UV)-induced segregations of heterozygous markers from both diploid and hybrid strains, and that hybrids also segregate through induced chromosome loss. Heterozygous diploid strains independently derived from the same wild-type diploid stock were alike in post-UV survival and segregational responses, and the organization of a four gene linkage group identified in diploids from the segregant products of reciprocal recombinations was transmitted intact to all hybrids from fusions between diploids of isogenic or nonisogenic backgrounds. However, hybrids arising independently from a given fusion cross differed significantly from each other in post-UV survival, absolute ability to segregate some parental markers, frequency of gene segregation, and proclivities for each of the three mechanisms of gene segregation. The bearings of the genetic backgrounds of parental strains and of growth temperatures during hybrid formation on each of these variables are described. The findings emphasize that awareness of the intrinsic heterogeneities of fusion hybrids is essential for reliable application of the protoplast fusion procedure to genetic analysis of C. albicans.

  14. Fusion hindrance and quasi-fission in heavy-ion induced reactions: disentangling the effect of different parameters

    SciTech Connect

    Fioretto, E.; Stefanini, A. M.; Behera, B. R.; Corradi, L.; Gadea, A.; Latina, A.; Trotta, M.; Beghini, S.; Montagnoli, G.; Scarlassara, F.; Chizhov, A. Yu.; Itkis, I. M.; Itkis, M. G.; Kniajeva, G. N.; Kondratiev, N. A.; Kozulin, E. M.; Pokrovsky, I. V.; Sagaidak, R. N.; Voskressensky, V. M.; Courtin, S.

    2006-04-26

    Experimental results on the fusion inhibition effect near the Coulomb barrier due to the onset of the quasi-fission mechanism are presented. The investigation was focused on reactions induced by 48Ca projectiles on different heavy targets and comparing them to reactions induced by light ions such as 12C and 16O leading to the same compound nuclei. Cross sections and angular distributions of evaporation residues and fission fragments have been measured.

  15. The relationship between Cd-induced autophagy and lysosomal activation in WRL-68 cells.

    PubMed

    Meng, Su-Fang; Mao, Wei-Ping; Wang, Fang; Liu, Xiao-Qian; Shao, Luan-Luan

    2015-11-01

    This study shows that Cd induces autophagy in the human's embryonic normal liver cell line (WRL-68). The expression of LC3B-II and the mature cathepsin L were analyzed by Western blotting. The autophagosomes and lysosomes were directly visualized by electron microscopy and confocal microscopy analysis in Cd-exposed WRL-68 cells. In this study, we first found that autophagy induced the activation of lysosomal function in WRL-68 cells. The lysosomal activation was markedly decreased when the cells were co-treated with 3-MA (an inhibitor of autophagy). Secondly, we provided the evidence that the activation of lysosomal function depended on autophagosome-lysosome fusion. The colocalization of lysosome-associated membrane protein-2 (LAMP2) and GFP-LC3 was significantly reduced, when they were treated with thapsigargin (an inhibitor of autophagosome-lysosome fusion). We demonstrated that deletion or blockage of the autophagosome-lysosome fusion process effectively diminished lysosomal activation, which suggests that lysosomal activation occurring in the course of autophagy is dependent on autophagosome-lysosome fusion. Thirdly, we provided evidence that the activation of lysosomal function was associated with lysosomal acid. We investigated the relationship between autophagosome-lysosome fusion and pH in acidic compartments by visualizing fusion process in WRL-68 cells. This suggests that increasing pH in acidic compartments in WRL-68 cells inhibits the autophagosome-lysosome fusion. Finally, we found that the activation of lysosomal function was associated with Ca(2+) stores and the intracellular Ca(2+) channels or pumps were possibly pH-dependent.

  16. Screening of cell cycle fusion proteins to identify kinase signaling networks.

    PubMed

    Trojanowsky, Michelle; Vidovic, Dusica; Simanski, Scott; Penas, Clara; Schurer, Stephan; Ayad, Nagi G

    2015-01-01

    Kinase signaling networks are well-established mediators of cell cycle transitions. However, how kinases interact with the ubiquitin proteasome system (UPS) to elicit protein turnover is not fully understood. We sought a means of identifying kinase-substrate interactions to better understand signaling pathways controlling protein degradation. Our prior studies used a luciferase fusion protein to uncover kinase networks controlling protein turnover. In this study, we utilized a similar approach to identify pathways controlling the cell cycle protein p27(Kip1). We generated a p27(Kip1)-luciferase fusion and expressed it in cells incubated with compounds from a library of pharmacologically active compounds. We then compared the relative effects of the compounds on p27(Kip1)-luciferase fusion stabilization. This was combined with in silico kinome profiling to identify potential kinases inhibited by each compound. This approach effectively uncovered known kinases regulating p27(Kip1) turnover. Collectively, our studies suggest that this parallel screening approach is robust and can be applied to fully understand kinase-ubiquitin pathway interactions.

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

    PubMed

    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

  18. Fusion of bone marrow-derived cells with cancer cells: metastasis as a secondary disease in cancer

    PubMed Central

    Pawelek, John M.

    2014-01-01

    This perspective article highlights the leukocyte-cancer cell hybrid theory as a mechanism for cancer metastasis. Beginning from the first proposal of the theory more than a century ago and continuing today with the first proof for this theory in a human cancer, the hybrid theory offers a unifying explanation for metastasis. In this scenario, leukocyte fusion with a cancer cell is a secondary disease superimposed upon the early tumor, giving birth to a new, malignant cell with a leukocyte-cancer cell hybrid epigenome. PMID:24589183

  19. Flow Cytometric Measurement of Blood Cells with BCR-ABL1 Fusion Protein in Chronic Myeloid Leukemia.

    PubMed

    Löf, Liza; Arngården, Linda; Olsson-Strömberg, Ulla; Siart, Benjamin; Jansson, Mattias; Dahlin, Joakim S; Thörn, Ingrid; Christiansson, Lisa; Hermansson, Monica; Larsson, Anders; Ahlstrand, Erik; Wålinder, Göran; Söderberg, Ola; Rosenquist, Richard; Landegren, Ulf; Kamali-Moghaddam, Masood

    2017-04-04

    Chronic myeloid leukemia (CML) is characterized in the majority of cases by a t(9;22)(q34;q11) translocation, also called the Philadelphia chromosome, giving rise to the BCR-ABL1 fusion protein. Current treatment with tyrosine kinase inhibitors is directed against the constitutively active ABL1 domain of the fusion protein, and minimal residual disease (MRD) after therapy is monitored by real-time quantitative PCR (RQ-PCR) of the fusion transcript. Here, we describe a novel approach to detect and enumerate cells positive for the BCR-ABL1 fusion protein by combining the in situ proximity ligation assay with flow cytometry as readout (PLA-flow). By targeting of the BCR and ABL1 parts of the fusion protein with one antibody each, and creating strong fluorescent signals through rolling circle amplification, PLA-flow allowed sensitive detection of cells positive for the BCR-ABL1 fusion at frequencies as low as one in 10,000. Importantly, the flow cytometric results correlated strongly to those of RQ-PCR, both in diagnostic testing and for MRD measurements over time. In summary, we believe this flow cytometry-based method can serve as an attractive approach for routine measurement of cells harboring BCR-ABL1 fusions, also allowing simultaneously assessment of other cell surface markers as well as sensitive longitudinal follow-up.

  20. Isolation of virus-cell fusion inhibitory components from the stem bark of Styrax japonica S. et Z.

    PubMed

    Lee, Dung Gun; Jin, Qinglong; Jin, Hong-Guang; Shin, Ji Eun; Choi, Eun Jin; Woo, Eun-Rhan

    2010-06-01

    Five compounds, styraxjaponoside A (1), matairesinoside (2), egonol glucoside (3), dihydrodehydrodiconiferyl alcohol 9'-O-glucoside (4), and styraxjaponoside B (5) were isolated from the stem bark of Styrax japonica. Among them, compounds 1 and 5 showed significantly high virus-cell fusion inhibitory activity. In addition, compound 5 exhibited almost equivalent virus-cell fusion inhibitory activity to that of dextran sulfate, which is used as a positive control.

  1. Detection of antistaphylococcal and toxic compounds by biological assay systems developed with a reporter Staphylococcus aureus strain harboring a heat inducible promoter - lacZ transcriptional fusion.

    PubMed

    Chanda, Palas Kumar; Ganguly, Tridib; Das, Malabika; Lee, Chia Yen; Luong, Thanh T; Sau, Subrata

    2007-11-30

    Previously it was reported that promoter of groES-groEL operon of Staphylococcus aureus is induced by various cell-wall active antibiotics. In order to exploit the above promoter for identifying novel antistaphylococcal drugs, we have cloned the promoter containing region (P(g)) of groES-groEL operon of S. aureus Newman and found that the above promoter is induced by sublethal concentrations of many antibiotics including cell-wall active antibiotics. A reporter S. aureus RN4220 strain (designated SAU006) was constructed by inserting the P(g)-lacZ transcriptional fusion into its chromosome. Agarose-based assay developed with SAU006 shows that P(g) in single-copy is also induced distinctly by different classes of antibiotics. Data indicate that ciprofloxacin, rifampicin, ampicillin, and cephalothin are strong inducers, whereas, tetracycline, streptomycin and vancomycin induce the above promoter weakly. Sublethal concentrations of ciprofloxacin and ampicilin even have induced P(g) efficiently in microtiter plate grown SAU006. Additional studies show for the first time that above promoter is also induced weakly by arsenate salt and hydrogen peroxide. Taken together, we suggest that our simple and sensitive assay systems with SAU006 could be utilized for screening and detecting not only novel antistaphylococcal compounds but also different toxic chemicals.

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

    PubMed

    Ozawa, Masayuki

    2015-10-09

    Myoblast fusion is a highly regulated process that is essential for skeletal muscle formation during muscle development and regeneration in mammals. Much remains to be elucidated about the molecular mechanism of myoblast fusion although cadherins, which are Ca(2+)-dependent cell-cell adhesion molecules, are thought to play a critical role in this process. Mouse myoblasts lacking either N-cadherin or M-cadherin can still fuse to form myotubes, indicating that they have no specific function in this process and may be functionally replaced by either M-cadherin or N-cadherin, respectively. In this study, we show that expressing the E-cadherin cytoplasmic domain ectopically in C2C12 myoblasts inhibits cell surface localization of endogenous M-cadherin and N-cadherin, as well as cell-cell fusion. This domain, however, does not inhibit myoblast differentiation according to microarray-based gene expression analysis. In contrast, expressing a dominant-negative β-catenin mutant ectopically, which suppresses Wnt/β-catenin signaling, did not inhibit cell-cell fusion. Therefore, the E-cadherin cytoplasmic domain inhibits cell-cell fusion by inhibiting cell surface localization of endogenous cadherins and not by inhibiting Wnt/β-catenin signaling.

  3. Efficiency and beam quality deterioration in double-cladding fiber amplifiers induced by core misalignment of fusion splices

    NASA Astrophysics Data System (ADS)

    Fu, Chen; Yan, Ping; Xiao, Qirong; li, Dan; Gong, Mali

    2015-09-01

    In this study, the efficiency and beam quality deteriorations in double-cladding fiber (DCF) amplifiers induced by core misalignment of series fusion splices are investigated. The losses of guide modes and power conversions between fundamental mode and high order modes (HOM) are well described according to mode coupling theory. Furthermore, a typical fiber amplifier with three fusion splices and bending mode-selection is established to discuss the mechanism of deteriorations in total systems. Mode competition theory is also introduced to describe different modes powers and beam quality changes with fiber length. Based on these calculations, the accumulations of deterioration caused by series fusion splices are intuitively illustrated. These simulations of laser performance show good accordance with the experimental results. An effective method to significantly enhance the performance of the fiber amplifier is also shown and discussed.

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

    NASA Technical Reports Server (NTRS)

    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.

  5. Regulation of cell protrusions by small GTPases during fusion of the neural folds

    PubMed Central

    Rolo, Ana; Savery, Dawn; Escuin, Sarah; de Castro, Sandra C; Armer, Hannah EJ; Munro, Peter MG; Molè, Matteo A; Greene, Nicholas DE; Copp, Andrew J

    2016-01-01

    Epithelial fusion is a crucial process in embryonic development, and its failure underlies several clinically important birth defects. For example, failure of neural fold fusion during neurulation leads to open neural tube defects including spina bifida. Using mouse embryos, we show that cell protrusions emanating from the apposed neural fold tips, at the interface between the neuroepithelium and the surface ectoderm, are required for completion of neural tube closure. By genetically ablating the cytoskeletal regulators Rac1 or Cdc42 in the dorsal neuroepithelium, or in the surface ectoderm, we show that these protrusions originate from surface ectodermal cells and that Rac1 is necessary for the formation of membrane ruffles which typify late closure stages, whereas Cdc42 is required for the predominance of filopodia in early neurulation. This study provides evidence for the essential role and molecular regulation of membrane protrusions prior to fusion of a key organ primordium in mammalian development. DOI: http://dx.doi.org/10.7554/eLife.13273.001 PMID:27114066

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

    PubMed Central

    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 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

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

    SciTech Connect

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

    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.

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

    SciTech Connect

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

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

    SciTech Connect

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

    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

  10. TMPRSS2-ERG Fusion Gene Expression in Prostate Tumor Cells and Its Clinical and Biological Significance in Prostate Cancer Progression

    PubMed Central

    St. John, Jason; Powell, Katelyn; Conley-LaComb, M. Katie; Chinni, Sreenivasa R.

    2012-01-01

    TMPRSS2-Ets gene fusions were identified in prostate cancers where the promoter of transmembrane protease, serine 2 (TMPRSS2) fused with coding sequence of the erythroblastosis virus E26 (Ets) gene family members. TMPRSS2 is an androgen responsive transmembrane serine protease. Ets family members are oncogenic transcription factors that contain a highly conserved Ets DNA binding domain and an N-terminal regulatory domain. Fusion of these gene results in androgen dependent transcription of Ets factor in prostate tumor cells. The ERG is the most common fusion partner with TMPRSS2 promoter in prostate cancer patients. The high prevalence of these gene fusions, in particular TMPRSS2-ERG, makes them attractive as potential diagnostic and prognostic indicators, as well as making them a potential target for tailored therapies. This review focuses on the clinical and biological significance of TMPRSS2-ERG fusions and their role in PC development and progression. PMID:23264855

  11. Membrane Cholesterol Regulates Lysosome-Plasma Membrane Fusion Events and Modulates Trypanosoma cruzi Invasion of Host Cells

    PubMed Central

    Hissa, Bárbara; Duarte, Jacqueline G.; Kelles, Ludmila F.; Santos, Fabio P.; del Puerto, Helen L.; Gazzinelli-Guimarães, Pedro H.; de Paula, Ana M.; Agero, Ubirajara; Mesquita, Oscar N.; Guatimosim, Cristina; Chiari, Egler; Andrade, Luciana O.

    2012-01-01

    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 lysosomes are

  12. Fusion Toxin BLyS-Gelonin Inhibits Growth of Malignant Human B Cell Lines In Vitro and In Vivo

    PubMed Central

    Luster, Troy A.; Mukherjee, Ipsita; Carrell, Jeffrey A.; Cho, Yun Hee; Gill, Jeffrey; Kelly, Lizbeth; Garcia, Andy; Ward, Christopher; Oh, Luke; Ullrich, Stephen J.; Migone, Thi-Sau; Humphreys, Robin

    2012-01-01

    B lymphocyte stimulator (BLyS) is a member of the TNF superfamily of cytokines. The biological activity of BLyS is mediated by three cell surface receptors: BR3/BAFF-R, TACI and BCMA. The expression of these receptors is highly restricted to B cells, both normal and malignant. A BLyS-gelonin fusion toxin (BLyS-gel) was generated consisting of the recombinant plant-derived toxin gelonin fused to the N-terminus of BLyS and tested against a large and diverse panel of B-NHL cell lines. Interestingly, B-NHL subtypes mantle cell lymphoma (MCL), diffuse large B cell lymphoma (DLBCL) and B cell precursor-acute lymphocytic leukemia (BCP-ALL) were preferentially sensitive to BLyS-gel mediated cytotoxicity, with low picomolar EC50 values. BLyS receptor expression did not guarantee sensitivity to BLyS-gel, even though the construct was internalized by both sensitive and resistant cells. Resistance to BLyS-gel could be overcome by treatment with the endosomotropic drug chloroquine, suggesting BLyS-gel may become trapped within endosomal/lysosomal compartments in resistant cells. BLyS-gel induced cell death was caspase-independent and shown to be at least partially mediated by the “ribotoxic stress response.” This response involves activation of p38 MAPK and JNK/SAPK, and BLyS-gel mediated cytotoxicity was inhibited by the p38/JNK inhibitor SB203580. Finally, BLyS-gel treatment was shown to localize to sites of disease, rapidly reduce tumor burden, and significantly prolong survival in xenograft mouse models of disseminated BCP-ALL, DLBCL, and MCL. Together, these findings suggest BLyS has significant potential as a targeting ligand for the delivery of cytotoxic “payloads” to malignant B cells. PMID:23056634

  13. Antibody–peptide–MHC fusion conjugates target non-cognate T cells to kill tumour cells

    PubMed Central

    King, Ben C.; Hamblin, Angela D.; Savage, Philip M.; Douglas, Leon R.; Hansen, Ted H.; Johnson, Peter W. M.; Glennie, Martin J.

    2013-01-01

    Attempts to generate robust anti-tumour cytotoxic T lymphocyte (CTL) responses using immunotherapy are frequently thwarted by exhaustion and anergy of CTL recruited to tumour. One strategy to overcome this is to retarget a population of virus-specific CTL to kill tumour cells. Here, we describe a proof-of-principle study using a bispecific conjugate designed to retarget ovalbumin (OVA)-specific CTL to kill tumour cells via CD20. A single-chain trimer (SCT) consisting of MHCI H-2Kb/SI-INFEKL peptide/beta 2 microglobulin/BirA was expressed in bacteria, refolded and chemically conjugated to one (1:1; F2) or two (2:1; F3) anti-hCD20 Fab′ fragments. In vitro, the [SCT × Fab′] (F2 and F3) redirected SIINFEKL-specific OT-I CTL to kill CD20+ target cells, and in the presence of CD20+ target cells to provide crosslinking, they were also able to induce proliferation of OT-I cells. In vivo, activated OT-I CTL could be retargeted to kill [SCT × Fab′]-coated B cells from hCD20 transgenic (hCD20 Tg) mice and also EL4 and B16 mouse tumour cells expressing human CD20 (hCD20). Importantly, in a hCD20 Tg mouse model, [SCT × Fab′] administered systemically were able to retarget activated OT-I cells to deplete normal B cells, and their performance matched that of a bispecific antibody (BsAb) comprising anti-CD3 and anti-CD20. [SCT × Fab′] were also active therapeutically in an EL4 tumour model. Furthermore, measurement of serum cytokine levels suggests that [SCT × Fab′] are associated with a lower level of inflammatory cytokine release than the BsAb and so may be advantageous clinically in terms of reduced toxicity. PMID:23604105

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

    SciTech Connect

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

    2016-07-15

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

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

    SciTech Connect

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

    2008-12-19

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

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

    SciTech Connect

    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 Yxx{phi} 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{sub 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

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

    PubMed

    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

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

    NASA Astrophysics Data System (ADS)

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

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

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

    DTIC Science & Technology

    2007-07-01

    IL-12 and IFNγ. In addition, fusion cells expressed CCR7 necessary for the migration of cells to sites of T cell traffic in the draining lymph...Conclusions Our clinical protocol has received approval by the FDA, NCI/CTEP (distributor of IL-12) and Dana-Farber/Harvard Cancer Center. We have also

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

    PubMed

    Foo, Chwan Hong; Rootes, Christina L; Cowley, Karla; Marsh, Glenn A; Gould, Cathryn M; Deffrasnes, Celine; Cowled, Christopher J; Klein, Reuben; Riddell, Sarah J; Middleton, Deborah; Simpson, Kaylene J; Wang, Lin-Fa; Bean, Andrew G D; Stewart, Cameron R

    2016-10-01

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

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

    PubMed Central

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

    2016-01-01

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

  2. Viral membrane fusion.

    PubMed

    Harrison, Stephen C

    2015-05-01

    Membrane fusion is an essential step when enveloped viruses enter cells. Lipid bilayer fusion requires catalysis to overcome a high kinetic barrier; viral fusion proteins are the agents that fulfill this catalytic function. Despite a variety of molecular architectures, these proteins facilitate fusion by essentially the same generic mechanism. Stimulated by a signal associated with arrival at the cell to be infected (e.g., receptor or co-receptor binding, proton binding in an endosome), they undergo a series of conformational changes. A hydrophobic segment (a "fusion loop" or "fusion peptide") engages the target-cell membrane and collapse of the bridging intermediate thus formed draws the two membranes (virus and cell) together. We know of three structural classes for viral fusion proteins. Structures for both pre- and postfusion conformations of illustrate the beginning and end points of a process that can be probed by single-virion measurements of fusion kinetics.

  3. Viral membrane fusion

    PubMed Central

    Harrison, Stephen C.

    2015-01-01

    Membrane fusion is an essential step when enveloped viruses enter cells. Lipid bilayer fusion requires catalysis to overcome a high kinetic barrier; viral fusion proteins are the agents that fulfill this catalytic function. Despite a variety of molecular architectures, these proteins facilitate fusion by essentially the same generic mechanism. Stimulated by a signal associated with arrival at the cell to be infected (e.g., receptor or co-receptor binding, proton binding in an endosome), they undergo a series of conformational changes. A hydrophobic segment (a “fusion loop” or “fusion peptide”) engages the target-cell membrane and collapse of the bridging intermediate thus formed draws the two membranes (virus and cell) together. We know of three structural classes for viral fusion proteins. Structures for both pre- and postfusion conformations of illustrate the beginning and end points of a process that can be probed by single-virion measurements of fusion kinetics. PMID:25866377

  4. R-spondin1 Controls Muscle Cell Fusion through Dual Regulation of Antagonistic Wnt Signaling Pathways.

    PubMed

    Lacour, Floriane; Vezin, Elsa; Bentzinger, C Florian; Sincennes, Marie-Claude; Giordani, Lorenzo; Ferry, Arnaud; Mitchell, Robert; Patel, Ketan; Rudnicki, Michael A; Chaboissier, Marie-Christine; Chassot, Anne-Amandine; Le Grand, Fabien

    2017-03-07

    Wnt-mediated signals are involved in many important steps in mammalian regeneration. In multiple cell types, the R-spondin (Rspo) family of secreted proteins potently activates the canonical Wnt/β-catenin pathway. Here, we identify Rspo1 as a mediator of skeletal muscle tissue repair. First, we show that deletion of Rspo1 results in global alteration of muscle regeneration kinetics following acute injury. We find that muscle progenitor cells lacking Rspo1 show delayed differentiation due to reduced activation of Wnt/β-catenin target genes. Furthermore, muscle cells lacking Rspo1 have a fusion phenotype leading to larger myotubes containing supernumerary nuclei both in vitro and in vivo. The increase in muscle fusion was dependent on downregulation of Wnt/β-catenin and upregulation of non-canonical Wnt7a/Fzd7/Rac1 signaling. We conclude that reciprocal control of antagonistic Wnt signaling pathways by Rspo1 in muscle stem cell progeny is a key step ensuring normal tissue architecture restoration following acute damage.

  5. Early Events in Chikungunya Virus Infection—From Virus Cell Binding to Membrane Fusion

    PubMed Central

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

    2015-01-01

    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. PMID:26198242

  6. A Fusion Algorithm for GFP Image and Phase Contrast Image of Arabidopsis Cell Based on SFL-Contourlet Transform

    PubMed Central

    Feng, Peng; Wang, Jing; Wei, Biao; Mi, Deling

    2013-01-01

    A hybrid multiscale and multilevel image fusion algorithm for green fluorescent protein (GFP) image and phase contrast image of Arabidopsis cell is proposed in this paper. Combining intensity-hue-saturation (IHS) transform and sharp frequency localization Contourlet transform (SFL-CT), this algorithm uses different fusion strategies for different detailed subbands, which include neighborhood consistency measurement (NCM) that can adaptively find balance between color background and gray structure. Also two kinds of neighborhood classes based on empirical model are taken into consideration. Visual information fidelity (VIF) as an objective criterion is introduced to evaluate the fusion image. The experimental results of 117 groups of Arabidopsis cell image from John Innes Center show that the new algorithm cannot only make the details of original images well preserved but also improve the visibility of the fusion image, which shows the superiority of the novel method to traditional ones. PMID:23476716

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

    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

  9. HIV Fusion Peptide Penetrates, Disorders, and Softens T-Cell Membrane Mimics

    PubMed Central

    Tristram-Nagle, Stephanie; Chan, Rob; Kooijman, Edgar; Uppamoochikkal, Pradeep; Qiang, Wei; Weliky, David P.; Nagle, John F.

    2010-01-01

    This work investigates the interaction of N-terminal gp41 fusion peptide (FP) of human immunodeficiency virus type 1 (HIV-1) with model membranes in order to elucidate how FP leads to fusion of HIV and T-cell membranes. FP constructs were (i) wild-type FP23 (23 N-terminal amino acids of gp41), (ii) water-soluble monomeric FP that adds six lysines on the C-terminus of FP23 (FPwsm), and (iii) the C-terminus covalently linked trimeric version (FPtri) of FPwsm. Model membranes were (i) LM3 (a T-cell mimic), (ii) 1,2-dioleoyl-sn-glycero-3-phosphocholine, (iii) 1,2-dioleoyl-sn-glycero-3-phosphocholine/30 mol% cholesterol, (iv) 1,2-dierucoyl-sn-glycero-3-phosphocholine, and (v) 1,2-dierucoyl-sn-glycero-3-phosphocholine/30 mol% cholesterol. Diffuse synchrotron low-angle x-ray scattering from fully hydrated samples, supplemented by volumetric data, showed that FP23 and FPtri penetrate into the hydrocarbon region and cause membranes to thin. Depth of penetration appears to depend upon a complex combination of factors including bilayer thickness, presence of cholesterol, and electrostatics. X-ray data showed an increase in curvature in hexagonal phase 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, which further indicates that FP23 penetrates into the hydrocarbon region rather than residing in the interfacial headgroup region. Low-angle x-ray scattering data also yielded the bending modulus KC, a measure of membrane stiffness, and wide-angle x-ray scattering yielded the Sxray orientational order parameter. Both FP23 and FPtri decreased KC and Sxray considerably, while the weak effect of FPwsm suggests that it did not partition strongly into LM3 model membranes. Our results are consistent with the HIV FP disordering and softening the T-cell membrane, thereby lowering the activation energy for viral membrane fusion. PMID:20655315

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

    PubMed

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

    2014-04-01

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

  11. Low-dose ionizing radiation induces mitochondrial fusion and increases expression of mitochondrial complexes I and III in hippocampal neurons

    PubMed Central

    Chang, Chuang-Rung; Kao, Mou-Chieh; Chen, Kuan-Wei; Chiu, Shih-Che; Hsu, Ming-Ling; Hsiang, I-Chou; Chen, Yu-Jen; Chen, Linyi

    2015-01-01

    High energy ionizing radiation can cause DNA damage and cell death. During clinical radiation therapy, the radiation dose could range from 15 to 60 Gy depending on targets. While 2 Gy radiation has been shown to cause cancer cell death, studies also suggest a protective potential by low dose radiation. In this study, we examined the effect of 0.2-2 Gy radiation on hippocampal neurons. Low dose 0.2 Gy radiation treatment increased the levels of MTT. Since hippocampal neurons are post-mitotic, this result reveals a possibility that 0.2 Gy irradiation may increase mitochondrial activity to cope with stimuli. Maintaining neural plasticity is an energy-demanding process that requires high efficient mitochondrial function. We thus hypothesized that low dose radiation may regulate mitochondrial dynamics and function to ensure survival of neurons. Our results showed that five days after 0.2 Gy irradiation, no obvious changes on neuronal survival, neuronal synapses, membrane potential of mitochondria, reactive oxygen species levels, and mitochondrial DNA copy numbers. Interestingly, 0.2 Gy irradiation promoted the mitochondria fusion, resulting in part from the increased level of a mitochondrial fusion protein, Mfn2, and inhibition of Drp1 fission protein trafficking to the mitochondria. Accompanying with the increased mitochondrial fusion, the expressions of complexes I and III of the electron transport chain were also increased. These findings suggest that, hippocampal neurons undergo increased mitochondrial fusion to modulate cellular activity as an adaptive mechanism in response to low dose radiation. PMID:26415228

  12. Low-dose ionizing radiation induces mitochondrial fusion and increases expression of mitochondrial complexes I and III in hippocampal neurons.

    PubMed

    Chien, Ling; Chen, Wun-Ke; Liu, Szu-Ting; Chang, Chuang-Rung; Kao, Mou-Chieh; Chen, Kuan-Wei; Chiu, Shih-Che; Hsu, Ming-Ling; Hsiang, I-Chou; Chen, Yu-Jen; Chen, Linyi

    2015-10-13

    High energy ionizing radiation can cause DNA damage and cell death. During clinical radiation therapy, the radiation dose could range from 15 to 60 Gy depending on targets. While 2 Gy radiation has been shown to cause cancer cell death, studies also suggest a protective potential by low dose radiation. In this study, we examined the effect of 0.2-2 Gy radiation on hippocampal neurons. Low dose 0.2 Gy radiation treatment increased the levels of MTT. Since hippocampal neurons are post-mitotic, this result reveals a possibility that 0.2 Gy irradiation may increase mitochondrial activity to cope with stimuli. Maintaining neural plasticity is an energy-demanding process that requires high efficient mitochondrial function. We thus hypothesized that low dose radiation may regulate mitochondrial dynamics and function to ensure survival of neurons. Our results showed that five days after 0.2 Gy irradiation, no obvious changes on neuronal survival, neuronal synapses, membrane potential of mitochondria, reactive oxygen species levels, and mitochondrial DNA copy numbers. Interestingly, 0.2 Gy irradiation promoted the mitochondria fusion, resulting in part from the increased level of a mitochondrial fusion protein, Mfn2, and inhibition of Drp1 fission protein trafficking to the mitochondria. Accompanying with the increased mitochondrial fusion, the expressions of complexes I and III of the electron transport chain were also increased. These findings suggest that, hippocampal neurons undergo increased mitochondrial fusion to modulate cellular activity as an adaptive mechanism in response to low dose radiation.

  13. Furin cleavage of the SARS coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry

    SciTech Connect

    Follis, Kathryn E.; York, Joanne; Nunberg, Jack H. . E-mail: jack.nunberg@umontana.edu

    2006-07-05

    The fusogenic potential of Class I viral envelope glycoproteins is activated by proteloytic cleavage of the precursor glycoprotein to generate the mature receptor-binding and transmembrane fusion subunits. Although the coronavirus (CoV) S glycoproteins share membership in this class of envelope glycoproteins, cleavage to generate the respective S1 and S2 subunits appears absent in a subset of CoV species, including that responsible for the severe acute respiratory syndrome (SARS). To determine whether proteolytic cleavage of the S glycoprotein might be important for the newly emerged SARS-CoV, we introduced a furin recognition site at single basic residues within the putative S1-S2 junctional region. We show that furin cleavage at the modified R667 position generates discrete S1 and S2 subunits and potentiates membrane fusion activity. This effect on the cell-cell fusion activity by the S glycoprotein is not, however, reflected in the infectivity of pseudotyped lentiviruses bearing the cleaved glycoprotein. The lack of effect of furin cleavage on virion infectivity mirrors that observed in the normally cleaved S glycoprotein of the murine coronavirus and highlights an additional level of complexity in coronavirus entry.

  14. Fusion and regenerative therapies: is immortality really recessive?

    PubMed

    Stolzing, Alexandra; Hescheler, Jürgen; Sethe, Sebastian

    2007-12-01

    Harnessing cellular fusion as a potential tool for regenerative therapy has been under tentative investigation for decades. A look back the history of fusion experiments in gerontology reveals that whereas some studies indicate that aging-related changes are conserved in fused cells, others have demonstrated that fusion can be used as a tool to revoke cellular senescence and induce tissue regeneration. Recent findings about the role of fusion processes in tissue homeostasis, replenishment, and repair link insights from fusion studies of previous decades with modern developments in stem cell biology and regenerative medicine. We suggest that age-associated loss of regenerative capacity is associated with a decline of effectiveness in stem cell fusion. We project how studies into the fusion of stem cells with tissue cells, or the fusion between activator stem cells and patient cells might help in the development of applications that "rejuvenate" certain target cells, thereby strategically reinstating a regeneration cascade. The outlook is concluded with a discussion of the next research milestones and the potential hazards of fusion therapies.

  15. Protective effects of a bacterially expressed NIF-KGF fusion protein against bleomycin-induced acute lung injury in mice.

    PubMed

    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.

  16. Rat-derived amniotic epithelial cells differentiate into mature hepatocytes in vivo with no evidence of cell fusion.

    PubMed

    Marongiu, Michela; Serra, Maria Paola; Contini, Antonella; Sini, Marcella; Strom, Stephen C; Laconi, Ezio; Marongiu, Fabio

    2015-06-15

    Amniotic epithelial cells (AEC) derived from human placenta represent a useful and noncontroversial source for liver-based regenerative medicine. Previous studies suggested that human- and rat-derived AEC differentiate into hepatocyte-like cells upon transplantation. In the retrorsine (RS) model of liver repopulation, clusters of donor-derived cells engrafted in the recipient liver and, importantly, showed characteristics of mature hepatocytes. The aim of the current study was to investigate the possible involvement of cell fusion in the emergence of hepatocyte clusters displaying a donor-specific phenotype. To this end, 4-week-old GFP(+)/DPP-IV(-) rats were treated with RS and then transplanted with undifferentiated AEC isolated from the placenta of DPP-IV(+) pregnant rats at 16-19 days of gestational age. Results indicated that clusters of donor-derived cells were dipeptidyl peptidase type IV (DPP-IV) positive, but did not express the green fluorescent protein (GFP), suggesting that rat amniotic epithelial cells (rAEC) did not fuse within the host parenchyma, as no colocalization of the two tags was observed. Moreover, rAEC-derived clusters expressed markers of mature hepatocytes (eg, albumin, cytochrome P450), but were negative for the expression of biliary/progenitor markers (eg, epithelial cell adhesion molecule [EpCAM]) and did not express the marker of preneoplastic hepatic nodules glutathione S-transferase P (GST-P). These results extend our previous findings on the potential of AEC to differentiate into mature hepatocytes and suggest that this process can occur in the absence of cell fusion with host-derived cells. These studies support the hypothesis that amnion-derived epithelial cells can be an effective cell source for the correction of liver disease.

  17. Sulforaphane induces differential modulation of mitochondrial biogenesis and dynamics in normal cells and tumor cells.

    PubMed

    Negrette-Guzmán, Mario; Huerta-Yepez, Sara; Vega, Mario I; León-Contreras, Juan Carlos; Hernández-Pando, Rogelio; Medina-Campos, Omar Noel; Rodríguez, Esteban; Tapia, Edilia; Pedraza-Chaverri, José

    2017-02-01

    Antioxidant-based chemotherapy has been intensely debated. Herein, we show that sulforaphane (SFN) induced mitochondrial biogenesis followed by mitochondrial fusion in a kidney cell line commonly used in nephroprotective models. At the same concentration and exposure time, SFN induced cell death in prostate cancer cells accompanied by mitochondrial biogenesis and fragmentation. Stabilization of the nuclear factor E2-related factor-2 (Nrf2) could be associated with these effects in the tumor cell line. An increase in the peroxisome proliferator-activated receptor-γ co-activator-1α (PGC1α) level and a decrease in the hypoxia-inducible factor-1α (HIF1α) level would suggest a possible metabolic shift. The knockdown in the nuclear respiratory factor-1 (NRF1) attenuated the SFN-induced effect on prostate cancer cells demonstrating that mitochondrial biogenesis plays an important role in cell death for this kind of tumor cells. This evidence supports SFN as a potential antineoplastic agent that could inhibit tumor development and could protect normal tissues by modulating common processes.

  18. Ewing sarcoma cells secrete EWS/Fli-1 fusion mRNA via microvesicles.

    PubMed

    Tsugita, Masanori; Yamada, Nami; Noguchi, Shunsuke; Yamada, Kazunari; Moritake, Hiroshi; Shimizu, Katsuji; Akao, Yukihiro; Ohno, Takatoshi

    2013-01-01

    Tumours defined as Ewing sarcoma (ES) constitute a group of highly malignant neoplasms that most often affect children and young adults in the first 2 decades of life. The EWS/Fli-1 fusion gene, a product of the translocation t(11;22) (q24; 12), is detected in 95% of ES patients. Recently, it was validated that cells emit a heterogeneous mixture of vesicular, organelle-like structures (microvesicles, MVs) into their surroundings including blood and body fluids, and that these MVs contain a selected set of tumor-related proteins and high levels of mRNAs and miRNAs. In this present study, we detected the Ewing sarcoma-specific EWS/Fli-1 mRNA in MVs from the culture medium of ES cell lines carrying t(11;22) (q24; 12). Also, we detected this fusion gene in approximately 40% of the blood samples from mice inoculated with xenografts of TC135 or A673 cells. These findings indicate the EWS/Fli-1 mRNA in MVs might be a new non-invasive diagnostic marker for specific cases of Ewing sarcoma.

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

    SciTech Connect

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

    2009-07-31

    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.

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

    PubMed

    Schiffer, S; Hansen, H P; Hehmann-Titt, G; Huhn, M; Fischer, R; Barth, S; Thepen, T

    2013-03-22

    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.

  1. SARS-CoV fusion peptides induce membrane surface ordering and curvature.

    PubMed

    Basso, Luis G M; Vicente, Eduardo F; Crusca, Edson; Cilli, Eduardo M; Costa-Filho, Antonio J

    2016-11-28

    Viral membrane fusion is an orchestrated process triggered by membrane-anchored viral fusion glycoproteins. The S2 subunit of the spike glycoprotein from severe acute respiratory syndrome (SARS) coronavirus (CoV) contains internal domains called fusion peptides (FP) that play essential roles in virus entry. Although membrane fusion has been broadly studied, there are still major gaps in the molecular details of lipid rearrangements in the bilayer during fusion peptide-membrane interactions. Here we employed differential scanning calorimetry (DSC) and electron spin resonance (ESR) to gather information on the membrane fusion mechanism promoted by two putative SARS FPs. DSC data showed the peptides strongly perturb the structural integrity of anionic vesicles and support the hypothesis that the peptides generate opposing curvature stresses on phosphatidylethanolamine membranes. ESR showed that both FPs increase lipid packing and head group ordering as well as reduce the intramembrane water content for anionic membranes. Therefore, bending moment in the bilayer could be generated, promoting negative curvature. The significance of the ordering effect, membrane dehydration, changes in the curvature properties and the possible role of negatively charged phospholipids in helping to overcome the high kinetic barrier involved in the different stages of the SARS-CoV-mediated membrane fusion are discussed.

  2. SARS-CoV fusion peptides induce membrane surface ordering and curvature

    PubMed Central

    Basso, Luis G. M.; Vicente, Eduardo F.; Crusca Jr., Edson; Cilli, Eduardo M.; Costa-Filho, Antonio J.

    2016-01-01

    Viral membrane fusion is an orchestrated process triggered by membrane-anchored viral fusion glycoproteins. The S2 subunit of the spike glycoprotein from severe acute respiratory syndrome (SARS) coronavirus (CoV) contains internal domains called fusion peptides (FP) that play essential roles in virus entry. Although membrane fusion has been broadly studied, there are still major gaps in the molecular details of lipid rearrangements in the bilayer during fusion peptide-membrane interactions. Here we employed differential scanning calorimetry (DSC) and electron spin resonance (ESR) to gather information on the membrane fusion mechanism promoted by two putative SARS FPs. DSC data showed the peptides strongly perturb the structural integrity of anionic vesicles and support the hypothesis that the peptides generate opposing curvature stresses on phosphatidylethanolamine membranes. ESR showed that both FPs increase lipid packing and head group ordering as well as reduce the intramembrane water content for anionic membranes. Therefore, bending moment in the bilayer could be generated, promoting negative curvature. The significance of the ordering effect, membrane dehydration, changes in the curvature properties and the possible role of negatively charged phospholipids in helping to overcome the high kinetic barrier involved in the different stages of the SARS-CoV-mediated membrane fusion are discussed. PMID:27892522

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

    PubMed Central

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

    2015-01-01

    HIV-1 Env mediates fusion of viral and target cell membranes, but it can also mediate fusion of infected (producer) and target cells, thus triggering the formation of multinucleated cells, so-called syncytia. Large, round, immobile syncytia are readily observable in cultures of HIV-1-infected T cells, but these fast growing “fusion sinks” are largely regarded as cell culture artifacts. In contrast, small HIV-1-induced syncytia were seen in the paracortex of peripheral lymph nodes and other secondary lymphoid tissue of HIV-1-positive individuals. Further, recent intravital imaging of lymph nodes in humanized mice early after their infection with HIV-1 demonstrated that a significant fraction of infected cells were highly mobile, small syncytia, suggesting that these entities contribute to virus dissemination. Here, we report that the formation of small, migratory syncytia, for which we provide further quantification in humanized mice, can be recapitulated in vitro if HIV-1-infected T cells are placed into 3D extracellular matrix (ECM) hydrogels rather than being kept in traditional suspension culture systems. Intriguingly, live-cell imaging in hydrogels revealed that these syncytia, similar to individual infected cells, can transiently interact with uninfected cells, leading to rapid virus transfer without cell-cell fusion. Infected cells were also observed to deposit large amounts of viral particles into the extracellular space. Altogether, these observations suggest the need to further evaluate the biological significance of small, T cell-based syncytia and to consider the possibility that these entities do indeed contribute to virus spread and pathogenesis. PMID:26703714

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

    PubMed

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

    2015-12-12

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

  5. Molecular Process Producing Oncogene Fusion in Lung Cancer Cells by Illegitimate Repair of DNA Double-Strand Breaks

    PubMed Central

    Seki, Yoshitaka; Mizukami, Tatsuji; Kohno, Takashi

    2015-01-01

    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. PMID:26437441

  6. RESOLFT Nanoscopy of Fixed Cells Using a Z-Domain Based Fusion Protein for Labelling.

    PubMed

    Ilgen, Peter; Grotjohann, Tim; Jans, Daniel C; Kilisch, Markus; Hell, Stefan W; Jakobs, Stefan

    2015-01-01

    RESOLFT super-resolution microscopy allows subdiffraction resolution imaging of living cells using low intensities of light. It relies on the light-driven switching of reversible switchable fluorescent proteins (RSFPs). So far, RESOLFT imaging was restricted to living cells, because chemical fixation typically affects the switching characteristics of RSFPs. In this study we created a fusion construct (FLASR) consisting of the RSFP rsEGFP2 and the divalent form of the antibody binding Z domain from protein A. FLASR can be used analogous to secondary antibodies in conventional immunochemistry, facilitating simple and robust sample preparation. We demonstrate RESOLFT super-resolution microscopy on chemically fixed mammalian cells. The approach may be extended to other super-resolution approaches requiring fluorescent proteins in an aqueous environment.

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

    SciTech Connect

    Zhang Xufeng; Wu Guoxiang; Chen, Jian-Quan; Zhang Aimin; Liu Siguo; Jiao Binghua . E-mail: jiaobh@uninet.com.cn; Cheng Guoxiang . E-mail: Chenggx@cngenon.com

    2005-07-22

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    NASA Astrophysics Data System (ADS)

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

    2016-10-01

    Beta-site APP cleaving enzyme 1 (BACE1) is a type I transmembrane aspartyl protease. In the amyloidogenic pathway, BACE1 provides β-secretase activity that cleaves the amyloid precursor protein (APP) that leads to amyloid beta (Aβ) peptides. The aggregation of these Aβ will ultimately results in amyloid plaque formation, a hallmark of Alzheimer's disease (AD). Amyloid aggregation leads to progressive memory impairment and neural loss. Recent detergent protein extraction studies suggest that the untreated BACE1 protein forms a dimer that has significantly higher catalytic activity than its monomeric counterpart. Here, we examine the dimerization hypothesis of BACE1 in cultured HEK293 cells using fluorescence correlation spectroscopy (FCS). Cells were transfected with a BACE1-EGFP fusion protein construct and imaged using confocal and DIC microscopy to monitor labeled BACE1 localization and distribution within the cell. Our one-photon fluorescence fluctuation autocorrelation of BACE1- EGFP on the plasma membrane of HEK cells is modeled using two diffusing species on the plasma membrane with estimated diffusion coefficients of 1.39 x 10-7 cm2/sec and 2.8 x 10-8 cm2/sec under resting conditions and STA-200 inhibition, respectively. Anomalous diffusion model also provided adequate description of the observed autocorrelation function of BACE1- EGFP on the plasma membrane with an estimate exponent (α) of 0.8 and 0.5 for resting and STA-200 treated cells, respectively. The corresponding hydrodynamic radius of this transmembrane fusion protein was estimated using the measured diffusion coefficients assuming both Stokes-Einstein and Saffman-Delbruck models. Our results suggest a complex diffusion pattern of BACE1-EGFP on the plasma membrane of HEK cells with the possibility for dimer formation, especially under STA-200 inhibition.

  10. Ribeye a-mCherry fusion protein: a novel tool for labeling synaptic ribbons of the hair cell.

    PubMed

    West, Megan C; McDermott, Brian M

    2011-04-30

    Synaptic ribbons are presynaptic cytomatrices that are required for efficient transfer of auditory information from hair cells to the central nervous system. In the hair cell, each electron-dense ribbon tethers numerous synaptic vesicles by fine filaments. The ribbon generally resides juxtaposed to the active zone plasma membrane. A dearth of appropriate tools to visualize the ribbon synapse has limited our knowledge of its development. Here we present the design and implementation of a method to visualize synaptic ribbons in hair cells. This scheme uses a tagged version of the protein Ribeye a, which is specific to ribbons. We generate the DNA construct Tg(pvalb3b:ribeye a-mCherry) to transgenically express the fusion protein Ribeye a-mCherry in zebrafish hair cells. The fusion protein localizes to the basolateral surface of the hair cell with a pattern similar to that of a hair cell labeled with an antiserum that recognizes ribeye proteins. Moreover, using this antiserum to label transgenics that express Ribeye a-mCherry, we demonstrate that the fusion protein and antibody-associated fluorescent signals overlap. In addition, ribbons labeled with the fusion protein are proximal to afferent nerve endings. Finally, the fusion protein labels hair-cell ribbons of zebrafish at different developmental time points. These findings indicate that the fusion protein is an effective tool to label ribbons in live and fixed hair cells, which will make it useful in the study of ribbon synapse development and to characterize zebrafish mutants with defects in synapse formation.

  11. The SNARE Protein Syp71 Is Essential for Turnip Mosaic Virus Infection by Mediating Fusion of Virus-Induced Vesicles with Chloroplasts

    PubMed Central

    Hou, Xilin; Sanfaçon, Hélène; Wang, Aiming

    2013-01-01

    All positive-strand RNA viruses induce the biogenesis of cytoplasmic membrane-bound virus factories for viral genome multiplication. We have previously demonstrated that upon plant potyvirus infection, the potyviral 6K2 integral membrane protein induces the formation of ER-derived replication vesicles that subsequently target chloroplasts for robust genome replication. Here, we report that following the trafficking of the Turnip mosaic potyvirus (TuMV) 6K2 vesicles to chloroplasts, 6K2 vesicles accumulate at the chloroplasts to form chloroplast-bound elongated tubular structures followed by chloroplast aggregation. A functional actomyosin motility system is required for this process. As vesicle trafficking and fusion in planta are facilitated by a superfamily of proteins known as SNAREs (soluble N-ethylmaleimide-sensitive-factor attachment protein receptors), we screened ER-localized SNARES or SNARE-like proteins for their possible involvement in TuMV infection. We identified Syp71 and Vap27-1 that colocalize with the chloroplast-bound 6K2 complex. Knockdown of their expression using a Tobacco rattle virus (TRV)-based virus-induced gene silencing vector showed that Syp71 but not Vap27-1 is essential for TuMV infection. In Syp71-downregulated plant cells, the formation of 6K2-induced chloroplast-bound elongated tubular structures and chloroplast aggregates is inhibited and virus accumulation is significantly reduced, but the trafficking of the 6K2 vesicles from the ER to chloroplast is not affected. Taken together, these data suggest that Syp71 is a host factor essential for successful virus infection by mediating the fusion of the virus-induced vesicles with chloroplasts during TuMV infection. PMID:23696741

  12. Toll-like Receptor 4 Engagement on Dendritic Cells Restrains Phago-Lysosome Fusion and Promotes Cross-Presentation of Antigens.

    PubMed

    Alloatti, Andrés; Kotsias, Fiorella; Pauwels, Anne-Marie; Carpier, Jean-Marie; Jouve, Mabel; Timmerman, Evy; Pace, Luigia; Vargas, Pablo; Maurin, Mathieu; Gehrmann, Ulf; Joannas, Leonel; Vivar, Omar I; Lennon-Duménil, Ana-Maria; Savina, Ariel; Gevaert, Kris; Beyaert, Rudi; Hoffmann, Eik; Amigorena, Sebastian

    2015-12-15

    The initiation of cytotoxic immune responses by dendritic cells (DCs) requires the presentation of antigenic peptides derived from phagocytosed microbes and infected or dead cells to CD8(+) T cells, a process called cross-presentation. Antigen cross-presentation by non-activated DCs, however, is not sufficient for the effective induction of immune responses. Additionally, DCs need to be activated through innate receptors, like Toll-like receptors (TLRs). During DC maturation, cross-presentation efficiency is first upregulated and then turned off. Here we show that during this transient phase of enhanced cross-presentation, phago-lysosome fusion was blocked by the topological re-organization of lysosomes into perinuclear clusters. LPS-induced lysosomal clustering, inhibition of phago-lysosome fusion and enhanced cross-presentation, all required expression of the GTPase Rab34. We conclude that TLR4 engagement induces a Rab34-dependent re-organization of lysosomal distribution that delays antigen degradation to transiently enhance cross-presentation, thereby optimizing the priming of CD8(+) T cell responses against pathogens.

  13. Parvovirus infection-induced cell death and cell cycle arrest

    PubMed Central

    Chen, Aaron Yun; Qiu, Jianming

    2011-01-01

    The cytopathic effects induced during parvovirus infection have been widely documented. Parvovirus infection-induced cell death is often directly associated with disease outcomes (e.g., anemia resulting from loss of erythroid progenitors during parvovirus B19 infection). Apoptosis is the major form of cell death induced by parvovirus infection. However, nonapoptotic cell death, namely necrosis, has also been reported during infection of the minute virus of mice, parvovirus H-1 and bovine parvovirus. Recent studies have revealed multiple mechanisms underlying the cell death during parvovirus infection. These mechanisms vary in different parvoviruses, although the large nonstructural protein (NS)1 and the small NS proteins (e.g., the 11 kDa of parvovirus B19), as well as replication of the viral genome, are responsible for causing infection-induced cell death. Cell cycle arrest is also common, and contributes to the cytopathic effects induced during parvovirus infection. While viral NS proteins have been indicated to induce cell cycle arrest, increasing evidence suggests that a cellular DNA damage response triggered by an invading single-stranded parvoviral genome is the major inducer of cell cycle arrest in parvovirus-infected cells. Apparently, in response to infection, cell death and cell cycle arrest of parvovirus-infected cells are beneficial to the viral cell lifecycle (e.g., viral DNA replication and virus egress). In this article, we will discuss recent advances in the understanding of the mechanisms underlying parvovirus infection-induced cell death and cell cycle arrest. PMID:21331319

  14. Genetic Control of Fusion Pore Expansion in the Epidermis of Caenorhabditis elegans

    PubMed Central

    Gattegno, Tamar; Mittal, Aditya; Valansi, Clari; Nguyen, Ken C.Q.; Hall, David H.; Chernomordik, Leonid V.

    2007-01-01

    Developmental cell fusion is found in germlines, muscles, bones, placentae, and stem cells. In Caenorhabditis elegans 300 somatic cells fuse during development. Although there is extensive information on the early intermediates of viral-induced and intracellular membrane fusion, little is known about late stages in membrane fusion. To dissect the pathway of cell fusion in C. elegans embryos, we use genetic and kinetic analyses using live-confocal and electron microscopy. We simultaneously monitor the rates of multiple cell fusions in developing embryos and find kinetically distinct stages of initiation and completion of membrane fusion in the epidermis. The stages of cell fusion are differentially blocked or retarded in eff-1 and idf-1 mutants. We generate kinetic cell fusion maps for embryos grown at different temperatures. Different sides of the same cell differ in their fusogenicity: the left and right membrane domains are fusion-incompetent, whereas the anterior and posterior membrane domains fuse with autonomous kinetics in embryos. All but one cell pair can initiate the formation of the largest syncytium. The first cell fusion does not trigger a wave of orderly fusions in either direction. Ultrastructural studies show that epidermal syncytiogenesis require eff-1 activities to initiate and expand membrane merger. PMID:17229888

  15. Gene expression profile of ewing sarcoma cell lines differing in their EWS-FLI1 fusion type.

    PubMed

    Bandrés, Eva; Malumbres, Raquel; Escalada, Alvaro; Cubedo, Elena; González, Iranzu; Honorato, Beatriz; Zarate, Ruth; García-Foncillas, Jesus; de Alava, Enrique

    2005-10-01

    The t(11;22)(q24;q12) translocation is present in up to 95% of Ewing tumor patients and results in the formation of an EWS-FLI-1 fusion gene that encodes a chimeric transcription factor. Many alternative forms of EWS-FLI-1 exist because of variations in the location of the EWS and FLI-1 genomic breakpoints. Previous reports have shown that the type 1 fusion is associated with a significantly better prognosis than the other fusion types. It has been suggested that the observed clinical discrepancies result from different transactivation potentials of the various EWS-FLI-1 fusion proteins. In an attempt to identify genes whose expression levels are differentially modulated by structurally different EWS-FLI-1 transcription factors, we have used microarray technology to interrogate 19,000 sequence genes to compare gene expression profile of type 1 or non-type 1 Ewing sarcoma cell lines. Data analysis showed few qualitative differences on gene expression; expression of only 41 genes (0.215% of possible sequences analyzed) differed significantly between Ewing tumor cell lines carrying EWS-FLI-1 fusion type 1 with respect to those with non-type 1 fusion.

  16. A toolkit for graded expression of green fluorescent protein fusion proteins in mammalian cells.

    PubMed

    Nalaskowski, Marcus M; Ehm, Patrick; Giehler, Susanne; Mayr, Georg W

    2012-09-01

    Green fluorescent protein (GFP) and GFP-like proteins of different colors are important tools in cell biology. In many studies, the intracellular targeting of proteins has been determined by transiently expressing GFP fusion proteins and analyzing their intracellular localization by fluorescence microscopy. In most vectors, expression of GFP is driven by the enhancer/promoter cassette of the immediate early gene of human cytomegalovirus (hCMV). This cassette generates high levels of protein expression in most mammalian cell lines. Unfortunately, these nonphysiologically high protein levels have been repeatedly reported to artificially alter the intracellular targeting of proteins fused to GFP. To cope with this problem, we generated a multitude of attenuated GFP expression vectors by modifying the hCMV enhancer/promoter cassette. These modified vectors were transiently expressed, and the expression levels of enhanced green fluorescent protein (EGFP) alone and enhanced yellow fluorescent protein (EYFP) fused to another protein were determined by fluorescence microscopy and/or Western blotting. As shown in this study, we were able to (i) clearly reduce the expression of EGFP alone and (ii) reduce expression of an EYFP fusion protein down to the level of the endogenous protein, both in a graded manner.

  17. Efficient killing of CD22{sup +} tumor cells by a humanized diabody-RNase fusion protein

    SciTech Connect

    Krauss, Juergen . E-mail: juergen.krauss@uni-essen.de; Arndt, Michaela A.E.; Vu, Bang K.; Newton, Dianne L.; Seeber, Siegfried; Rybak, Susanna M.

    2005-06-03

    We report on the generation of a dimeric immunoenzyme capable of simultaneously delivering two ribonuclease (RNase) effector domains on one molecule to CD22{sup +} tumor cells. As targeting moiety a diabody derived from the previously humanized scFv SGIII with grafted specificity of the murine anti-CD22 mAb RFB4 was constructed. Further engineering the interface of this construct (V{sub L}36{sub Leu{yields}}{sub Tyr}) resulted in a highly robust bivalent molecule that retained the same high affinity as the murine mAb RFB4 (K{sub D} 0.2 nM). A dimeric immunoenzyme comprising this diabody and Rana pipiens liver ribonuclease I (rapLRI) was generated, expressed as soluble protein in bacteria, and purified to homogeneity. The dimeric fusion protein killed several CD22{sup +} tumor cell lines with high efficacy (IC{sub 50} = 3-20 nM) and exhibited 9- to 48-fold stronger cytotoxicity than a monovalent rapLRI-scFv counterpart. Our results demonstrate that engineering of dimeric antibody-ribonuclease fusion proteins can markedly enhance their biological efficacy.

  18. Laser fusion

    SciTech Connect

    Smit, W.A.; Boskma, P.

    1980-12-01

    Unrestricted laser fusion offers nations an opportunity to circumvent arms control agreements and develop thermonuclear weapons. Early laser weapons research sought a clean radiation-free bomb to replace the fission bomb, but this was deceptive because a fission bomb was needed to trigger the fusion reaction and additional radioactivity was induced by generating fast neutrons. As laser-implosion experiments focused on weapons physics, simulating weapons effects, and applications for new weapons, the military interest shifted from developing a laser-ignited hydrogen bomb to more sophisticated weapons and civilian applications for power generation. Civilian and military research now overlap, making it possible for several countries to continue weapons activities and permitting proliferation of nuclear weapons. These countries are reluctant to include inertial confinement fusion research in the Non-Proliferation Treaty. 16 references. (DCK)

  19. Oligophrenin-1 Connects Exocytotic Fusion to Compensatory Endocytosis in Neuroendocrine Cells.

    PubMed

    Houy, Sébastien; Estay-Ahumada, Catherine; Croisé, Pauline; Calco, Valérie; Haeberlé, Anne-Marie; Bailly, Yannick; Billuart, Pierre; Vitale, Nicolas; Bader, Marie-France; Ory, Stéphane; Gasman, Stéphane

    2015-08-05

    Oligophrenin-1 (OPHN1) is a protein with multiple domains including a Rho family GTPase-activating (Rho-GAP) domain, and a Bin-Amphiphysin-Rvs (BAR) domain. Involved in X-linked intellectual disability, OPHN1 has been reported to control several synaptic functions, including synaptic plasticity, synaptic vesicle trafficking, and endocytosis. In neuroendocrine cells, hormones and neuropeptides stored in large dense core vesicles (secretory granules) are released through calcium-regulated exocytosis, a process that is tightly coupled to compensatory endocytosis, allowing secretory granule recycling. We show here that OPHN1 is expressed and mainly localized at the plasma membrane and in the cytosol in chromaffin cells from adrenal medulla. Using carbon fiber amperometry, we found that exocytosis is impaired at the late stage of membrane fusion in Ophn1 knock-out mice and OPHN1-silenced bovine chromaffin cells. Experiments performed with ectopically expressed OPHN1 mutants indicate that OPHN1 requires its Rho-GAP domain to control fusion pore dynamics. On the other hand, compensatory endocytosis assessed by measuring dopamine-β-hydroxylase (secretory granule membrane) internalization is severely inhibited in Ophn1 knock-out chromaffin cells. This inhibitory effect is mimicked by the expression of a truncated OPHN1 mutant lacking the BAR domain, demonstrating that the BAR domain implicates OPHN1 in granule membrane recapture after exocytosis. These findings reveal for the first time that OPHN1 is a bifunctional protein that is able, through distinct mechanisms, to regulate and most likely link exocytosis to compensatory endocytosis in chromaffin cells.

  20. The Epstein-Barr virus (EBV) glycoprotein B cytoplasmic C-terminal tail domain regulates the energy requirement for EBV-induced membrane fusion.

    PubMed

    Chen, Jia; Zhang, Xianming; Jardetzky, Theodore S; Longnecker, Richard

    2014-10-01

    The entry of enveloped viruses into host cells is preceded by membrane fusion, which in Epstein-Barr virus (EBV) is thought to be mediated by the refolding of glycoprotein B (gB) from a prefusion to a postfusion state. In our current studies, we characterized a gB C-terminal tail domain (CTD) mutant truncated at amino acid 843 (gB843). This truncation mutant is hyperfusogenic as monitored by syncytium formation and in a quantitative fusion assay and is dependent on gH/gL for fusion activity. gB843 can rescue the fusion function of other glycoprotein mutants that have null or decreased fusion activity in epithelial and B cells. In addition, gB843 requires less gp42 and gH/gL for fusion, and can function in fusion at a lower temperature than wild-type gB, indicating a lower energy requirement for fusion activation. Since a key step in fusion is the conversion of gB from a prefusion to an active postfusion state by gH/gL, gB843 may access this activated gB state more readily. Our studies indicate that the gB CTD may participate in the fusion function by maintaining gB in an inactive prefusion form prior to activation by receptor binding. Importance: Diseases resulting from Epstein-Barr virus (EBV) infection in humans range from the fairly benign disease infectious mononucleosis to life-threatening cancer. As an enveloped virus, EBV must fuse with a host cell membrane for entry and infection by using glycoproteins gH/gL, gB, and gp42. Among these glycoproteins, gB is thought to be the protein that executes fusion. To further characterize the function of the EBV gB cytoplasmic C-terminal tail domain (CTD) in fusion, we used a previously constructed CTD truncation mutant and studied its fusion activity in the context of other EBV glycoprotein mutants. From these studies, we find that the gB CTD regulates fusion by altering the energy requirements for the triggering of fusion mediated by gH/gL or gp42. Overall, our studies may lead to a better understanding of EBV fusion

  1. Fusions of Breast Carcinoma and Dendritic Cells as a Vaccine for the Treatment of Metatastic Breast Cancer

    DTIC Science & Technology

    2010-07-31

    well as the stimulatory cytokines, IL-12 and IFNγ. In addition, fusion cells expressed CCR7 necessary for the migration of cells to sites of T cell...a marked expansion of anti-tumor effector cells. Body Our clinical protocol had received approval by the FDA, NCI/CTEP (distributor of IL...followed at the Dana-Farber Cancer Institute, and was receiving chemotherapy for metastatic disease. She was seen by our vaccine group on 3/23/10 to

  2. Stromal Cell-Derived Growth Factor-1 Alpha-Elastin Like Peptide Fusion Protein Promotes Cell Migration and Revascularization of Experimental Wounds in Diabetic Mice

    PubMed Central

    Yeboah, Agnes; Maguire, Tim; Schloss, Rene; Berthiaume, Francois; Yarmush, Martin L.

    2017-01-01

    Objective: In previous work, we demonstrated the development of a novel fusion protein containing stromal cell-derived growth factor-1 alpha juxtaposed to an elastin-like peptide (SDF1-ELP), which has similar bioactivity, but is more stable in elastase than SDF1. Herein, we compare the ability of a single topical application of SDF1-ELP to that of SDF1 in healing 1 × 1 cm excisional wounds in diabetic mice. Approach: Human Leukemia-60 cells were used to demonstrate the chemotactic potential of SDF1-ELP versus SDF1 in vitro. Human umbilical vascular endothelial cells were used to demonstrate the angiogenic potential of SDF1-ELP versus SDF1 in vitro. The bioactivity of SDF1-ELP versus SDF1 after incubation in ex-vivo diabetic wound fluid was compared. The in-vivo effectiveness of SDF1-ELP versus SDF1 was compared in diabetic mice wound model by monitoring for the number of CD31+ cells in harvested wound tissues. Results: SDF1-ELP promotes the migration of cells and induces vascularization similar to SDF1 in vitro. SDF1-ELP is more stable in wound fluids compared to SDF1. In vivo, SDF1-ELP induced a higher number of vascular endothelial cells (CD31+ cells) compared to SDF1 and other controls, suggesting increased vascularization. Innovation: While growth factors have been shown to improve wound healing, this strategy is largely ineffective in chronic wounds. In this work, we show that SDF1-ELP is a promising agent for the treatment of chronic skin wounds. Conclusion: The superior in vivo performance and stability of SDF1-ELP makes it a promising agent for the treatment of chronic skin wounds. PMID:28116224

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

    SciTech Connect

    Lattuada, D.; Barbarino, M.; 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.

    2016-04-19

    In this paper, 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 2H(d,n)3He. The experiment was performed with the Texas Petawatt Laser, which delivered 150–270 fs pulses of energy ranging from 90 to 180 J to D2 or CD4 molecular clusters (where D denotes 2H). 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 2H(d,n)3He and 3He(d,p)4He cases discussed here, it is very important to apply the background subtraction for the energetic ions and to measure the fusion yields with high precision. While the available data on both ion distribution and fusion yields allow us to determine with good precision the S factor in the d+d case (lower Gamow energies), for the d+3He case the data are not precise enough to obtain the S factor using this method. Our results agree with other experiments within the experimental error, even though smaller values of the S factor were obtained. This might be due to the plasma environment differing from the beam target conditions in a conventional accelerator experiment.

  4. Expression of Bcl-2 and Bcl-2-Ig fusion transcripts in normal and neoplastic cells.

    PubMed Central

    Graninger, W B; Seto, M; Boutain, B; Goldman, P; Korsmeyer, S J

    1987-01-01

    We examined the expression of the Bcl-2 gene at chromosome segment 18q21, that is translocated into the Ig heavy chain gene locus in t(14;18) bearing lymphomas. Bcl-2, while B cell associated, is expressed in a variety of hematopoietic lineages including T cells. Bcl-2 mRNA levels are high during pre-B cell development, the time at which the t(14;18) translocation occurs, but are down regulated with maturation. Like certain other oncogenes, Bcl-2 is quiescent in resting B cells but up-regulated with B cell activation. Mature B cell lymphomas with a t(14;18) have log-folds more mRNA than matched counterparts without the translocation. A sensitive S1 protection assay revealed that all transcripts in t(14;18) B cells were Bcl-2-Ig fusion mRNAs and originated from the translocated allele. Thus, there is a marked deregulation of Bcl-2 when it is introduced into the Ig locus in t(14;18) lymphomas. Images PMID:3500184

  5. Effect of electrical stimulation-induced resistance exercise on mitochondrial fission and fusion proteins in rat skeletal muscle.

    PubMed

    Kitaoka, Yu; Ogasawara, Riki; Tamura, Yuki; Fujita, Satoshi; Hatta, Hideo

    2015-11-01

    It is well known that resistance exercise increases muscle protein synthesis and muscle strength. However, little is known about the effect of resistance exercise on mitochondrial dynamics, which is coupled with mitochondrial function. In skeletal muscle, mitochondria exist as dynamic networks that are continuously remodeling through fusion and fission. The purpose of this study was to investigate the effect of acute and chronic resistance exercise, which induces muscle hypertrophy, on the expression of proteins related to mitochondrial dynamics in rat skeletal muscle. Resistance exercise consisted of maximum isometric contraction, which was induced by percutaneous electrical stimulation of the gastrocnemius muscle. Our results revealed no change in levels of proteins that regulate mitochondrial fission (Fis1 and Drp1) or fusion (Opa1, Mfn1, and Mfn2) over the 24-h period following acute resistance exercise. Phosphorylation of Drp1 at Ser616 was increased immediately after exercise (P < 0.01). Four weeks of resistance training (3 times/week) increased Mfn1 (P < 0.01), Mfn2 (P < 0.05), and Opa1 (P < 0.01) protein levels without altering mitochondrial oxidative phosphorylation proteins. These observations suggest that resistance exercise has little effect on mitochondrial biogenesis but alters the expression of proteins involved in mitochondrial fusion and fission, which may contribute to mitochondrial quality control and improved mitochondrial function.

  6. End-products diacylglycerol and ceramide modulate membrane fusion induced by a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa

    PubMed Central

    Ibarguren, Maitane; Bomans, Paul H. H.; Frederik, Peter M.; Stonehouse, Martin; Vasil, Michael L.; Alonso, Alicia; Goñi, Félix M.

    2009-01-01

    A phospholipase C/ sphingomyelinase from Pseudomonas aeruginosa has been assayed on vesicles containing phosphatidylcholine, sphingomyelin, phosphatidylethanolamine and cholesterol, at equimolar ratios. The enzyme activity modifies the bilayer chemical composition giving rise to diacylglycerol (DAG) and ceramide (Cer). Assays of enzyme activity, enzyme-induced aggregation and fusion have been performed. Ultrastructural evidence of vesicle fusion at various stages of the process is presented, based on cryo-EM observations. The two enzyme lipidic end-products, DAG and Cer, have opposite effects on the bilayer physical properties, the former abolishes lateral phase separation, while the latter generates a new gel phase [Sot et al., FEBS Lett. 582, 3230–3236 (2008)]. Addition of either DAG, or Cer, or both to the liposome mixture causes an increase in enzyme binding to the bilayers and a decrease in lag time of hydrolysis. These two lipids also have different effects on the enzyme activity, DAG enhancing enzyme-induced vesicle aggregation and fusion, Cer inhibiting the hydrolytic activity. These effects are explained in terms of the different physical properties of the two lipids. DAG increases bilayers fluidity and decreases lateral separation of lipids, thus increasing enzyme activity and substrate accessibility to the enzyme. Cer has the opposite effect mainly because of its tendency to sequester sphingomyelin, an enzyme substrate, into rigid domains, presumably less accessible to the enzyme. PMID:19891956

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

    SciTech Connect

    Eom, Young Woo; Lee, Jong Eun; Yang, Mal Sook; Jang, In Keun; Kim, Hyo Eun; Lee, Doo Hoon; Kim, Young Jin; Park, Won Jin; Kong, Jee Hyun; Shim, Kwang Yong; Lee, Jong In; Kim, Hyun Soo

    2011-04-29

    Highlights: {yields} hASCs were differentiated into skeletal muscle cells by treatment with 5-azacytidine, FGF-2, and the supernatant of cultured hASCs. {yields} Dystrophin and MyHC were expressed in late differentiation step by treatment with the supernatant of cultured hASCs. {yields} hASCs expressing dystrophin and MyHC contributed to myotube formation during co-culture with mouse myoblast C2C12 cells. -- Abstract: Stem cell therapy for muscular dystrophies requires stem cells that are able to participate in the formation of new muscle fibers. However, the differentiation steps that are the most critical for this process are not clear. We investigated the myogenic phases of human adipose tissue-derived stem cells (hASCs) step by step and the capability of myotube formation according to the differentiation phase by cellular fusion with mouse myoblast C2C12 cells. In hASCs treated with 5-azacytidine and fibroblast growth factor-2 (FGF-2) for 1 day, the early differentiation step to express MyoD and myogenin was induced by FGF-2 treatment for 6 days. Dystrophin and myosin heavy chain (MyHC) expression was induced by hASC conditioned medium in the late differentiation step. Myotubes were observed only in hASCs undergoing the late differentiation step by cellular fusion with C2C12 cells. In contrast, hASCs that were normal or in the early stage were not involved in myotube formation. Our results indicate that stem cells expressing dystrophin and MyHC are more suitable for myotube formation by co-culture with myoblasts than normal or early differentiated stem cells expressing MyoD and myogenin.

  8. 5-Fluorocytosine combined with Fcy-hEGF fusion protein targets EGFR-expressing cancer cells

    SciTech Connect

    Lan, Keng-Hsueh; Shih, Yi-Sheng; Chang, Cheng Allen; Yen, Sang-Hue; Lan, Keng-Li

    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

  9. Application of optical tweezers and excimer laser to study protoplast fusion

    NASA Astrophysics Data System (ADS)

    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.

  10. Identification of Nafamostat as a Potent Inhibitor of Middle East Respiratory Syndrome Coronavirus S Protein-Mediated Membrane Fusion Using the Split-Protein-Based Cell-Cell Fusion Assay.

    PubMed

    Yamamoto, Mizuki; Matsuyama, Shutoku; Li, Xiao; Takeda, Makoto; Kawaguchi, Yasushi; Inoue, Jun-Ichiro; Matsuda, Zene

    2016-11-01

    Middle East respiratory syndrome (MERS) is an emerging infectious disease associated with a relatively high mortality rate of approximately 40%. MERS is caused by MERS coronavirus (MERS-CoV) infection, and no specific drugs or vaccines are currently available to prevent MERS-CoV infection. MERS-CoV is an enveloped virus, and its envelope protein (S protein) mediates membrane fusion at the plasma membrane or endosomal membrane. Multiple proteolysis by host proteases, such as furin, transmembrane protease serine 2 (TMPRSS2), and cathepsins, causes the S protein to become fusion competent. TMPRSS2, which is localized to the plasma membrane, is a serine protease responsible for the proteolysis of S in the post-receptor-binding stage. Here, we developed a cell-based fusion assay for S in a TMPRSS2-dependent manner using cell lines expressing Renilla luciferase (RL)-based split reporter proteins. S was stably expressed in the effector cells, and the corresponding receptor for S, CD26, was stably coexpressed with TMPRSS2 in the target cells. Membrane fusion between these effector and target cells was quantitatively measured by determining the RL activity. The assay was optimized for a 384-well format, and nafamostat, a serine protease inhibitor, was identified as a potent inhibitor of S-mediated membrane fusion in a screening of about 1,000 drugs approved for use by the U.S. Food and Drug Administration. Nafamostat also blocked MERS-CoV infection in vitro Our assay has the potential to facilitate the discovery of new inhibitors of membrane fusion of MERS-CoV as well as other viruses that rely on the activity of TMPRSS2.

  11. Discovery and characterization of a novel CCND1/MRCK gene fusion in mantle cell lymphoma.

    PubMed

    Masamha, Chioniso Patience; Albrecht, Todd R; Wagner, Eric J

    2016-03-29

    The t(11;14) translocation resulting in constitutive cyclin D1 expression is an early event in mantle cell lymphoma (MCL) transformation. Patients with a highly proliferative phenotype produce cyclin D1 transcripts with truncated 3'UTRs that evade miRNA regulation. Here, we report the recurrence of a novel gene fusion in MCL cell lines and MCL patient isolates that consists of the full protein coding region of cyclin D1 (CCND1) and a 3'UTR consisting of sequences from both the CCND1 3'UTR and myotonic dystrophy kinase-related Cdc42-binding kinase's (MRCK) intron one. The resulting CCND1/MRCK mRNA is resistant to CCND1-targeted miRNA regulation, and targeting the MRCK region of the chimeric 3'UTR with siRNA results in decreased CCND1 levels.

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

    PubMed

    Schoeman, Rogier M; Kemna, Evelien W M; Wolbers, Floor; van den Berg, Albert

    2014-02-01

    In this article, we present a microfluidic device capable of successive high-yield single-cell encapsulation in droplets, with additional droplet pairing, fusion, and shrinkage. Deterministic single-cell encapsulation is realized using Dean-coupled inertial ordering of cells in a Yin-Yang-shaped curved microchannel using a double T-junction, with a frequency over 2000 Hz, followed by controlled droplet pairing with a 100% success rate. Subsequently, droplet fusion is realized using electrical actuation resulting in electro-coalescence of two droplets, each containing a single HL60 cell, with 95% efficiency. Finally, volume reduction of the fused droplet up to 75% is achieved by a triple pitchfork structure. This droplet volume reduction is necessary to obtain close cell-cell membrane contact necessary for final cell electrofusion, leading to hybridoma formation, which is the ultimate aim of this research.

  13. Acquisition of an oncogenic fusion protein serves as an initial driving mutation by inducing aneuploidy and overriding proliferative defects

    PubMed Central

    Maggi, Elaine C.; Vijayaraghavan, Jyothi; Zabaleta, Jovanny; Taylor, Christopher M.; Tsien, Fern; Crabtree, Judy S.; Hollenbach, Andrew D.

    2016-01-01

    While many solid tumors are defined by the presence of a particular oncogene, the role that this oncogene plays in driving transformation through the acquisition of aneuploidy and overcoming growth arrest are often not known. Further, although aneuploidy is present in many solid tumors, it is not clear whether it is the cause or effect of malignant transformation. The childhood sarcoma, Alveolar Rhabdomyosarcoma (ARMS), is primarily defined by the t(2;13)(q35;q14) translocation, creating the PAX3-FOXO1 fusion protein. It is unclear what role PAX3-FOXO1 plays in the initial stages of tumor development through the acquisition and persistence of aneuploidy. In this study we demonstrate that PAX3-FOXO1 serves as a driver mutation to initiate a cascade of mRNA and miRNA changes that ultimately reprogram proliferating myoblasts to induce the formation of ARMS. We present evidence that cells containing PAX3-FOXO1 have changes in the expression of mRNA and miRNA essential for maintaining proper chromosome number and structure thereby promoting aneuploidy. Further, we demonstrate that the presence of PAX3-FOXO1 alters the expression of growth factor related mRNA and miRNA, thereby overriding aneuploid-dependent growth arrest. Finally, we present evidence that phosphorylation of PAX3-FOXO1 contributes to these changes. This is one of the first studies describing how an oncogene and post-translational modifications drive the development of a tumor through the acquisition and persistence of aneuploidy. This mechanism has implications for other solid tumors where large-scale genomics studies may elucidate how global alterations contribute to tumor phenotypes allowing the development of much needed multi-faceted tumor-specific therapeutic regimens. PMID:27588498

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

    PubMed

    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.

  15. Transfer of herpes simplex virus thymidine kinase synthesized in bacteria by a high-expression plasmid to tissue culture cells by protoplast fusion

    SciTech Connect

    Waldman, A.S.; Milman, G.

    1984-08-01

    The introduction of a protein into living tissue culture cells may permit the in vivo study of functions of the protein. The authors have previously described a high-efficiency-expression plasmid, pHETK2, containing the herpes simplex virus type 1 thymidine kinase (TK) gene which, upon temperature induction, causes TK to be synthesized as greater than 4% of the bacterial protein. In this report it is shown that enzymatically active TK was transferred to mouse Ltk- cells by polyethylene glycol-mediated fusion with protoplasts prepared from bacteria containing induced levels of TK. The presence of TK in the Ltk- cells was detected by the incorporation of (/sup 3/H)thymidine into cell nuclei as measured by autoradiography.

  16. High-level production of human interleukin-10 fusions in tobacco cell suspension cultures

    PubMed Central

    Kaldis, Angelo; Ahmad, Adil; Reid, Alexandra; McGarvey, Brian; Brandle, Jim; Ma, Shengwu; Jevnikar, Anthony; Kohalmi, Susanne E; Menassa, Rima

    2013-01-01

    The production of pharmaceutical proteins in plants has made much progress in recent years with the development of transient expression systems, transplastomic technology and humanizing glycosylation patterns in plants. However, the first therapeutic proteins approved for administration to humans and animals were made in plant cell suspensions for reasons of containment, rapid scale-up and lack of toxic contaminants. In this study, we have investigated the production of human interleukin-10 (IL-10) in tobacco BY-2 cell suspension and evaluated the effect of an elastin-like polypeptide tag (ELP) and a green fluorescent protein (GFP) tag on IL-10 accumulation. We report the highest accumulation levels of hIL-10 obtained with any stable plant expression system using the ELP fusion strategy. Although IL-10-ELP has cytokine activity, its activity is reduced compared to unfused IL-10, likely caused by interference of ELP with folding of IL-10. Green fluorescent protein has no effect on IL-10 accumulation, but examining the trafficking of IL-10-GFP over the cell culture cycle revealed fluorescence in the vacuole during the stationary phase of the culture growth cycle. Analysis of isolated vacuoles indicated that GFP alone is found in vacuoles, while the full-size fusion remains in the whole-cell extract. This indicates that GFP is cleaved off prior to its trafficking to the vacuole. On the other hand, IL-10-GFP-ELP remains mostly in the ER and accumulates to high levels. Protein bodies were observed at the end of the culture cycle and are thought to arise as a consequence of high levels of accumulation in the ER. PMID:23297698

  17. Single-cell RNA-seq reveals activation of unique gene groups as a consequence of stem cell-parenchymal cell fusion.

    PubMed

    Freeman, Brian T; Jung, Jangwook P; Ogle, Brenda M

    2016-03-21

    Fusion of donor mesenchymal stem cells with parenchymal cells of the recipient can occur in the brain, liver, intestine and heart following transplantation. The therapeutic benefit or detriment of resultant hybrids is unknown. Here we sought a global view of phenotypic diversification of mesenchymal stem cell-cardiomyocyte hybrids and associated time course. Using single-cell RNA-seq, we found hybrids consistently increase ribosome components and decrease genes associated with the cell cycle suggesting an increase in protein production and decrease in proliferation to accommodate the fused state. But in the case of most other gene groups, hybrids were individually distinct. In fact, though hybrids can express a transcriptome similar to individual fusion partners, approximately one-third acquired distinct expression profiles in a single day. Some hybrids underwent reprogramming, expressing pluripotency and cardiac precursor genes latent in parental cells and associated with developmental and morphogenic gene groups. Other hybrids expressed genes associated with ontologic cancer sets and two hybrids of separate experimental replicates clustered with breast cancer cells, expressing critical oncogenes and lacking tumor suppressor genes. Rapid transcriptional diversification of this type garners consideration in the context of cellular transplantation to damaged tissues, those with viral infection or other microenvironmental conditions that might promote fusion.

  18. Protein body-inducing fusions for high-level production and purification of recombinant proteins in plants.

    PubMed

    Conley, Andrew J; Joensuu, Jussi J; Richman, Alex; Menassa, Rima

    2011-05-01

    For the past two decades, therapeutic and industrially important proteins have been expressed in plants with varying levels of success. The two major challenges hindering the economical production of plant-made recombinant proteins include inadequate accumulation levels and the lack of efficient purification methods. To address these limitations, several fusion protein strategies have been recently developed to significantly enhance the production yield of plant-made recombinant proteins, while simultaneously assisting in their subsequent purification. Elastin-like polypeptides are thermally responsive biopolymers composed of a repeating pentapeptide 'VPGXG' sequence that are valuable for the purification of recombinant proteins. Hydrophobins are small fungal proteins capable of altering the hydrophobicity of their respective fusion partner, thus enabling efficient purification by surfactant-based aqueous two-phase systems. Zera, a domain of the maize seed storage protein γ-zein, can induce the formation of protein storage bodies, thus facilitating the recovery of fused proteins using density-based separation methods. These three novel protein fusion systems have also been shown to enhance the accumulation of a range of different recombinant proteins, while concurrently inducing the formation of protein bodies. The packing of these fusion proteins into protein bodies may exclude the recombinant protein from normal physiological turnover. Furthermore, these systems allow for quick, simple and inexpensive nonchromatographic purification of the recombinant protein, which can be scaled up to industrial levels of protein production. This review will focus on the similarities and differences of these artificial storage organelles, their biogenesis and their implication for the production of recombinant proteins in plants and their subsequent purification.

  19. A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells

    PubMed Central

    Chaikind, Brian; Bessen, Jeffrey L.; Thompson, David B.; Hu, Johnny H.; Liu, David R.

    2016-01-01

    We describe the development of ‘recCas9’, an RNA-programmed small serine recombinase that functions in mammalian cells. We fused a catalytically inactive dCas9 to the catalytic domain of Gin recombinase using an optimized fusion architecture. The resulting recCas9 system recombines DNA sites containing a minimal recombinase core site flanked by guide RNA-specified sequences. We show that these recombinases can operate on DNA sites in mammalian cells identical to genomic loci naturally found in the human genome in a manner that is dependent on the guide RNA sequences. DNA sequencing reveals that recCas9 catalyzes guide RNA-dependent recombination in human cells with an efficiency as high as 32% on plasmid substrates. Finally, we demonstrate that recCas9 expressed in human cells can catalyze in situ deletion between two genomic sites. Because recCas9 directly catalyzes recombination, it generates virtually no detectable indels or other stochastic DNA modification products. This work represents a step toward programmable, scarless genome editing in unmodified cells that is independent of endogenous cellular machinery or cell state. Current and future generations of recCas9 may facilitate targeted agricultural breeding, or the study and treatment of human genetic diseases. PMID:27515511

  20. 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.

    PubMed Central

    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 constants for fusion pore formation and closure. An Arrhenius plot of the rate of fusion pore formation shows a simple linear relationship with an apparent activation energy of 23 kcal/mol. In contrast, the Arrhenius plot of the rate of closure of the fusion pore is discontinuous, with the break at approximately 13 degrees C. Above the break point, the rate of closure has a weak temperature dependence (7 kcal/mol), whereas below 13 degrees C the rate of closure is temperature independent. This type of temperature dependency is characteristic of events that depend on diffusion in a lipid phase that undergoes a fluid-solid phase transition. We propose that the formation of the fusion pore is regulated by the conformational change of a molecular structure with a high activation energy, whereas the closure of the fusion pore is regulated by lipids that become phase separated at 13 degrees C. PMID:1504250

  1. HIV transcription is induced in dying cells

    SciTech Connect

    Woloschak, G.E.; Chang-Liu, Chin-Mei; Schreck, S. |; Panozzo, J.; Libertin, C.R.

    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.

  2. Effects of an adenoviral vector containing a suicide gene fusion on growth characteristics of breast cancer cells.

    PubMed

    Kong, Heng; Liu, Chunli; Zhu, Ting; Huang, Zonghai; Yang, Liucheng; Li, Qiang

    2014-12-01

    The herpes simplex virus thymidine kinase/ganciclovir (HSV‑TK/GCV) and the cytosine deaminase/5‑fluorocytosine (CD/5‑FC) systems have been widely applied in suicide gene therapy for cancer. Although suicide gene therapy has been successfully used in vitro and in vivo studies, the number of studies on the effects of recombinant adenoviruses (Ads) containing suicide genes on target cancer cells is limited. The aim of this study was to examine whether recombinant Ads containing the CD/TK fusion gene affect cell proliferation of breast cancer cells in vitro. In the present study, we explored the use of a recombinant adenoviral vector to deliver the CD/TK fusion gene to the breast cancer cell line MCF‑7. We found that the recombinant adenoviral vector efficiently infected MCF‑7 cells. Western blot analysis revealed that CD and TK proteins are expressed in the infected cells. The infected breast cancer cells did not show any significant changes in morphology, ultrastructure, cell growth, and cell‑cycle distribution compared to the uninfected cells. This study revealed that the Ad‑vascular endothelial growth factor promoter (VEGFp)‑CD/TK vector is non‑toxic to MCF‑7 cells at the appropriate titer. Our results indicate that it is feasible to use a recombinant adenoviral vector containing the CD/TK fusion gene in suicide gene therapy to target breast cancer cells.

  3. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells

    PubMed Central

    Kim, Dae In; Raida, Manfred; Burke, Brian

    2012-01-01

    We have developed a new technique for proximity-dependent labeling of proteins in eukaryotic cells. Named BioID for proximity-dependent biotin identification, this approach is based on fusion of a promiscuous Escherichia coli biotin protein ligase to a targeting protein. BioID features proximity-dependent biotinylation of proteins that are near-neighbors of the fusion protein. Biotinylated proteins may be isolated by affinity capture and identified by mass spectrometry. We apply BioID to lamin-A (LaA), a well-characterized intermediate filament protein that is a constituent of the nuclear lamina, an important structural element of the nuclear envelope (NE). We identify multiple proteins that associate with and/or are proximate to LaA in vivo. The most abundant of these include known interactors of LaA that are localized to the NE, as well as a new NE-associated protein named SLAP75. Our results suggest BioID is a useful and generally applicable method to screen for both interacting and neighboring proteins in their native cellular environment. PMID:22412018

  4. Search for cold fusion using Pd-D2O cells and Ti-D mixtures

    NASA Astrophysics Data System (ADS)

    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.

  5. Posterior spinal fusion in adolescent idiopathic scoliosis with or without intraoperative cell salvage system: a retrospective comparison.

    PubMed

    Ersen, Omer; Ekıncı, Safak; Bılgıc, Serkan; Kose, Ozkan; Oguz, Erbil; Sehırlıoglu, Ali

    2012-08-01

    This study investigates efficacy and safety of routine cell salvage system use in adolescent idiopathic scoliosis patients undergoing primary posterior spinal fusion surgery with segmental spinal instrumentation. Forty-five consecutive adolescent idiopathic scoliosis patients undergoing posterior spinal fusion by two surgeons at a single hospital were studied. Intraoperative cell salvage system was used in 23 patients, and the control group was 22 patients who underwent surgery without cell salvage system. The cell salvage system was the Haemonetics Cell Saver 5. The primary outcome measures were intraoperative and perioperative allogeneic transfusion rate, difference between preoperative and discharge Hg and Hct levels. Average patient age was 14.65 ± 1.49 in cell saver group and 13.86 ± 2.0 in control group. In cell saver group, average intraoperative autotransfusion was 382.1 ± 175 ml. Average perioperative allogeneic blood transfusion need was 1.04 ± 0.7 unit in cell saver group and 2.5 ± 1.14 unit in control group. No transfusion reactions occurred in either group. Average hemoglobin level in cell saver group was 10.7 ± 0.86 and average hemoglobin level in control group was 10.7 ± 0.82 on discharge. Cell saver reduces perioperative transfusion rate in patients undergoing posterior spinal fusion for adolescent idiopathic scoliosis.

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

    PubMed Central

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

    2015-01-01

    Myogenesis is defined as growth, differentiation and repair of muscles where cell fusion of myoblasts to multinucleated myofibers is one major characteristic. Other cell fusion events in humans are found with bone resorbing osteoclasts and placental syncytiotrophoblasts. No unifying gene regulation for natural cell fusions has been found. We analyzed skeletal muscle biopsies of competitive cyclists for muscle-specific attributes and expression of human endogenous retrovirus (ERV) envelope genes due to their involvement in cell fusion of osteoclasts and syncytiotrophoblasts. Comparing muscle biopsies from post- with the pre-competitive seasons a significant 2.25-fold increase of myonuclei/mm fiber, a 2.38-fold decrease of fiber area/nucleus and a 3.1-fold decrease of satellite cells (SCs) occurred. We propose that during the pre-competitive season SC proliferation occurred following with increased cell fusion during the competitive season. Expression of twenty-two envelope genes of muscle biopsies demonstrated a significant increase of putative muscle-cell fusogenic genes Syncytin-1 and Syncytin-3, but also for the non-fusogenic erv3. Immunohistochemistry analyses showed that Syncytin-1 mainly localized to the sarcolemma of myofibers positive for myosin heavy-chain isotypes. Cellular receptors SLC1A4 and SLC1A5 of Syncytin-1 showed significant decrease of expression in post-competitive muscles compared with the pre-competitive season, but only SLC1A4 protein expression localized throughout the myofiber. Erv3 protein was strongly expressed throughout the myofiber, whereas envK1-7 localized to SC nuclei and myonuclei. Syncytin-1 transcription factors, PPARγ and RXRα, showed no protein expression in the myofiber, whereas the pCREB-Ser133 activator of Syncytin-1 was enriched to SC nuclei and myonuclei. Syncytin-1, Syncytin-3, SLC1A4 and PAX7 gene regulations along with MyoD1 and myogenin were verified during proliferating or actively-fusing human primary myoblast cell

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

    PubMed

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

    2015-01-01

    Myogenesis is defined as growth, differentiation and repair of muscles where cell fusion of myoblasts to multinucleated myofibers is one major characteristic. Other cell fusion events in humans are found with bone resorbing osteoclasts and placental syncytiotrophoblasts. No unifying gene regulation for natural cell fusions has been found. We analyzed skeletal muscle biopsies of competitive cyclists for muscle-specific attributes and expression of human endogenous retrovirus (ERV) envelope genes due to their involvement in cell fusion of osteoclasts and syncytiotrophoblasts. Comparing muscle biopsies from post- with the pre-competitive seasons a significant 2.25-fold increase of myonuclei/mm fiber, a 2.38-fold decrease of fiber area/nucleus and a 3.1-fold decrease of satellite cells (SCs) occurred. We propose that during the pre-competitive season SC proliferation occurred following with increased cell fusion during the competitive season. Expression of twenty-two envelope genes of muscle biopsies demonstrated a significant increase of putative muscle-cell fusogenic genes Syncytin-1 and Syncytin-3, but also for the non-fusogenic erv3. Immunohistochemistry analyses showed that Syncytin-1 mainly localized to the sarcolemma of myofibers positive for myosin heavy-chain isotypes. Cellular receptors SLC1A4 and SLC1A5 of Syncytin-1 showed significant decrease of expression in post-competitive muscles compared with the pre-competitive season, but only SLC1A4 protein expression localized throughout the myofiber. Erv3 protein was strongly expressed throughout the myofiber, whereas envK1-7 localized to SC nuclei and myonuclei. Syncytin-1 transcription factors, PPARγ and RXRα, showed no protein expression in the myofiber, whereas the pCREB-Ser133 activator of Syncytin-1 was enriched to SC nuclei and myonuclei. Syncytin-1, Syncytin-3, SLC1A4 and PAX7 gene regulations along with MyoD1 and myogenin were verified during proliferating or actively-fusing human primary myoblast cell

  8. Baculovirus expression of the respiratory syncytial virus fusion protein using Trichoplusia ni insect cells.

    PubMed

    Parrington, M; Cockle, S; Wyde, P; Du, R P; Snell, E; Yan, W Y; Wang, Q; Gisonni, L; Sanhueza, S; Ewasyshyn, M; Klein, M

    1997-01-01

    Respiratory syncytial virus (RSV) is a major viral pathogen responsible for severe respiratory tract infections in infants, young children, and the elderly. The RSV fusion (F) protein is highly conserved among RSV subgroups A and B and is the major protective immunogen. A genetically-engineered version of the RSV F protein was produced in insect cells using the baculovirus expression system. To express a secreted form of this protein, the transmembrane domain was eliminated by removing the region of the gene encoding 48 amino acids at the C-terminus. Production of the truncated RSV F protein (RSV-Fs) was compared in two different insect cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five). The yield of RSV-Fs secreted from High Five insect cells was over 7-fold higher than that from Sf9 insect cells. Processing of the RSV-Fs protein was also different in the two insect cell lines. N-terminal sequencing demonstrated that while most of the RSV-Fs protein secreted by High Five cells was correctly processed at the F2-F1 proteolytic cleavage site, most of the RSV-Fs protein secreted by Sf9 cells was unprocessed or incorrectly processed. Antigenicity of the major RSV F neutralization epitopes was maintained in the RSV-Fs protein secreted from High Five cells. The RSV-specific neutralizing antibody titres in the sera of cotton rats immunized with the RSV-Fs protein were equivalent to those in the sera of animals intranasally inoculated with live RSV. Animals immunized with either live RSV or the immunoaffinity purified RSV-Fs protein from High Five cells were completely protected against live virus challenge.

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

    PubMed Central

    Simonte, Giacoma; Di Vicino, Umberto; Romo, Neus; Pinilla, Isabel; Nicolás, Marta

    2016-01-01

    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

  10. Productive infection of human immunodeficiency virus type 1 in dendritic cells requires fusion-mediated viral entry

    SciTech Connect

    Janas, Alicia M.; Dong, Chunsheng; Wang Jianhua; Wu Li

    2008-06-05

    Human immunodeficiency virus type 1 (HIV-1) enters dendritic cells (DCs) through endocytosis and viral receptor-mediated fusion. Although endocytosis-mediated HIV-1 entry can generate productive infection in certain cell types, including human monocyte-derived macrophages, productive HIV-1 infection in DCs appears to be dependent on fusion-mediated viral entry. It remains to be defined whether endocytosed HIV-1 in DCs can initiate productive infection. Using HIV-1 infection and cellular fractionation assays to measure productive viral infection and entry, here we show that HIV-1 enters monocyte-derived DCs predominately through endocytosis; however, endocytosed HIV-1 cannot initiate productive HIV-1 infection in DCs. In contrast, productive HIV-1 infection in DCs requires fusion-mediated viral entry. Together, these results provide functional evidence in understanding HIV-1 cis-infection of DCs, suggesting that different pathways of HIV-1 entry into DCs determine the outcome of viral infection.

  11. Involvement of ROS in Curcumin-induced Autophagic Cell Death.

    PubMed

    Lee, Youn Ju; Kim, Nam-Yi; Suh, Young-Ah; Lee, Chuhee

    2011-02-01

    Many anticancer agents as well as ionizing radiation have been shown to induce autophagy which is originally described as a protein recycling process and recently reported to play a crucial role in various disorders. In HCT116 human colon cancer cells, we found that curcumin, a polyphenolic phytochemical extracted from the plant Curcuma longa, markedly induced the conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II and degradation of sequestome-1 (SQSTM1) which is a marker of autophagosome degradation. Moreover, we found that curcumin caused GFP-LC3 formation puncta, a marker of autophagosome, and decrease of GFP-LC3 and SQSTM1 protein level in GFP-LC3 expressing HCT116 cells. It was further confirmed that treatment of cells with hydrogen peroxide induced increase of LC3 conversion and decrease of GFP-LC3 and SQSTM1 levels, but these changes by curcumin were almost completely blocked in the presence of antioxidant, N-acetylcystein (NAC), indicating that curcumin leads to reactive oxygen species (ROS) production, which results in autophagosome development and autolysosomal degradation. In parallel with NAC, SQSTM1 degradation was also diminished by bafilomycin A, a potent inhibitor of autophagosome-lysosome fusion, and cell viability assay was further confirmed that cucurmin-induced cell death was partially blocked by bafilomycin A as well as NAC. We also observed that NAC abolished curcumin-induced activation of extracelluar signal-regulated kinases (ERK) 1/2 and p38 mitogen-activated protein kinases (MAPK), but not Jun N-terminal kinase (JNK). However, the activation of ERK1/2 and p38 MAPK seemed to have no effect on the curcumin-induced autophagy, since both the conversion of LC3 protein and SQSTM1 degradation by curcumin was not changed in the presence of NAC. Taken together, our data suggest that curcumin induced ROS production, which resulted in autophagic activation and concomitant cell death in HCT116 human colon cancer cell

  12. Involvement of ROS in Curcumin-induced Autophagic Cell Death

    PubMed Central

    Lee, Youn Ju; Kim, Nam-Yi; Suh, Young-Ah

    2011-01-01

    Many anticancer agents as well as ionizing radiation have been shown to induce autophagy which is originally described as a protein recycling process and recently reported to play a crucial role in various disorders. In HCT116 human colon cancer cells, we found that curcumin, a polyphenolic phytochemical extracted from the plant Curcuma longa, markedly induced the conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II and degradation of sequestome-1 (SQSTM1) which is a marker of autophagosome degradation. Moreover, we found that curcumin caused GFP-LC3 formation puncta, a marker of autophagosome, and decrease of GFP-LC3 and SQSTM1 protein level in GFP-LC3 expressing HCT116 cells. It was further confirmed that treatment of cells with hydrogen peroxide induced increase of LC3 conversion and decrease of GFP-LC3 and SQSTM1 levels, but these changes by curcumin were almost completely blocked in the presence of antioxidant, N-acetylcystein (NAC), indicating that curcumin leads to reactive oxygen species (ROS) production, which results in autophagosome development and autolysosomal degradation. In parallel with NAC, SQSTM1 degradation was also diminished by bafilomycin A, a potent inhibitor of autophagosome-lysosome fusion, and cell viability assay was further confirmed that cucurmin-induced cell death was partially blocked by bafilomycin A as well as NAC. We also observed that NAC abolished curcumin-induced activation of extracelluar signal-regulated kinases (ERK) 1/2 and p38 mitogen-activated protein kinases (MAPK), but not Jun N-terminal kinase (JNK). However, the activation of ERK1/2 and p38 MAPK seemed to have no effect on the curcumin-induced autophagy, since both the conversion of LC3 protein and SQSTM1 degradation by curcumin was not changed in the presence of NAC. Taken together, our data suggest that curcumin induced ROS production, which resulted in autophagic activation and concomitant cell death in HCT116 human colon cancer cell

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

    PubMed

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

    2016-02-01

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

  14. Electron emission from tungsten induced by slow, fusion-relevant ions

    NASA Astrophysics Data System (ADS)

    Kowarik, Gregor; Brunmayr, Michael; Aumayr, Friedrich

    2009-08-01

    Tungsten has recently been introduced as a new wall material for fusion, because it exhibits favourably low sputtering yields and a very low tritium retention as compared to the commonly used graphite wall and divertor tiles. We measure total electron emission yields due to impact of slow singly and multiply charged ions (deuterium, helium and carbon) on sputter-cleaned polycrystalline tungsten surfaces by using a current method in combination with a retarding grid. Results are presented in the eV to keV impact energy region as typical for fusion edge plasma conditions and discussed in terms of potential and kinetic electron emission.

  15. Local anesthetics induce human renal cell apoptosis.

    PubMed

    Lee, H Thomas; Xu, Hua; Siegel, Cory D; Krichevsky, Igor E

    2003-01-01

    Renal cell apoptosis contributes significantly to the pathogenesis of acute renal failure. Local anesthetics induce apoptosis in neuronal and lymphocytic cell lines. We examined the effects of chronic (48 h) local anesthetic treatment (lidocaine, bupivacaine and tetracaine) on human proximal tubular (HK-2) cells. Apoptosis induction was assessed by detecting poly(ADP)-ribose polymerase fragmentation, caspase activation, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining, DNA laddering and by cellular morphology. Cell death was quantified by measuring neutral red dye uptake and lactate dehydrogenase released into the cell culture medium. All 3 local anesthetics caused concentration-dependent cell death, induced HK-2 cell apoptosis and potentiated TNF-alpha induced apoptosis. Local anesthetics induced HK-2 cell apoptosis by activation of caspases 3, 6, 7, 8 and 9. ZVAD-fmk, a pan-caspase inhibitor, blocked the local anesthetic induced HK-2 cell apoptosis. Local anesthetics also inhibited the activities of anti-apoptotic kinases protein kinase B (Akt) and extracellular signal regulated mitrogen-activated protein kinase. Local anesthetic's pro-apoptotic effects are independent of sodium channel inhibition as tetrodotoxin, a selective voltage-gated sodium channel blocker, failed to mimic local anesthetic-mediated induction or potentiation of HK-2 cell apoptosis. We conclude that local anesthetics induce human renal cell apoptotic signaling by caspase activation and via inhibition of pro-survival signaling pathways.

  16. Reactivity of murine cytokine fusion toxin, diphtheria toxin390-murine interleukin-3 (DT390-mIL-3), with bone marrow progenitor cells.

    PubMed

    Chan, C H; Blazar, B R; Greenfield, L; Kreitman, R J; Vallera, D A

    1996-08-15

    Myeloid leukemias can express interleukin-3 receptors (IL-3R). Therefore, as an antileukemia drug, a fusion immunotoxin was synthesized consisting of the murine IL-3 (mIL-3) gene spliced to a truncated form of the diphtheria toxin (DT390) gene coding for a molecule that retained full enzymatic activity, but excluded the native binding domain. The DT390-mIL-3 hybrid gene was cloned into a vector under the control of an inducible promote. The fusion protein was expressed in Escherichia coli and then purified from inclusion bodies. The fusion toxin was potent because it inhibited FDC-P1, an IL-3R-expressing murine myelomonocytic tumor line (IC50 = 0.025 nmol/L or 1.5 ng/mL). Kinetics were rapid and cell-free studies showed that DT390-mIL-3 was as toxic as native DT. DT390-mIL-3 was selective because anti-mIL-3 monoclonal antibody, but not irrelevant antibody, inhibited its ability to kill. Cell lines not expressing IL-3R were not inhibited by the fusion protein. Because the use of DT390-mIL-3 as an antileukemia agent could be restricted by its reactivity with committed and/or primitive progenitor cells, bone marrow (BM) progenitor assays were performed. DT390-mIL-3 selectively inhibited committed BM progenitor cells as measured by in vitro colony-forming unit-granulocyte-macrophage and in vivo colony-forming unit-spleen colony assays. To determine if this fusion protein was reactive against BM progenitor cells required to rescue lethally irradiated recipients, adoptive transfer experiments were performed. Eight million DT390-mIL-3-treated C57BL/6 Ly5.2 BM cells, but not 4 million, were able to rescue lethally irradiated congenic C57BL/6 Ly5.1 recipients, suggesting that progenitor cells might be heterogenous in their expression of IL-3R. This idea was supported in competitive repopulation experiments in which DT390-mIL-3 treated C57BL/6 Ly5.2 BM cells were mixed with nontreated C57BL/6 Ly5.1 BM cells and used to reconstitute C57BL/6 Ly5.1 mice. A significant reduction

  17. Viral membrane fusion

    SciTech Connect

    Harrison, Stephen C.

    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.

  18. BMP-2-induced Neuroforaminal Bone Growth in the Setting of a Minimally Invasive Transforaminal Lumbar Interbody Fusion.

    PubMed

    Ahn, Junyoung; Tabaraee, Ehsan; Singh, Kern

    2015-06-01

    Minimally invasive transforaminal lumbar interbody fusion (MIS-TLIF) has become a popular alternative to traditional methods of lumbar decompression and fusion. When compared with the open technique, the minimally invasive approach can result in decreased pain and blood loss as well as a shorter length of hospitalization. However, the narrower working channel through the tubular retractor increases the difficulty of decortication and bone grafting. Therefore, recombinant human bone morphogenetic proteins (rhBMP-2) is often utilized (although this is off-label) to create a more favorable interbody fusion environment. Recently, the use of rhBMP-2 has been associated with excessive bone growth in an MIS-TLIF. If this bone growth compresses the neighboring neural structures, patients may present with either new or recurrent radicular pain. Computed tomographic (CT) imaging can demonstrate heterotopic bone growth extending from the disk space into either the ipsilateral neuroforamen or lateral recess, which may result in the compression of the exiting or traversing root, respectively. The purpose of this article and the accompanying video is to demonstrate a technique for defining and resecting rhBMP-2-induced heterotopic bone growth following a previous MIS-TLIF.

  19. Starvation-induced MTMR13 and RAB21 activity regulates VAMP8 to promote autophagosome-lysosome fusion.

    PubMed

    Jean, Steve; Cox, Sarah; Nassari, Sonya; Kiger, Amy A

    2015-03-01

    Autophagy, the process for recycling cytoplasm in the lysosome, depends on membrane trafficking. We previously identified Drosophila Sbf as a Rab21 guanine nucleotide exchange factor (GEF) that acts with Rab21 in endosomal trafficking. Here, we show that Sbf/MTMR13 and Rab21 have conserved functions required for starvation-induced autophagy. Depletion of Sbf/MTMR13 or Rab21 blocked endolysosomal trafficking of VAMP8, a SNARE required for autophagosome-lysosome fusion. We show that starvation induces Sbf/MTMR13 GEF and RAB21 activity, as well as their induced binding to VAMP8 (or closest Drosophila homolog, Vamp7). MTMR13 is required for RAB21 activation, VAMP8 interaction and VAMP8 endolysosomal trafficking, defining a novel GEF-Rab-effector pathway. These results identify starvation-responsive endosomal regulators and trafficking that tunes membrane demands with changing autophagy status.

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

    SciTech Connect

    Nordlund, Henri R. . E-mail: henri.nordlund@uta.fi; Laitinen, Olli H.; Uotila, Sanna T.H.; Kulmala, Minna; Kalkkinen, Nisse; Kulomaa, Markku S.

    2005-10-14

    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.

  1. Separating myoblast differentiation from muscle cell fusion using IGF-I and the p38 MAP kinase inhibitor SB202190

    PubMed Central

    Gardner, Samantha; Gross, Sean M.; David, Larry L.; Klimek, John E.

    2015-01-01

    The p38 MAP kinases play critical roles in skeletal muscle biology, but the specific processes regulated by these kinases remain poorly defined. Here we find that activity of p38α/β is important not only in early phases of myoblast differentiation, but also in later stages of myocyte fusion and myofibrillogenesis. By treatment of C2 myoblasts with the promyogenic growth factor insulin-like growth factor (IGF)-I, the early block in differentiation imposed by the p38 chemical inhibitor SB202190 could be overcome. Yet, under these conditions, IGF-I could not prevent the later impairment of muscle cell fusion, as marked by the nearly complete absence of multinucleated myofibers. Removal of SB202190 from the medium of differentiating myoblasts reversed the fusion block, as multinucleated myofibers were detected several hours later and reached ∼90% of the culture within 30 h. Analysis by quantitative mass spectroscopy of proteins that changed in abundance following removal of the inhibitor revealed a cohort of upregulated muscle-enriched molecules that may be important for both myofibrillogenesis and fusion. We have thus developed a model system that allows separation of myoblast differentiation from muscle cell fusion and should be useful in identifying specific steps regulated by p38 MAP kinase-mediated signaling in myogenesis. PMID:26246429

  2. Separating myoblast differentiation from muscle cell fusion using IGF-I and the p38 MAP kinase inhibitor SB202190.

    PubMed

    Gardner, Samantha; Gross, Sean M; David, Larry L; Klimek, John E; Rotwein, Peter

    2015-10-01

    The p38 MAP kinases play critical roles in skeletal muscle biology, but the specific processes regulated by these kinases remain poorly defined. Here we find that activity of p38α/β is important not only in early phases of myoblast differentiation, but also in later stages of myocyte fusion and myofibrillogenesis. By treatment of C2 myoblasts with the promyogenic growth factor insulin-like growth factor (IGF)-I, the early block in differentiation imposed by the p38 chemical inhibitor SB202190 could be overcome. Yet, under these conditions, IGF-I could not prevent the later impairment of muscle cell fusion, as marked by the nearly complete absence of multinucleated myofibers. Removal of SB202190 from the medium of differentiating myoblasts reversed the fusion block, as multinucleated myofibers were detected several hours later and reached ∼90% of the culture within 30 h. Analysis by quantitative mass spectroscopy of proteins that changed in abundance following removal of the inhibitor revealed a cohort of upregulated muscle-enriched molecules that may be important for both myofibrillogenesis and fusion. We have thus developed a model system that allows separation of myoblast differentiation from muscle cell fusion and should be useful in identifying specific steps regulated by p38 MAP kinase-mediated signaling in myogenesis.

  3. Construction of Recombinant Bacmid Containing M2e-Ctxb and Producing the Fusion Protein in Insect Cell Lines

    PubMed Central

    Mirzaei, Nima; Mokhtari Azad, Talat; Nategh, Rakhshandeh; Soleimanjahi, Hoorieh; Amirmozafari, Nour

    2014-01-01

    Background: Sequence variations in glycoproteins of influenza virus surface impel us to design new candidate vaccines yearly. Ectodomain of influenza M2 protein is a surface and highly conserved protein. M2e in influenza vaccines may eliminate the need for changing vaccine formulation every year. Objectives: In this study, a recombinant baculovirus containing M2e and cholera toxin subunit B fusion gene was generated with transposition process to express in large amounts in insect cell lines. Materials and Methods: M2e-ctxB fusion gene was created and cloned into pFastBac HT. The recombinant vector was transformed into DH10Bac cells to introduce the fusion gene into the bacmid DNA via a site-specific transposition process. The recombinant bacmid was then extracted from white colonies and further analyzed using PCR, DNA sequence analyzing, and indirect immunofluorescence assay. Results: PCR and DNA sequence analyzing results showed that the fusion gene was constructed as a single open reading frame and was successfully inserted into bacmid DNA. Moreover, indirect immunofluorescence results showed that the fusion gene was successfully expressed. Conclusions: Baculovirus expression vector system is valuable to produce M2e based influenza vaccines due to its simple utilization and ease of target gene manipulation. The expressed protein in such systems can improve the evaluating process of new vaccination strategies. PMID:24719728

  4. Human T-cell lymphotropic virus type 1 (HTLV-1)-induced syncytium formation mediated by vascular cell adhesion molecule-1: evidence for involvement of cell adhesion molecules in HTLV-1 biology.

    PubMed Central

    Hildreth, J E; Subramanium, A; Hampton, R A

    1997-01-01

    While studying the potential role of vascular cell adhesion molecule-1 (VCAM-1) in infection of endothelial cells by human immunodeficiency virus (HIV), we found that VCAM-1 can mediate human T-cell lymphotropic virus type 1 (HTLV-1)-induced syncytium formation. Both expression-vector-encoded and endogenously expressed VCAM-1 supported fusion of uninfected cells with HTLV-1-infected cells. Fusion was obtained with cell lines carrying the HTLV-1 genome and expressing viral proteins but not with an HTLV-1-transformed cell line that does not express viral proteins. In clones of VCAM-1-transfected cells, the degree of syncytium formation observed directly reflected the level of VCAM-1 expression. Syncytium formation between HTLV-1-expressing cells and VCAM-1+ cells could be blocked with antiserum against HTLV-1 gp46 and with a monoclonal antibody (MAb) against VCAM-1. Fusion was not blocked by antiserum against HIV or a MAb against VLA-4, the physiological counter-receptor for VCAM-1. The results indicate that VCAM-1 can serve as an accessory molecule or potential coreceptor for HTLV-1-induced cell fusion and provide direct evidence of a role for cell adhesion molecules in the biology of HTLV-1. PMID:8995639

  5. The dynamics of autophagy visualized in live cells: from autophagosome formation to fusion with endo/lysosomes.

    PubMed

    Bampton, Edward T W; Goemans, Christoph G; Niranjan, Dhevahi; Mizushima, Noboru; Tolkovsky, Aviva M

    2005-04-01

    Autophagy has been implicated in a range of disorders and hence is of major interest. However, imaging autophagy in real time has been hampered by lack of suitable markers. We have compared the potential of monodansylcadaverine, widely used as an autophagosomal marker, and the Atg8 homologue LC3, to follow autophagy by fluorescence microscopy whilst labelling late endosomes and lysosomes simultaneously using EGFP-CD63. Monodansylcadaverine labelled only acidic CD63-positive compartments in response to a range of autophagic inducers in various live or post-fixed cells, staining being identical in atg5(+/+) and atg5(-/-) MEFs in which autophagosome formation is disabled. Monodansylcadaverine staining was essentially indistinguishable from that of LysoTracker Red, LAMP-1 or LAMP-2. In contrast, 60-90% of EGFP-LC3-positive punctate organelles did not colocalise with LAMP-1/LAMP-2/CD63 and were monodansylcadaverine-negative while EGFP-LC3 puncta that did colocalise with LAMP-1/LAMP-2/CD63 were also monodansylcadaverine-positive. Hence monodansylcadaverine is no different from other markers of acidic compartments and it cannot be used to follow autophagosome formation. In contrast, fusion of mRFP-LC3-labelled autophagosomes with EGFP-CD63-positive endosomes and lysosomes and sequestration of dsRed-labelled mitochondria by EGFP-LC3- and EGFP-CD63-positive compartments could be visualized in real time. Moreover, transition of EGFP-LC3-I (45 kDa) to EGFP-LC3-II (43 kDa)-traced by immunoblotting and verified by [(3)H]ethanolamine labelling-revealed novel insights into the dynamics of autophagosome homeostasis, including the rapid activation of autophagy by the apoptotic inducer staurosporine prior to apoptosis proper. Use of fluorescent LC3 and a counter-fluorescent endosomal/lysosomal protein clearly allows the entire autophagic process to be followed by live cell imaging with high fidelity.

  6. Significant differences in cell-cell fusion and viral entry between strains revealed by scanning mutagenesis of the C-heptad repeat of HIV gp41.

    PubMed

    Diaz-Aguilar, Barbara; Dewispelaere, Karen; Yi, Hyun Ah; Jacobs, Amy

    2013-05-21

    The transmembrane subunit, gp41, of the HIV envelope mediates the viral fusion step of entry into the host cell. The protein consists of an extracellular domain, a transmembrane domain, and a cytoplasmic tail. The extracellular domain contains a fusion peptide, an N-terminal heptad repeat, a loop region, a C-terminal heptad repeat (CHR), and a membrane-proximal external region. For this study, we examined each amino acid in the CHR (residues 623-659) by alanine scanning mutagenesis in two HIV strains: one CCR5-utilizing strain (JRFL) and one CXCR4-utilizing strain (HXB2). We studied the functional importance of each amino acid residue by measuring mutational effects in both cell-cell fusion and viral entry and assessing envelope expression and gp120-gp41 proteolytic processing. The transmembrane subunit of the HIV envelope, gp41, is very sensitive to subtle changes, like alanine substitution, which severely affect envelope function at multiple sites. Two important general findings are apparent when the entire data set from this study is taken into account. (1) Strain HXB2 is much more stable to mutagenesis than strain JRFL, and (2) viral entry is much more stable to mutagenesis than cell-cell fusion. These findings strengthen our notion that gp41 is a vulnerable target for therapeutic and prophylactic intervention. Further structural studies aimed at gaining a full understanding of the intermediate states that drive HIV membrane fusion are imperative.

  7. Recombinant TAT-Gelonin Fusion Toxin: Synthesis and Characterization of Heparin/Protamine-Regulated Cell Transduction

    PubMed Central

    Zhang, Jian; Huang, Yongzhuo; He, Huining; Wang, Mei; Min, Kyoung Ah; Yang, Victor C.

    2014-01-01

    Protein toxins, such as gelonin, are highly desirable anti-cancer drug candidates due to their unparalleled potency and repetitive reaction mechanism in inhibiting protein translation. However, for its potential application in cancer therapy, there remains the cell membrane barrier that allows permeation of only small molecules, which must be overcome. To address this challenge, we conjugated gelonin with a protein transduction domain (PTD), the TAT peptide, via genetic recombination. The chimeric TAT-gelonin fusion protein (TAT-Gel) retained equipotent N-glycosidase activity yet displayed greater cell uptake than unmodified recombinant gelonin (rGel), thereby yielding a significantly augmented cytotoxic activity. Remarkably, TATGel displayed up to 177-fold lower IC50 (avg. 54.3 nM) than rGel (avg. IC50: 3640 nM) in tested cell lines. This enhanced cytotoxicity, however, also raised potential toxicity concerns due to the non-selectivity of PTD in its mediated cell transduction. To solve this problem, we investigated the plausibility of regulating the cell transduction of TAT-Gel via a reversible masking using heparin and protamine. Here, we demonstrated, both in vitro and in vivo, that the cell transduction of TAT-Gel can be completely curbed with heparin and yet this heparin block can be efficiently reversed by the addition of protamine. This reversible tight regulation of the cell transduction of TAT-Gel by heparin and protamine sheds light of possible application of TATGel in achieving a highly effective yet safe drug therapy for the treatment of tumors. PMID:24733757

  8. Human metapneumovirus Induces Reorganization of the Actin Cytoskeleton for Direct Cell-to-Cell Spread

    PubMed Central

    El Najjar, Farah; Cifuentes-Muñoz, Nicolás; Zhu, Haining; Buchholz, Ursula J.; Moncman, Carole L.; Dutch, Rebecca Ellis

    2016-01-01

    Paramyxovirus spread generally involves assembly of individual viral particles which then infect target cells. We show that infection of human bronchial airway cells with human metapneumovirus (HMPV), a recently identified paramyxovirus which causes significant respiratory disease, results in formation of intercellular extensions and extensive networks of branched cell-associated filaments. Formation of these structures is dependent on actin, but not microtubule, polymerization. Interestingly, using a co-culture assay we show that conditions which block regular infection by HMPV particles, including addition of neutralizing antibodies or removal of cell surface heparan sulfate, did not prevent viral spread from infected to new target cells. In contrast, inhibition of actin polymerization or alterations to Rho GTPase signaling pathways significantly decreased cell-to-cell spread. Furthermore, viral proteins and viral RNA were detected in intercellular extensions, suggesting direct transfer of viral genetic material to new target cells. While roles for paramyxovirus matrix and fusion proteins in membrane deformation have been previously demonstrated, we show that the HMPV phosphoprotein extensively co-localized with actin and induced formation of cellular extensions when transiently expressed, supporting a new model in which a paramyxovirus phosphoprotein is a key player in assembly and spread. Our results reveal a novel mechanism for HMPV direct cell-to-cell spread and provide insights into dissemination of respiratory viruses. PMID:27683250

  9. Lipopolysaccharide induces multinuclear cell from RAW264.7 line with increased phagocytosis activity

    SciTech Connect

    Nakanishi-Matsui, Mayumi; Yano, Shio; Matsumoto, Naomi; Futai, Masamitsu

    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.

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

    SciTech Connect

    Lyu, Qing; Tou, Fangfang; Su, Hong; Wu, Xiaoyong; Chen, Xinyi; Zheng, Zhi

    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.

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

    PubMed Central

    Suh, Jin Sook; Lee, Jue Yeon; Choi, Yoon Jung; You, Hyung Keun; Hong, Seong-Doo; Chung, Chong Pyoung; Park, Yoon Jeong

    2014-01-01

    Protein-transduction technology has been attempted to deliver macromolecular materials, including protein, nucleic acids, and polymeric drugs, for either diagnosis or therapeutic purposes. Herein, fusion protein composed of an arginine-rich cell-penetrating peptide, termed low-molecular-weight protamine (LMWP), and a transcriptional coactivator with a PDZ-binding motif (TAZ) protein was prepared and applied in combination with biomaterials to increase bone-forming capacity. TAZ has been recently identified as a specific osteogenic stimulating transcriptional coactivator in human mesenchymal stem cell (hMSC) differentiation, while simultaneously blocking adipogenic differentiation. However, TAZ by itself cannot penetrate the cells, and thus needs a transfection tool for translocalization. The LMWP-TAZ fusion proteins were efficiently translocalized into the cytosol of hMSCs. The hMSCs treated with cell-penetrating LMWP-TAZ exhibited increased expression of osteoblastic genes and protein, producing significantly higher quantities of mineralized matrix compared to free TAZ. In contrast, adipogenic differentiation of the hMSCs was blocked by treatment of LMWP-TAZ fusion protein, as reflected by reduced marker-protein expression, adipocyte fatty acid-binding protein 2, and peroxisome proliferator-activated receptor-γ messenger ribonucleic acid levels. LMWP-TAZ was applied in alginate gel for the purpose of localization and controlled release. The LMWP-TAZ fusion protein-loaded alginate gel matrix significantly increased bone formation in rabbit calvarial defects compared with alginate gel matrix mixed with free TAZ protein. The protein transduction of TAZ fused with cell-penetrating LMWP peptide was able selectively to stimulate osteogenesis in vitro and in vivo. Taken together, this fusion protein-transduction technology for osteogenic protein can thus be applied in combination with biomaterials for tissue regeneration and controlled release for tissue

  12. Listeria monocytogenes induces mast cell extracellular traps.

    PubMed

    Campillo-Navarro, Marcia; Leyva-Paredes, Kahiry; Donis-Maturano, Luis; González-Jiménez, Marco; Paredes-Vivas, Yuriria; Cerbulo-Vázquez, Arturo; Serafín-López, Jeanet; García-Pérez, Blanca; Ullrich, Stephen E; Flores-Romo, Leopoldo; Pérez-Tapia, Sonia M; Estrada-Parra, Sergio; Estrada-García, Iris; Chacón-Salinas, Rommel

    2017-02-01

    Mast cells play an essential role in different immunological phenomena including allergy and infectious diseases. Several bacteria induce mast cell activation leading to degranulation and the production of several cytokines and chemokines. However, mast cells also have different microbicidal activities such as phagocytosis and the release of DNA with embedded granular proteins known as Mast Cell Extracellular Traps (MCETs). Although previous reports indicate that extracellular bacteria are able to induce MCETs little is known if intracellular bacteria can induce these structures. In this work, we evaluated MCETs induction by the intracellular bacteria Listeria monocytogenes. We found that mast cells released DNA after stimulation with L. monocytogenes, and this DNA was complexed to histone and tryptase. Before extracellular DNA release, L. monocytogenes induced modifications to the mast cell nuclear envelope and DNA was detected outside the nucleus. L. monocytogenes stimulated mast cells to produce significant amounts of reactive oxygen species (ROS) and blocking NADPH oxidase diminished DNA release by mast cells. Finally, MCETs showed antimicrobial activity against L. monocytogenes that was partially blocked when β-hexosaminidase activity was inhibited. These results show that L. monocytogenes induces mast cells to produce microbicidal MCETs, suggesting a role for mast cells in containing infection beyond the induction of inflammation.

  13. Protection of macaques from vaginal SHIV challenge by vaginally delivered inhibitors of virus-cell fusion.

    PubMed

    Veazey, Ronald S; Klasse, Per Johan; Schader, Susan M; Hu, Qinxue; Ketas, Thomas J; Lu, Min; Marx, Preston A; Dufour, Jason; Colonno, Richard J; Shattock, Robin J; Springer, Martin S; Moore, John P

    2005-11-03

    Human immunodeficiency virus type 1 (HIV-1) continues to spread, principally by heterosexual sex, but no vaccine is available. Hence, alternative prevention methods are needed to supplement educational and behavioural-modification programmes. One such approach is a vaginal microbicide: the application of inhibitory compounds before intercourse. Here, we have evaluated the microbicide concept using the rhesus macaque 'high dose' vaginal transmission model with a CCR5-receptor-using simian-human immunodeficiency virus (SHIV-162P3) and three compounds that inhibit different stages of the virus-cell attachment and entry process. These compounds are BMS-378806, a small molecule that binds the viral gp120 glycoprotein and prevents its attachment to the CD4 and CCR5 receptors, CMPD167, a small molecule that binds to CCR5 to inhibit gp120 association, and C52L, a bacterially expressed peptide inhibitor of gp41-mediated fusion. In vitro, all three compounds inhibit infection of T cells and cervical tissue explants, and C52L acts synergistically with CMPD167 or BMS-378806 to inhibit infection of cell lines. In vivo, significant protection was achieved using each compound alone and in combinations. CMPD167 and BMS-378806 were protective even when applied 6 h before challenge.

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

    PubMed

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

    2008-08-08

    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.

  15. Determination of the minimal fusion peptide of bovine leukemia virus gp30

    SciTech Connect

    Lorin, Aurelien; Lins, Laurence; Stroobant, Vincent; Brasseur, Robert . E-mail: brasseur.r@fsagx.ac.be; Charloteaux, Benoit

    2007-04-13

    In this study, we determined the minimal N-terminal fusion peptide of the gp30 of the bovine leukemia virus on the basis of the tilted peptide theory. We first used molecular modelling to predict that the gp30 minimal fusion peptide corresponds to the 15 first residues. Liposome lipid-mixing and leakage assays confirmed that the 15-residue long peptide induces fusion in vitro and that it is the shortest peptide inducing optimal fusion since longer peptides destabilize liposomes to the same extent but not shorter ones. The 15-residue long peptide can thus be considered as the minimal fusion peptide. The effect of mutations reported in the literature was also investigated. Interestingly, mutations related to glycoproteins unable to induce syncytia in cell-cell fusion assays correspond to peptides predicted as non-tilted. The relationship between obliquity and fusogenicity was also confirmed in vitro for one tilted and one non-tilted mutant peptide.

  16. New model for cardiomyocyte sheet transplantation using a virus-cell fusion technique

    PubMed Central

    Takahashi, Yuto; Tomotsune, Daihachiro; Takizawa, Sakiko; Yue, Fengming; Nagai, Mika; Yokoyama, Tadayuki; Hirashima, Kanji; Sasaki, Katsunori

    2015-01-01

    AIM: To facilitate close contacts between transplanted cardiomyocytes and host skeletal muscle using cell fusion mediated by hemagglutinating virus of Japan envelope (HVJ-E) and tissue maceration. METHODS: Cardiomyocytes (1.5 × 106) from fetal rats were first cultured. After proliferation, some cells were used for fusion with adult muscle fibers using HVJ-E. Other cells were used to create cardiomyocyte sheets (area: about 3.5 cm2 including 2.1 × 106 cells), which were then treated with Nile blue, separated, and transplanted between the latissimus dorsi and intercostal muscles of adult rats with four combinations 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 beating domains, motion images using moving target analysis software, action potentials, gene expression of MLC-2v and Mesp1 by reverse transcription-polymerase chain reaction, hematoxylin-eosin staining, and immunostaining for cardiac troponin and skeletal myosin. RESULTS: In vitro cardiomyocytes were fused with skeletal muscle fibers using HVJ-E. Cardiomyocyte sheets remained in the primary transplanted sites for 2 wk. Although beating domains were detected in G1, G2, and G3 rats, G1 rats prevailed in the number, size, motion image amplitudes, and action potential compared with G2 and G3 rats. Close contacts were only found in G1 rats. At 1 wk after transplantation, the cardiomyocyte sheets showed adhesion at various points to the myoblast layer in the latissimus dorsi muscle. At 2 wk after transplantation, close contacts were seen over a broad area. Part of the skeletal muscle sarcoplasma seemed to project into the myocardiocyte plasma and some nuclei appeared to share both sarcoplasmas. CONCLUSION: The present results show that close contacts were acquired and facilitated

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

    DOE PAGES

    Jonkers, Wilfried; Leeder, Abigail C.; Ansong, Charles; ...

    2014-11-20

    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 NRC1, MEK2 and MAK2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every 4 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a protein of unknown biochemical function. How this 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) thatmore » can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM5-GFP co-localized with NRC1, MEK2 and MAK2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK2 activity influences HAM5 function/localization. However, MAK2-GFP showed only cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta, as observed in wild type germlings. Via co-immunoprecipitation experiments, HAM5 was shown to physically interact with MAK2, MEK2 and NRC1, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members during oscillation and chemotropic interactions during both germling and hyphal fusion in N. crassa. The identification of HAM5 as a scaffold-like protein will help to link the activation of MAK2 to upstream factors and other proteins involved in this intriguing process of fungal

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

    PubMed

    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.

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

    DTIC Science & Technology

    2009-07-01

    adhesion, and maturation markers as well as the stimulatory cytokines, IL-12 and IFNγ. In addition, fusion cells expressed CCR7 necessary for the...stimulation with anti-CD3/CD28 results in a marked expansion of anti-tumor effector cells. Body Our clinical protocol had received approval by...34, was received by the Cancer’therapy Evaluation Program of the Division of Cancer Treatment and Diagnosis, National Cancer Institute on 071) 612009

  20. Evaluating Osteogenic Potential of Ligamentum Flavum Cells Cultivated in Photoresponsive Hydrogel that Incorporates Bone Morphogenetic Protein-2 for Spinal Fusion

    PubMed Central

    Chiang, Chih-Wei; Chen, Wei-Chuan; Liu, Hsia-Wei; Wang, I-Chun; Chen, Chih-Hwa

    2015-01-01

    Regenerative medicine is increasingly important in clinical practice. Ligamentum flava (LF) are typically removed during spine-related surgeries. LF may be a source of cells for spinal fusion that is conducted using tissue engineering techniques. In this investigation, LF cells of rabbits were isolated and then characterized by flow cytometry, morphological observation, and immunofluorescence staining. The LF cells were also cultivated in polyethylene (glycol) diacrylate (PEGDA) hydrogels that incorporated bone morphogenetic protein-2 (BMP-2) growth factor, to evaluate their proliferation and secretion of ECM and differentiation in vitro. The experimental results thus obtained that the proliferation, ECM secretion, and differentiation of the PEGDA-BMP-2 group exceeded those of the PEGDA group during the period of cultivation. The mineralization and histological staining results differed similarly. A nude mice model was utilized to prove that LF cells on hydrogels could undergo osteogenic differentiation in vivo. These experimental results also revealed that the PEGDA-BMP-2 group had better osteogenic effects than the PEGDA group following a 12 weeks after transplantation. According to all of these experimental results, LF cells are a source of cells for spinal fusion and PEGDA-BMP-2 hydrogel is a candidate biomaterial for spinal fusion by tissue engineering. PMID:26426006

  1. Over-expression of TSC-22 (TGF-beta stimulated clone-22) markedly enhances 5-fluorouracil-induced apoptosis in a human salivary gland cancer cell line.

    PubMed

    Uchida, D; Kawamata, H; Omotehara, F; Miwa, Y; Hino, S; Begum, N M; Yoshida, H; Sato, M

    2000-06-01

    We have recently isolated TSC-22 (transforming growth factor-beta-stimulated clone-22) cDNA as an anticancer, drug-inducible (with vesnarinone) gene in a human salivary gland cancer cell line, TYS. We have also reported that TSC-22 negatively regulates the growth of TYS cells and that down-regulation of TSC-22 in TYS cells plays a major role in salivary gland tumorigenesis (Nakashiro et al, 1998). In this study, we transfected TYS cells with an expression vector encoding the TSC-22-GFP (green fluorescent protein) fusion protein, and we established TSC-22-GFP-expressing TYS cell clones. Next, we examined (a) the subcellular localization of the fusion protein, (b) the sensitivity of the transfectants to several anticancer drugs (5-fluorouracil, cis-diaminedichloroplatinum, peplomycin), and (c) induction of apoptotic cell death in the transfectants by 5-fluorouracil treatment. The TSC-22-GFP fusion protein was clearly localized to the cytoplasm, but not to the nucleus. Over-expression of the TSC-22-GFP fusion protein did not affect cell growth, but significantly increased the sensitivity of the cells to the anticancer drugs (p < 0.01; one-way ANOVA). Furthermore, over-expression of the TSC-22-GFP fusion protein markedly enhanced 5-fluorouracil-induced apoptosis. These findings suggest that over-expression of TSC-22-GFP protein in TYS cells enhances the chemosensitivity of the cells via induction of apoptosis.

  2. Perspective for special Gurdon issue for differentiation: can cell fusion inform nuclear reprogramming?

    PubMed

    Burns, David; Blau, Helen M

    2014-07-01

    Nuclear reprogramming was first shown to be possible by Sir John Gurdon over a half century ago. The process has been revolutionized by the production of induced pluripotent cells by overexpression of the four transcription factors discovered by Shinya Yamanaka, which now enables mammalian applications. Yet, reprogramming by a few transcription factors remains incomplete and inefficient, whether to pluripotent or differentiated cells. We propose that a better understanding of mechanistic insights based on developmental principles gained from heterokaryon studies may inform the process of directing cell fate, fundamentally and clinically.

  3. Coupled-channel effects in elastic scattering and near-barrier fusion induced by weakly bound nuclei and exotic halo nuclei

    SciTech Connect

    Beck, C.; Keeley, N.

    2007-05-15

    The influence on fusion of coupling to the breakup process is investigated for reactions where at least one of the colliding nuclei has a sufficiently low binding energy for breakup to become an important process. Elastic scattering, excitation functions for sub- and near-barrier fusion cross sections, and breakup yields are analyzed for {sup 6,7}Li+{sup 59}Co. Continuum-discretized coupled-channels (CDCC) calculations describe well the data at and above the barrier. Elastic scattering with {sup 6}Li (as compared to {sup 7}Li) indicates the significant role of breakup for weakly bound projectiles. A study of {sup 4,6}He induced fusion reactions with a three-body CDCC method for the {sup 6}He halo nucleus is presented. The relative importance of breakup and bound-state structure effects on total fusion is discussed.

  4. Differential roles of SS18-SSX fusion gene and insulin-like growth factor-1 receptor in synovial sarcoma cell growth

    SciTech Connect

    Toernkvist, Maria; Natalishvili, Natalia; Xie Yuntao; Girnita, Ada; D'Arcy, Padraig; Brodin, Bertha; Axelson, Magnus; Girnita, Leonard

    2008-04-11

    Recently we demonstrated that the synovial sarcoma specific fusion gene SS18-SSX is crucial for cyclin D1 expression and is linked to cell proliferation. In this report we explore the role of SS18-SSX and IGF-1R for their potential functions in cellular proliferation and survival in cultured synovial sarcoma cells. We found that targeting of SS18-SSX mRNA by antisense oligonucleotide treatment drastically and rapidly decreased cell proliferation but caused only a slight increase of apoptosis. The synovial sarcoma cells were confirmed to express IGF-1R, and treatment with an IGF-1R inhibitor resulted in substantially reduced cell viability by inducing apoptosis in these cells. Conversely, inhibition of the IGF-1R resulted only in a slight to moderate decrease in DNA synthesis. In conclusion, SS18-SSX and IGF-1R seem to play important but different roles in maintaining malignant growth of synovial sarcoma cells. Whereas SS18-SSX maintains cyclin D1 and cell proliferation, IGF-1R protects from apoptosis.

  5. HIV transcription is induced with cell killing

    SciTech Connect

    Woloschak, G.E.; Schreck, S.; Chang-Liu, Chin-Mei; Panozzo, J.; Libertin, C.R.

    1993-11-01

    In this report, we demonstrate that this induction of HIV-LTR transcription occurs when stably transfected HeLa cells are exposed to agents which mediate cell killing, such as UV radiation, electroporation of sucrose buffer, prolonged heating, and low and high pH. Cells cultured following UV exposure demonstrated a peak in CAT expression that is evident in viable (but not necessarily cell division-competent) cells 24 h after exposure; this inductive response continued until at least 72 h after exposure. HIV-LTR induction was dose-dependent, and the amount of CAT transcription induced was correlated with the amount of cell killing that occurred in the culture.

  6. Liposome reconstitution of a minimal protein-mediated membrane fusion machine

    PubMed Central

    Top, Deniz; de Antueno, Roberto; Salsman, Jayme; Corcoran, Jennifer; Mader, Jamie; Hoskin, David; Touhami, Ahmed; Jericho, Manfred H; Duncan, Roy

    2005-01-01

    Biological membrane fusion is dependent on protein catalysts to mediate localized restructuring of lipid bilayers. A central theme in current models of protein-mediated membrane fusion involves the sequential refolding of complex homomeric or heteromeric protein fusion machines. The structural features of a new family of fusion-associated small transmembrane (FAST) proteins appear incompatible with existing models of membrane fusion protein function. While the FAST proteins function to induce efficient cell–cell fusion when expressed in transfected cells, it was unclear whether they function on their own to mediate membrane fusion or are dependent on cellular protein cofactors. Using proteoliposomes containing the purified p14 FAST protein of reptilian reovirus, we now show via liposome–cell and liposome–liposome fusion assays that p14 is both necessary and sufficient for membrane fusion. Stoichiometric and kinetic analyses suggest that the relative efficiency of p14-mediated membrane fusion rivals that of the more complex cellular and viral fusion proteins, making the FAST proteins the simplest known membrane fusion machines. PMID:16079913